Peptide screening of cerebrospinal fluid in patients with glioblastoma multiforme.

AIMS: To apply modern mass spectrometry based technology to identify possible CSF peptide markers of glioblastoma multiforme (GBM). METHODS: Mass spectrometry based peptidomics technology enables a systematic and comprehensive screening of cerebrospinal fluid (CSF) with regard to its peptide composition. Differential Peptide Display (DPD) allows the identification of single marker peptides for a target disease. Using both, we analyzed CSF samples of 11 patients harbouring a glioblastoma multiforme in comparison to 13 normal controls. RESULTS: Four CSF peptides which significantly distinguished GBM from controls in all applied statistic tests could be identified out of more than 2,000 detected CSF peptides. They were specific C-terminal fragments of alpha-1-antichymotrypsin, osteopontin, and transthyretin as well as a N-terminal residue of albumin. All molecules are constituents of normal CSF, but none has previously been reported to be significantly elevated in CSF of GBM patients. CONCLUSION: The study showed that peptidomics technology is able to identify possible biomarkers of neoplastic CNS disease. It remains to be determined if the identified elevated CSF peptides are specific for GBM. With regard to GBM, however, the more important role of CSF peptide biomarkers than aiding initial diagnosis might be early recognition of disease recurrence or monitoring of efficacy of adjuvant therapy protocols.

Multiple vascular abnormalities and a paradoxical combination of vitamin B12 deficiency and thrombocytosis in a case with POEMS syndrome.

POEMS/Crow-Fukase syndrome is a rare multisystem disorder associated with elevated vascular endothelial growth factor (VEGF), which clinically presents with polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes. We report a case of POEMS syndrome due to a gammopathy of undetermined significance with thrombocytosis, vitamin B(12) deficiency, highly elevated VEGF and in addition to glomeruloid angiomas two previously undescribed proliferative vascular lesions: a spinal arteriovenous fistula and a plexogenic pulmonary arteriopathy, which ultimately resulted in lethal pulmonary hypertension. We assume that the high VEGF levels caused the vascular abnormalities observed in our patient.

Plasma concentrations of 5-HT, 5-HIAA, norepinephrine, epinephrine and dopamine in ecstasy users.

Recreational use of the synthetic methamphetamine derivative MDMA (3,4-methylenedioxymethamphetamine), the main constituent of the illegal drug "ecstasy", has increased dramatically in recent years. The reasons for ecstasy-associated cardiovascular complications like tachycardia, arrhythmias and hypertensive crises and psychiatric symptoms like psychotic episodes are not well understood. We have measured the plasma concentrations of 5-HIAA, 5-HT, norepinephrine, epinephrine and dopamine in 159 ecstasy users and controls. Ecstasy users showed elevated resting sympathetic activity, reflected in increased norepinephrine, epinephrine and dopamine levels. The levels of these catecholamines correlated positively with the cumulative dose and also with consumption during the last 30 days and 12 months. Although it is known that significant changes in 5-HT and 5-HIAA appear in the cerebrospinal fluid in ecstasy users, we could not detect alterations in serotonergic neurotransmitters in plasma in this large sample of subjects. Thus, in the drug-free interval, ecstasy users show lowered central serotonergic activity (lowered 5-HT and 5-HIAA concentrations in CSF) along with unchanged central noradrenergic and dopaminergic activity (HVA and MHPG unchanged in CSF) and elevated peripheral noradrenergic, dopaminergic and adrenergic activity along with unchanged peripheral serotonergic activity (plasma levels). We conclude, that the data presented here could argue for a noradrenergic hyperreactivity in the drug-free interval in ecstasy users resulting from previous ecstasy consumption. Also for an association with psychotic episodes and cardiovascular complications like tachycardia, arrhythmias.

Matrix metalloproteinases in inflammatory myopathies: enhanced immunoreactivity near atrophic myofibers.

OBJECTIVES: To further examine the role of proteolytic enzyme expression of matrix metalloproteinases (MMP) and T-cell markers in inflammatory myopathies and controls. MATERIAL AND METHODS: We studied the expression of MMP-2, MMP-7, and MMP-9 in 19 cases of inflammatory myopathies and controls using immunocytochemistry. RESULTS: Inflammatory myopathies showed distinct patterns of up-regulation of MMP. MMP-9 was strongly expressed in atrophic myofibers in all inflammatory myopathies. MMP-2 immunoreactivity was similar in its distribution, however, to a weaker intensity. In dermatomyositis the perifascicular atrophy showed pronounced MMP-9 immunoreactivity, probably reflecting denervated patterns of myofibers. Moreover, MMP-7 strongly immunolabeled invaded myofibers in polymyositis cases only. CONCLUSION: These patterns confirm, that MMP-7 up-regulation is prominent in PM, while MMP-2 immunoreactivity is only slightly elevated in inflamed muscle. In general, MMP-9 up-regulation appears to be an important additional molecular event in the multistep process of all inflammatory myopathies.

COMT-inhibition increases serum levels of dihydroxyphenylacetic acid (DOPAC) in patients with advanced Parkinson's disease.

UNLABELLED: Inhibition of the catechol-O-methyltransferase (COMT) is an effective treatment for end-of-dose fluctuations in advanced Parkinson's disease. The aim of the present investigation was to analyse the consequences of subsequent alterations in levodopa metabolism under common treatment conditions when the levodopa dose is adjusted due to the occurrence of dyskinesias after initiation of the COMT-inhibitor. Ten patients with advanced Parkinson's disease (Hoehn & Yahr stage IV) were medicated with tolcapone. Prior to and five to ten days after the initiation of tolcapone 300 mg/d, serum level profiles of levodopa and its metabolites (3-O-methyldopa (3-OMD), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)) were performed. The mean daily levodopa dose was reduced from 894 +/- 248 mg to 646 +/- 252 mg (p = 0.003). There was a significant increase in the area under the curve (AUC) of DOPAC during COMT-inhibition compared to the baseline profile (p = 0.009). There were significant decreases of the AUC of HAV (p = 0.001) and the ratios of the AUC HVA / AUC DOPAC (p = 0.0001) and AUC 3-OMD / AUC levodopa (p = 0.0001). CONCLUSION: The elevation of DOPAC and the decrease of HVA and HVA / DOPAC reflect a shift of the levodopa metabolism towards the MAO-B dependent oxidative pathway. This might contribute to production of hydroxyl radicals and induction of oxidative stress.

Characterization of four lipoprotein classes in human cerebrospinal fluid.

Lipoprotein metabolism in brain has not yet been fully elucidated, although there are a few reports concerning lipids in the brain and lipoproteins and apolipoproteins in the cerebrospinal fluid (CSF). To establish normal levels of lipoproteins in human CSF, total cholesterol, phospholipids, and fatty acids as well as apolipoprotein E (apoE) and apoA-I levels were determined in CSF samples from 216 individuals. For particle characterization, lipoproteins from human CSF were isolated by affinity chromatography and analyzed for size, lipid and apolipoprotein composition. Two consecutive immunoaffinity columns with antibodies, first against apoE and subsequently against apoA-I, were used to define four distinct lipoprotein classes. The major lipoprotein fraction consisted of particles of 13;-20 nm containing apoE and apoA-I as well as apoA-IV, apoD, apoH, and apoJ. In the second particle class (13;-18 nm) mainly apoA-I and apoA-II but no apoE was detected. Third, there was a small number of large particles (18;-22 nm) containing no apoA-I but apoE associated with apoA-IV, apoD, and apoJ. In the unbound fraction we detected small particles (10;-12 nm) with low lipid content containing apoA-IV, apoD, apoH, and apoJ. In summary, we established lipid and apolipoprotein levels in CSF in a large group of individuals and described four distinct lipoprotein classes in human CSF, differing in their apolipoprotein pattern, lipid composition, and size. On the basis of our own data and previous findings from other groups, we propose a classification of CSF lipoproteins.

The roles of carnosine in aging of skeletal muscle and in neuromuscular diseases.

Skeletal muscles undergo specific alterations that are related to the aging process. The incidence of several neuromuscular diseases (e.g., amyotrophic lateral sclerosis (ALS), myasthenia gravis, polymyositis, drug-induced myopathies, late-onset mitochondrial myopathy) is age-related. The increased sensitivity to disease of aging muscle represents an additional age-related negative influence in the presence of existing risk factors (such as a genetic predisposition). The potential significance of carnosine lies on one hand in its possible influence on specific physiological changes in muscle associated with the aging process, and on the other in its effect on oxidative stress and the antioxidative system in specific neuromuscular diseases such as ALS or polymyositis.

Rapid clearance of human immunodeficiency virus type 1 from ventricular cerebrospinal fluid during antiretroviral treatment.

To understand the pathogenesis of human immunodeficiency virus-induced neuropathology, it is critical to know the dynamics of viral replication in the central nervous system. Viral decay kinetics were mathematically analyzed from multiple serial specimens of ventricular cerebrospinal fluid and plasma during antiretroviral therapy in a patient with asymptomatic human immunodeficiency virus infection and an external ventricular catheter for hydrocephalus. A rapid exponential decay of virus with an elimination half-life of 4.2 days in ventricular cerebrospinal fluid and 2.3 days in plasma was found. Sequencing the V3 loop-encoding envelope gene of virus in both compartments revealed high sequence homology. The combined data suggest that virus in ventricular cerebrospinal fluid is at least partly contributed by rapidly replicating virus-producing cells recruited from the circulation.

CSF copper concentrations, blood-brain barrier function, and coeruloplasmin synthesis during the treatment of Wilson's disease.

During the treatment of four patients with cerebral manifestation of Wilson's disease, we measured the copper concentration in the cerebrospinal fluid (CSF) and serum, the serum coeruloplasmin concentration, the free copper concentration in the serum, and the albumin ratio CSF/serum (AR). These measurements were treated as indicators of the copper-related toxic effects on the brain and the blood-brain barrier (BBB). The half-life of the decrease in the CSF copper concentration during therapy was 23.5 +/- 5.78 months (mean +/- S.E.M.). The therapeutic-target-copper concentration in the CSF (mean normal concentration) is below 20 microg/l. The average length of therapy needed to normalize CSF-copper values in our patients with an average initial value of 76.25 microg/L was 47 month. During the first 10 month of treatment there was an increase in all cases of the measured disturbance in the blood-brain barrier (measured as the ratio of albumin in CSF to albumin in serum, AR). All patients showed an initial worsening of the neurological condition, on average after 1.75 +/- 0.25 months. The maximal rise in AR, from the initial values, was on average 18.4 +/- 5.08%; this maximum was reached after an average of 6.9 +/- 1.5 months. The AR normalized during therapy, indicating a reduction in toxicity in the blood-brain barrier region. The extent of the AR increases in individual patients did not correlate significantly with CSF copper half-life, serum copper half-life, the initial half-life of the reduction in the ratio (copper in serum)/(coeruloplasmin in serum), the initial copper concentration in CSF or serum, the initial free copper concentration in serum, or the initial dose of penicillamine (within the first 2 months). We conclude that the normalization of the CSF copper concentration in patients with the cerebral manifestation of Wilson's disease is a slow process, even if therapy is sufficient. The initial worsening of the neurological condition which has often been reported may be reflected in the disturbance of blood-brain barrier function, which we have measured here for the first time (using the parameter of the albumin ratio CSF/serum). Based on repeated measurements of the AR during the course of treatment it seems that the brain toxicity of mobilized copper can be assessed and the therapy adjusted.

Glucocorticoid receptor concentrations in muscle biopsies from patients with neuromuscular diseases.

Increases in circulating glucocorticoids promote catabolism, particularly in skeletal muscle. The sensitivity of the muscle to glucocorticoids can be altered by a change in the number of glucocorticoid receptors in the muscle, or by a change in the proportions of activated receptors (between binders IB and II). We have investigated the concentration of glucocorticoid receptors, and the proportions of types IB and II, in healthy and diseased muscle. We found significantly reduced concentrations of glucocorticoid receptors in the group of inflammatory myopathies (51% reduction; P < 0.05, Wilcoxon signed rank test). No significant changes in the relative proportions of binders IB and II were found in pathological muscle, although the proportion of binder IB tended towards elevated values (especially in the diabetic neuropathies, with a 17% increase). We conclude that the sensitivity of muscle to glucocorticoids can be reduced in neuromuscular diseases, especially in myositis, by a reduction in the number of glucocorticoid receptors in the tissue, but that no relevant shift in the relation between activated receptor types is present. This could be important in relation to the risk of a secondary steroid myopathy and catabolism of skeletal muscle in the treatment of inflammatory myopathies with glucocorticoids.

Age effects on growth hormone and testosterone responses to endurance exercise in patients with neuromuscular diseases.

We have investigated the effect of age on the changes in growth hormone (GH) and testosterone induced by muscular exercise in patients with neuromuscular diseases, by means of a test procedure which maintained compatibility. We investigated 21 control subjects and 36 patients. We found a significant increase in GH concentrations (+329%; p<0.0001) after 20 min of exercise on a bicycle ergometer. Testosterone concentrations showed no significant change. There was a significant negative correlation between the GH level after exercise and the age of the patients (R=-0.3, p=0.015). The influence of increasing age on the GH reaction in patients with neuromuscular disease is substantial and could have a significant influence on muscle mass, muscle strength and the time at which decompensation occurs in elderly patients with neuromuscular diseases.

Influence of age on metabolism of testosterone, dihydrotestosterone, and 3-alpha-androstanediol in muscle biopsies from patients with neuromuscular diseases.

We investigated the possible effect of age on the metabolism of androgens in muscle biopsies from patients with neuromuscular diseases. The conversion of testosterone, dihydrotestosterone (DHT) and 5-alpha-androstane-3-alpha-17-beta-diol (3-alpha-androstanediol) was measured in muscle biopsies from 24 patients with neuromuscular diseases and seven controls. The reductive metabolism of 3-alpha-HSDH was significantly higher than the oxidative metabolism. Significant metabolism of testosterone to DHT was not found. Only the age of the patients emerged as a significant negative predictor in a stepwise multiple linear regression model for V(max) and K(m) (Lineweaver-Burke plots) of the reductive metabolism of 3-alpha-HSDH. Therefore, altered metabolism of anabolic androgens in skeletal muscles could be demonstrated. We conclude that this could alter the androgenic catabolic/anabolic balance in the (androgenic target organ) skeletal muscle.

Concentrations of free carnosine (a putative membrane-protective antioxidant) in human muscle biopsies and rat muscles.

Carnosine has possible functional effects on skeletal muscle contractility, along with membrane-protective, antioxidant effects. We determined tissue free carnosine concentrations in skeletal muscles from patients with neuromuscular diseases and in skeletal and heart muscles from rats of various ages. The effects of age, gender and diagnostic category on free carnosine levels in patients with neuromuscular diseases were analyzed by a stepwise multiple linear regression model. The age of the patients emerged as a significant negative predictor of carnosine concentrations (R=-0.40, P<0.05). Free carnosine concentrations in rat skeletal muscles also showed a significant negative correlation with the ages of the rats (male rats: R=-0.49, P<0.05; female rats: R=-0.56, P<0.05). Only the diagnostic category amyotrophic lateral sclerosis (ALS) emerged as a significant negative predictor compared to the control group in the stepwise regression model, this was confirmed by Wilcoxon Signed Rank Test (P<0.05). We conclude that the age-related decline in muscle mass, strength and function is associated with decreased tissue concentrations of the putative membrane-protective antioxidant carnosine. In addition we found decreased carnosine tissue concentrations in ALS. The reduction in carnosine content might be caused by progressive denervation processes.

Age effects on serum amino acids in endurance exercise at the aerobic/anaerobic threshold in patients with neuromuscular diseases.

We have measured concentrations of 26 serum amino acids in 46 subjects (aged 17-75 years), with the following neurological diseases: amyotrophic lateral sclerosis, n=7; peripheral neuropathy, n=5; muscular dystrophy, n=7; mitochondriopathy, n=3; metabolic myopathy (others), n=2; inflammatory myopathy, n=4; mononeuropathy, n=3; controls (patients with symptoms suggesting neuromuscular system dysfunction without objective evidence of neuromuscular disease), n=15, before and after prolonged muscular effort. Tests were done on a bicycle ergometer at the individual aerobic/anaerobic threshold determined for each subject in preliminary tests. Using a stepwise multiple linear regression model, age emerged as a significant negative predictor (P<0.05) of the post/before ratio of the levels of five amino acids. We conclude that an increase in recovery time and a reduction in training capacity with aging could be linked to these changes. The cause is assumed to be principally a reduction in glycogen storage in muscle with increasing age; this situation could possibly be improved by consumption of carbohydrate before or during exercise, or also during rehabilitation exercise or training in neuromuscular or other diseases.

Tissue concentrations of nerve growth factor in aging rat heart and skeletal muscle.

In order to examine the association between adult nerve growth factor (NGF) levels and age-related changes in skeletal and heart muscle mass, we determined NGF concentrations in both tissues. NGF concentrations in rat heart muscle were significantly higher than those in skeletal muscle. NGF concentrations in heart muscle had a significant positive correlation with heart muscle wet weight. A causal association may exist between age-related changes in adult heart muscle mass and tissue NGF levels (in contrast to skeletal muscle). Among the potential clinical implications for skeletal muscle, it appears that age-related delay or deterioration in regeneration processes in neuromuscular diseases, or age-related decline in skeletal muscle mass, are not caused by reduced tissue NGF concentrations.