Functional mutation analysis provides evidence for a role of REEP1 in lipid droplet biology.

Hereditary axonopathies are frequently caused by mutations in proteins that reside in the endoplasmic reticulum (ER). Which of the many ER functions are pathologically relevant, however, remains to be determined. REEP1 is an ER protein mutated in hereditary spastic paraplegia (HSP) and hereditary motor neuropathy (HMN). We found that HSP-associated missense variants at the N-terminus of REEP1 abolish ER targeting, whereas two more central variants are either rare benign SNPs or confer pathogenicity via a different mechanism. The mis-targeted variants accumulate at lipid droplets (LDs). N-terminal tagging, deletion of the N-terminus, and expression of a minor REEP1 isoform had the same effect. We also confirmed an increase in LD size upon cooverexpression of atlastins and REEP1. Neither wild-type REEP1, LD-targeted HSP variants, nor a non-LD-targeted HMN variant reproduced this effect when expressed alone. We conclude that the N-terminus of REEP1 is necessary for proper targeting to and/or retention in the ER. The protein's potential to also associate with LDs corroborates a synergistic effect with atlastins on LD size. Interestingly, LD size is also altered upon knockdown of seipin, mutations of which also cause HSP and HMN. Regulation of LDs may thus be an ER function critical for long-term axonal maintenance.

Immediate diagnosis of ventriculits: evaluation of parameters independent of microbiological culture.

BACKGROUND: Generally accepted reference values in CSF diagnostics are not valid in cerebrospinal fluid (CSF) containing large amounts of blood. Residual blood may obscure ventriculitis as diagnostics largely depend on parameters such as cell count, lactic acid and total protein measurement. We sought to improve the diagnostics by evaluating a cytokine panel and soluble CD62L as markers of ventriculitis. In addition, we tested an algorithm of established parameters to predict ventriculitis in a specific patient collective. METHODS: Analysis was performed on ventricular CSF samples from 50 consecutive patients. Gram staining, microbiological culture, total cell count, total protein and CD62L expression on neutrophil granulocytes were analysed immediately. Cytokines and soluble CD62L were measured by flow cytometry. FINDINGS: Positive culture was detected in ten patients. Of all parameters tested only IL1-beta, IL8 and CD62L on neutrophils were significantly different between culture-positive and -negative patients. The highest predictive value was obtained when analysing IL1-beta. The predictive value of a parameter combination (IL6 in CSF, C-reactive protein and leukocytes in periphereal blood) was comparable to IL1-beta. Half of the patients in this analysis were identified as culture positive because of the lack of inflammatory response. CONCLUSIONS: IL1-beta and perhaps also IL8 provide very good analytical performance when looking for ventriculitis in patients with residual blood in CSF. Turn-around time is short, and results could be reported within 1 h for 24 h a day. In some patients application of glucocorticoids may result in restricted inflammatory response. Even in these patients IL1-beta provides a reliable parameter for the immediate diagnosis of ventriculitis.

No influence of 5-HTTLPR gene polymorphism on migraine symptomatology, comorbid depression, and chronification.

BACKGROUND: The serotonergic system is thought to play an important role for mediating susceptibility to migraine and depression, which is frequently found comorbid in migraine. The functional polymorphism in the serotonin transporter gene linked polymorphic region (5-HTTLPR/SLC6A4) was previously associated with attack frequency and, thus, possibly with chronification. OBJECTIVE: We hypothesized that patients with the "s" allele have higher attack frequency and, paralleling results in depression research, higher scores of depression. METHODS: Genetic analysis of the SLC6A4 44 bp insertion/deletion polymorphism (5-HTTLPR) was performed in 293 patients with migraine with and without aura. Self-rating questionnaires were used for assessment of depression. RESULTS: Multinomial logistic regression analysis found no evidence for association of the 5-HTTLPR polymorphism with either depression or migraine attack frequency. CONCLUSION: We were not able to demonstrate any influence of the serotonin transporter 5-HTTLPR polymorphism on migraine phenomenology (attack frequency or comorbid depression), thereby excluding this variant to be a common genetic denominator for chronic migraine and depression.

Analysis of CYP7B1 in non-consanguineous cases of hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a neurodegenerative condition defined clinically by lower limb spasticity and weakness. Homozygous mutations in CYP7B1 have been identified in several consanguineous families that represented HSP type 5 (SPG5), one of the many genetic forms of the disease. We used direct sequencing and multiplex ligation-dependent probe amplification to screen for CYP7B1 alterations in apparently sporadic HSP patients (n = 12) as well as index patients from non-consanguineous families with recessive (n = 8) and dominant (n = 8) transmission of HSP. One sporadic patient showing HSP as well as optic atrophy carried a homozygous nonsense mutation. Compound heterozygosity was observed in a recessive family with a clinically pure phenotype. A heterozygous missense change segregated in a small dominant family. We also found a significant association of a known coding polymorphism with cerebellar signs complicating a primary HSP phenotype. Our findings suggest CYP7B1 alterations to represent a rather frequent cause of HSP that should be considered in patients with various clinical presentations.

Approaching clinical proteomics: current state and future fields of application in cellular proteomics.

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

Quantitation of serum free light chains does not compensate for serum immunofixation only when screening for monoclonal gammopathies.

BACKGROUND: Detection of plasma cell dyscrasias (PCD) requires screening of serum and urine for monoclonal proteins. Several studies have demonstrated increased sensitivity and specificity when measurement of serum free light chain (SFLC) is part of the screening protocol. In addition, omission of immunofixation (IFE) in the standard work-up that includes SFLC assay has been proposed. This study attempts to define the role of the SFLC assay in a screening strategy limited to serum only. It compares outcomes to a serum-only screening strategy that omits serum IFE. METHODS: Serum from 691 patients was analysed for the presence of monoclonal protein using standard serum IFE, serum protein electrophoresis (SPE) and measurement of SFLC. Data were analysed retrospectively. RESULTS: Specificity and sensitivity of abnormal SFLC-ratios for the detection of monoclonal protein using IFE were 96% and 41%, respectively. Eighteen patients with negative monospecific and Bence Jones IFE, but abnormal SFLC-ratios were identified. In most cases, this could be attributed to kidney and inflammatory disease or haematological disorders. In four cases, this resulted in further diagnostic investigation and light chain disease was later detected in two cases. Light chain disease was confirmed in one case but not confirmed in the other patient. In 14 patients, Bence Jones IFE was negative, although the concentrations of SFLC suggested the presence of monoclonal Bence Jones protein at concentrations detectable by IFE. Thus, either the anti-serum failed at detection, there was polymerisation of the free light chains or the SFLC assay overestimated protein concentrations. Simulating a work-up that included IFE only if abnormalities were detected by SPE or the SFLC assay would have resulted in 26% fewer IFEs being performed, but three patients with monoclonal proteins by IFE would have been missed. CONCLUSIONS: Abnormal SFLC concentrations are neither sensitive nor specific for the detection of monoclonal proteins by IFE. Not all PCD are accompanied by excessive production of SFLC, and several other conditions, such as renal disease are associated with increased SFLC concentrations. An abnormal SFLC-ratio is a specific marker for PCD, and occurs primarily in patients with haematological disease. If renal and inflammatory diseases are excluded, this should prompt further diagnostic investigation. Screening of serum without performing an IFE as a standard procedure leads to a reduction of sensitivity when compared to screening of serum that includes IFE.

REEP1 mutation spectrum and genotype/phenotype correlation in hereditary spastic paraplegia type 31.

Mutations in the receptor expression enhancing protein 1 (REEP1) have recently been reported to cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG31. In a large collaborative effort, we screened a sample of 535 unrelated HSP patients for REEP1 mutations and copy number variations. We identified 13 novel and 2 known REEP1 mutations in 16 familial and sporadic patients by direct sequencing analysis. Twelve out of 16 mutations were small insertions, deletions or splice site mutations. These changes would result in shifts of the open-reading-frame followed by premature termination of translation and haploinsufficiency. Interestingly, we identified two disease associated variations in the 3'-UTR of REEP1 that fell into highly conserved micro RNA binding sites. Copy number variation analysis in a subset of 133 HSP index patients revealed a large duplication of REEP1 that involved exons 2-7 in an Irish family. Clinically most SPG31 patients present with a pure spastic paraplegia; rare complicating features were restricted to symptoms or signs of peripheral nerve involvement. Interestingly, the distribution of age at onset suggested a bimodal pattern with the appearance of initial symptoms of disease either before the age of 20 years or after the age of 30 years. The overall mutation rate in our clinically heterogeneous sample was 3.0%; however, in the sub-sample of pure HSP REEP1 mutations accounted for 8.2% of all patients. These results firmly establish REEP1 as a relatively frequent autosomal dominant HSP gene for which genetic testing is warranted. We also establish haploinsufficiency as the main molecular genetic mechanism in SPG31, which should initiate and guide functional studies on REEP1 with a focus on loss-of-function mechanisms. Our results should be valid as a reference for mutation frequency, spectrum of REEP1 mutations, and clinical phenotypes associated with SPG31.

Synthesis and biological evaluation of NO-donor-tacrine hybrids as hepatoprotective anti-Alzheimer drug candidates.

In search of safer anti-Alzheimer drugs, 14 NO-donor-tacrine hybrids (1- 14) were synthesized and evaluated for their ability to inhibit cholinesterases and for vasorelaxation effects. Compounds 1- 13 showed good cholinesterases inhibitory activities in vitro, while 14, particularly, was highly selective, preferring butyrylcholinesterase rather than acetylcholinesterase. Four selected compounds (1, 9, 11, and 14) moderately relaxed the porcine pulmonary arteries in organ bath. In the hepatotoxicity study, significant hepatotoxicity was caused by tacrine but not by 9.

Comparison of the ecarin chromogenic assay and different aPTT assays for the measurement of argatroban concentrations in plasma from healthy individuals and from coagulation factor deficient patients.

INTRODUCTION: The aim of this study was to assess the usefulness of different aPTT assays and the ecarin chromogenic assay reaction time (ECA) for measurement of argatroban concentration in plasma from healthy persons as well as in different patient subgroups. METHODS: We spiked plasma samples from healthy individuals, patients under oral anticoagulation (OAT) or with liver dysfunction (LD) with increasing argatroban concentrations (0-2000 ng/ml) and performed 4 different aPTTs assays and the ECA. RESULTS: Depending on argatroban concentrations aPTTs increased in a curvilinear fashion; in plasma from healthy individuals means of calculated argatroban concentration at 2-fold aPTT differed extensively depending on the aPTT reagent used (725 ng/ml to 1136 ng/ml) and were even more pronounced in plasma from coagulation factor deficient patients (460 ng/ml in patients with LD vs. 1172 ng/ml in patients with OAT), whereas ECA showed linear argatroban influence and reliable results in all subgroups. CONCLUSIONS: Because of wide differences in aPTT measurements depending on the aPTT reagent used, interindividual variations and different clinical conditions the aPTT is not the method of choice for monitoring argatroban and the ECA should be preferred.

Screening of hereditary spastic paraplegia patients for alterations at NIPA1 mutational hotspots.

Mutations in NIPA1 cause hereditary spastic paraplegia type 6 (SPG6 HSP). Sequencing of the whole gene has revealed alterations of either of two nucleotides in eight of nine SPG6 HSP families reported to date. By analysing CpG methylation, we provide a mechanistic explanation for a mutational hotspot to underlie frequent alteration of one of these nucleotides. We also developed PCR RFLP assays to detect recurrent NIPA1 changes and screened 101 independent HSP patients, including 45 index patients of autosomal dominant HSP families. Our negative finding in this cohort for which several other causes of HSP had been excluded suggests NIPA1 alterations at mutational hotspots to be less frequent than previously thought. Nevertheless, the assays introduced represent a valid pre-screen easily implementable in the molecular diagnosis of HSP.

Analysis of single blood cells for CSF diagnostics via a combination of fluorescence staining and micro-Raman spectroscopy.

This contribution provides a new approach for single blood cell analysis in cerebrospinal fluid (CSF) with the possibility of utilizing simultaneously on the same sample the unique capabilities of the two methods fluorescence staining and Raman spectroscopy. By doing so this technique enables the potential of accurate and rapid cell identification in order to determine cell parameters immediately (e.g. the study of the level of activation or phagocytosis activity of single blood cells). Fluorescence labeling of blood cells offers the great possibility of differentiating easily between the subtypes of white blood cells, while Raman spectroscopy reveals molecular fingerprint information with a spatial resolution down to the diffraction limit. Compared to an unstained cell, the presented results nicely demonstrate that the selected fluorescence dye does not influence the Raman spectrum of a labeled blood cell notably. By the combined application of Raman spectroscopy and statistical data analysis a distinction between white blood cell substructures could be performed. Since several blood cell types also differ in the amount of their cell components, differentiation between several blood cell types is also possible when one blood cell is described in the database by several Raman spectra according their presented sub-microscopic structures. This capability with the possibility of accurate and rapid blood cell identification in cerebrospinal fluid is extremely promising for implementation in clinical diagnostics.

Serum concentration of NSE and S-100b during LVAD in non-resuscitated patients.

BACKGROUND AND OBJECTIVES: Increased serum concentrations of brain-derived proteins neuron-specific enolase (NSE) and protein S-100beta (S-100b) are used as early predictors of long-term outcome in unconscious survivors after cardiopulmonary resuscitation (CPR). We investigated whether use of short-term Left Ventricular Assist Devices (LVAD) in patients undergoing percutaneous coronary intervention (PCI) effect serum concentrations of NSE and S-100b, because use of such devices in resuscitated cardiogenic shock patients increased during the last years. METHOD: We analysed data from 80 consecutive non-resuscitated patients who received LVAD support. 43 patients with uncomplicated myocardial infarction (AMI) without LVAD support after PCI formed the reference group. RESULTS: 69 patients (86%) with LVAD support survived and were discharged from hospital. We observed an increase in NSE serum levels in 93.6% and in S-100b serum levels in 58.6% of these patients during LVAD support. This increase was significant in comparison to the upper limit of normal (ULN) of both biomarkers and to the reference group. Cardiogenic shock patients showed significantly higher serum concentrations of both neuroproteins than patients after high-risk PCI, and after AMI during LVAD support. The use of axial flow pumps led to significantly higher serum concentrations of NSE compared to patients on IABP, but not of S-100b. Thrombocytes and haemoglobin (Hb) concentrations declined significantly during LVAD support. Surprisingly, we also observed a significant increase in NSE in the reference group. CONCLUSIONS: LVAD support after PCI is associated with a significant increase in NSE serum concentration as well as in S-100b. We therefore postulate an overestimation of the extent of hypoxic brain damage in unconscious survivors after CPR if treatment include LVAD support or PCI or both procedures. The increase in NSE can be partly explained by alteration of thrombocytes and other blood cells. However, the increase in S-100b remains unexplained since S-100b does not occur in peripheral blood cells. An additional release of both biomarkers from ischemic myocardium or cerebral microembolism should be drawn into consideration.

Influence of direct thrombin inhibitor argatroban on coagulation assays in healthy individuals, patients under oral anticoagulation therapy and patients with liver dysfunction.

We assumed that argatroban, a direct thrombin inhibitor, has a strong influence on different coagulation tests which is even more pronounced in patients with an established reduced factor activity like those under oral anticoagulation therapy or with liver dysfunction. To validate this influence we spiked plasma samples from healthy individuals, patients under oral anticoagulation therapy or with liver dysfunction with increasing argatroban concentrations (0-2000 ng/ml) and performed routine laboratory coagulation tests. Consequently, prothrombin time, activated partial thromboplastin time, thrombin time, batroxobin time, coagulation factor activity (FII-FXIII), protein S (activity), protein C (chromogen) and fibrinogen (derived and Clauss fibrinogen method) were measured. Furthermore, the influence of argatroban on the induced platelet aggregation was evaluated. Argatroban interference on standard coagulation assays differed markedly depending on the different subgroups of patients investigated. Prolongation of prothrombin time by argatroban (at 2000 ng/ml 2.7-fold in healthy persons) was significantly higher in oral anticoagulation therapy (3.9-fold) and even more pronounced in liver dysfunction (6.0-fold). The fibrinogen concentration was determined falsely even at low-argatroban concentrations using functional methods in healthy persons and all patient subgroups. The influence of argatroban on standard laboratory coagulation tests is significantly increased by a preexisting factor deficiency. Functional fibrinogen measurement may be helpful to assess in-vivo fibrinogen function but should be avoided to evaluate fibrinogen concentration in argatroban treated patients. Argatroban had no influence on chromogenic protein C measurement, batroxobin time and induced platelet aggregation. Knowledge of argatroban interference is a prerequisite for the reliable interpretation of coagulation assays.

A multi-exonic SPG4 duplication underlies sex-dependent penetrance of hereditary spastic paraplegia in a large Brazilian pedigree.

SPG4 mutations are the most frequent cause of autosomal-dominant hereditary spastic paraplegia (HSP). SPG4 HSP is characterized by large inter- and intrafamilial variability in age at onset (AAO) and disease severity. The broad spectrum of SPG4 mutations has recently been further extended by the finding of large genomic deletions in SPG4-linked pedigrees negative for 'small' mutations. We had previously reported a very large pedigree, linked to the SPG4 locus with many affected members, which showed gender difference in clinical manifestation. Screening for copy number aberrations revealed the first case of a multi-exonic duplication (exon10_12dup) in the SPG4 gene. The mutation leads to a premature stop codon, suggesting that the protein product is not functional. The analysis of 30 individuals who carry the mutation showed that males have on average an earlier AAO and are more severely affected. The present family suggests that this HSP pathogenesis may be modulated by factors related to individual background and gender as observed for other autosomal dominant conditions, such as facio-scapulohumeral muscular dystrophy or amyloidosis. Understanding why some individuals, particularly women, are 'partially protected' from the effects of this and other pathogenic mutations is of utmost importance.

Automated monitoring of C2 and C0 blood levels of mycophenolic acid and cyclosporine on the Abbott Architect c8000.

OBJECTIVES: Evaluation of the performance of the EMIT 2000 Cyclosporin assay using 2 sets of assay calibrators and the EMIT 2000 mycophenolic acid assay to measure C0 and C2 concentrations on the Abbott Architect c8000 analyzer. DESIGN AND METHODS: Imprecision studies were performed. Cyclosporin concentration was assayed by EMIT on the c8000, by ACMIA on the Dimension and by LC-MS/MS while mycophenolic acid was analyzed by EMIT on c8000 and on Dimension and by HPLC. RESULTS: Agreement between cyclosporin and mycophenolic acid concentrations assayed on the c8000 and on the Dimension was very good. Method comparison between the c8000 and LC-MS/MS resulted in a relative bias of 15.7% for C0 and 11.5% for C2 concentrations. Relative bias of the mycophenolic acid concentrations assayed on the c8000 and the HPLC was 37.7%. CONCLUSIONS: When reported properly to the clinician mycophenolic acid and cyclosporine blood levels can be monitored using the EMIT assays on the c8000 consolidating standard routine workflow and reducing reagent costs significantly.

Outcome-based profiling of astrocytic tumours identifies prognostic gene expression signatures which link molecular and morphology-based pathology.

Astrocytomas are intracranial malignancies for which invasive growth and high motility of tumour cells preclude total resection; the tumours usually recur in a more aggressive and, eventually, lethal form. Clinical outcome is highly variable and the accuracy of morphology-based prognostic statements is limited. In order to identify novel molecular markers for prognosis we obtained expression profiles of: i) tumours associated with particularly long recurrence-free intervals, ii) tumours which led to rapid patient death, and iii) tumour-free control brain. Unsupervised data analysis completely separated the three sample entities indicating a strong impact of the selection criteria on general gene expression. Consequently, significant numbers of specifically expressed genes could be identified for each entity. An extended set of tumours was then investigated by RT-PCR targeting 12 selected genes. Data from these experiments were summarised into a sample-specific index which assigns tumours to high- and low-risk groups as successfully as does morphology-based grading. Moreover, this index directly correlates with definite survival suggesting that integrated gene expression data allow individualised prognostic statements. We also analysed localisation of selected marker transcripts by in situ hybridization. Our finding of cell-specificity for some of these outcome-determining genes relates global expression data to the presence of morphological correlates of tumour behaviour and, thus, provides a link between morphology-based and molecular pathology. Our identification of expression signatures that are associated individually with clinical outcome confirms the prognostic relevance of gene expression data and, thus, represents a step towards eventually implementing molecular diagnosis into clinical practice in neuro-oncology.

Application of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 high volume clinical chemistry analyzer.

OBJECTIVES: Evaluation of the performance of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 analyzer. DESIGN AND METHODS: Imprecision studies were performed and patient samples were assayed by EMIT assay and by LC-MS/MS. RESULTS: Limit of quantification was established at 2.8 microg/L. A positive bias of 17.5% between results measured on EMIT and on LC-MS/MS was detected. CONCLUSIONS: EMIT 2000 Tacrolimus assay is suitable for automated analyses of Tacrolimus on the Architect c8000.

A polymorphic Alu insertion that mediates distinct disease-associated deletions.

Large deletions that are associated with insertions of Alu-derived sequence represent a rare, but potentially unique class of alterations. Whether they form by a one-step mechanism or by a primary insertion step followed by an independent secondary deletion step is not clear. We resolved two disease-associated SPAST deletions, which involve distinct exons by long range PCR. Alu-derived sequence was observed between the breakpoints in both cases. The intronic regions that represent the targets of potentially involved Alu retrotransposition events overlapped. Microsatellite- and SNP-based haplotyping indicated that both deletions originated on one and the same founder allele. Our data suggest that the deletions are best explained by two-step insertion-deletion scenarios for which a single Alu retrotransposition event represents the shared primary step. This Alu then mediated one of the deletions by non-homologous end joining and the other by non-allelic homologous recombination. Our findings thus strongly argue for temporal separation of insertion and deletion in Alu insertion-associated deletions. They also suggest that certain Alu integrations confer a general increase in local genomic instability, and that this explains why they are usually not detected during the probably short time that precedes the rearrangements they mediate.

Low expression but infrequent genomic loss of the putative tumour suppressor DBCCR1 in astrocytoma.

DBCCR1 (deleted in bladder cancer chromosomal region 1) has been reported as the gene functionally affected by frequent loss of 9q32-33 in transitional cell carcinomas of the urinary bladder. For these particular tumours, its proposed role in tumour suppression is supported both by the observation of methylation-based silencing of DBCCR1 in a large fraction of bladder cancers and by re-expression studies in bladder cancer-derived cell lines. A more general involvement of DBCCR1 in tumour development might be inferred from recent chip-based expression studies in other tumours. The present study addressed expression of DBCCR1 in gliomas, specifically in astrocytomas, using semi-quantitative RT-PCR on 25 tumours of different malignancy grade and on 5 control brain tissue samples. Genomic deletion of the DBCCR1 locus at 9q32-33 was also investigated, together with the CDKN2A locus at 9p21, by loss of heterozygosity analysis in a second series of 26 astrocytic tumours. We found that DBCCR1 mRNA expression is markedly reduced in the majority of tumour samples compared to controls, and that this reduction significantly correlates with tumour grade. Genomic loss of the DBCCR1 region was found in only 5 of 24 (21%) informative samples, with no obvious correlation to tumour grade, while loss of the CDKN2A locus was observed in 13 of 21 (62%) informative samples with high-grade tumours being affected more often. If present, LOH at 9q coincided with LOH at 9p and is then likely to reflect loss of the entire chromosome rather than a specific, potentially causative event. In contrast to the situation in bladder cancer, the prevalent inactivation of DBCCR1 seen at the expression level in astrocytomas is not primarily caused by genomic loss of the gene. Our findings support a more general role for DBCCR1 in tumour suppression with mechanisms of inactivation differing between tumour types.

Single and chronic administration of ciprofibrate or of ciprofibrate-glycinate in male Fischer 344 rats: comparison of the effects on morphological and biochemical parameters in liver and blood.

Fibrates lead to a reduction of serum triglycerides and cholesterol in hyperlipidemic patients. Their therapeutic use, however, can be associated with adverse effects like gastrointestinal disorders, myalgia, myositis and hepatotoxicity. Large doses can even cause hepatocellular carcinoma in rodents. Additionally, interactions with the biotransformation of other compounds at the cytochrome P450 (CYP) system have been observed. Thus, the discovery of new derivatives with less of these side effects is of great interest. In the present study a single (10 mg/kg body weight) or a 4-week (1 or 10 mg/kg body weight daily) oral administration of ciprofibrate or of the newly synthesized ciprofibrate-glycinate was investigated in adult male Fischer 344 rats. Serum lipid concentrations were distinctly decreased after single but only slightly after chronic administration of the two fibrates, whereas liver parameters revealed a slight concentration and time dependent hepatotoxicity. Histologically, a hypertrophy, an eosinophilia, a reduced glycogen content and also an apoptosis of the hepatocytes was observed. Effects were more pronounced after chronic treatment and after application of the higher dosage. All CYP enzymes investigated were induced in a time and concentration dependent manner. Resulting CYP mediated monooxygenase and oxidase activities showed a dependency both on enzyme induction and hepatotoxic effects. With no parameter investigated major differences were seen between ciprofibrate and ciprofibrate-glycinate. Thus, the present investigations revealed no noticeable advantages of ciprofibrate-glycinate over its parent compound ciprofibrate.