Anti-apolipoprotein A-1 IgG in patients with myocardial infarction promotes inflammation through TLR2/CD14 complex.

OBJECTIVES: Toll-like receptor (TLR)-mediated vascular inflammation, inducible by - amongst other factors - auto-antibodies, is increasingly recognized as a potential mediator of cardiovascular disease. We investigated whether anti-apolipoprotein (Apo)A-1 IgG was associated with a pro-inflammatory cytokine profile in myocardial infarction (MI) patients and whether anti-ApoA-1 IgG elicited a pro-inflammatory response by activating TLRs. METHODS: As surrogate markers of atherosclerotic plaque vulnerability, interleukin (IL)-6, tumour necrosis factor (TNF)-α, matrix metalloproteinase (MMP)-9 and MMP-3 levels were assessed in 221 consecutive MI patients. Using human monocyte-derived macrophages (HMDMs) we investigated (i) the anti-ApoA-1 IgG interaction with TLRs using proximity ligation assay and (ii) anti-ApoA-1 IgG-dependent IL-6/TNF-α production. TLR involvement was further confirmed using HEK293-Blue TLR-2/-4 cells and by computational docking simulations. RESULTS: In MI patients, anti-ApoA-1 IgG positivity was associated with higher levels of IL-6, TNF-α and MMP-9, but lower MMP-3 levels. In in vitro experiments, anti-ApoA-1 antibodies bound to HDMDs in a TLR2-dependent manner, resulting in nuclear translocation of NFκB and a significant increase in TNF-α and IL-6 production. Subsequent functional studies highlighted the importance of CD14 as co-receptor in the anti-ApoA-1 IgG-TLR2-induced cytokine production. Additional bioinformatic studies identified structural homologies between TLR2 and ApoA-1, which may explain the observed cross-reactivity between antibodies against these two molecules. CONCLUSIONS: Anti-ApoA-1 IgG positivity in MI is associated with a high-risk cytokine profile. These auto-antibodies promote inflammation by stimulating the TLR2/CD14 receptor complex, probably because of molecular mimicry, which may contribute to atherosclerosis-related complications in patients.

Autoantibodies against apolipoprotein A-1 and phosphorylcholine for diagnosis of non-ST-segment elevation myocardial infarction.

OBJECTIVES: To explore the diagnostic accuracies of anti-apolipoproteinA-1 (anti-ApoA-1) IgG and anti-phosphorylcholine (anti-PC) IgM alone, expressed as a ratio (anti-ApoA-1 IgG/anti-PC IgM), and combined with the Thrombolysis In Myocardial Infarction (TIMI) score for non-ST-segment elevation myocardial infarction (NSTEMI) (NSTEMI-TIMI score) to create a new diagnostic algorithm - the Clinical Autoantibody Ratio (CABR) score - for the diagnosis of NSTEMI and subsequent cardiac troponin I (cTnI) elevation in patients with acute chest pain (ACP). METHODS: In this single-centre prospective study, 138 patients presented at the emergency department with ACP without ST-segment elevation myocardial infarction. Anti-ApoA-1 IgG and anti-PC IgM were assessed by enzyme-linked immunosorbent assay on admission. Post hoc determination of the CABR score cut-off was performed by receiver operating characteristics analyses. RESULTS: The adjudicated final diagnosis was NSTEMI in 17% (24/138) of patients. Both autoantibodies alone were found to be significant predictors of NSTEMI diagnosis, but the CABR score had the best diagnostic accuracy [area under the curve (AUC): 0.88; 95% confidence interval (CI): 0.82-0.95]. At the optimal cut-off of 3.3, the CABR score negative predictive value (NPV) was 97% (95% CI: 90-99). Logistic regression analysis showed that a CABR score >3.3 increased the risk of subsequent NSTEMI diagnosis 19-fold (odds ratio: 18.7; 95% CI: 5.2-67.3). For subsequent cTnI positivity, only anti-ApoA-1 IgG and CABR score displayed adequate predictive accuracies with AUCs of 0.80 (95% CI: 0.68-0.91) and 0.82 (95% CI: 0.70-0.94), respectively; the NPVs were 95% (95% CI: 90-98) and 99% (95% CI: 94-100), respectively. CONCLUSION: The CABR score, derived from adding the anti-ApoA-1 IgG/anti-PC IgM ratio to the NSTEMI-TIMI score, could be a useful measure to rule out NSTEMI in patients presenting with ACP at the emergency department without electrocardiographic changes.

A multi-target screening analysis in human plasma using fast liquid chromatography-hybrid tandem mass spectrometry (Part II).

OBJECTIVES: Perform a comparison of results obtained with a LC-MS/MS method and a Remedi® instrument on clinical serum samples. DESIGN AND METHODS: Results obtained on 146 selected plasma samples were compared between the two methods. RESULTS: On the 336 positive identifications, 89% were obtained using the LC-MS/MS technique and 57% by the LC-DAD. Benzodiazepines were well recognized by LC-MS/MS. For some compounds such as antidepressant agents, sensitivity was improved using LC-MS/MS. Moreover, this method extended the panel of drugs detected in clinical toxicology. CONCLUSION: The new software platform developed for screening and identification of small molecules (SmileMS) allows an easy and reproducible detection of drugs and toxic compounds in blood for general unknown screening. It offers automated generation of reports, which makes the LC-MS/MS easier to use without having specialised skills in mass spectrometry. This LC-MS/MS screening method will be a reliable alternative to the Remedi® instrument in the global process of screening in emergency clinical toxicology laboratories.

A multi-target screening analysis in human plasma using fast liquid chromatography-hybrid tandem mass spectrometry (Part I).

OBJECTIVES: Evaluate a new LC-MS/MS screening method for drugs and drugs of abuse as an alternative to the existing methods used in clinical toxicology laboratories. DESIGN AND METHODS: The work was divided in two parts. The first part was dedicated to the technical development and evaluation of the method for which a set of 97 drugs and relevant metabolites was used to perform a complete investigation of matrix effects and lower limit of identification (LOI). The second part was a comparison of identified drugs between LC-MS/MS and Remedi® instrument on clinical serum samples. RESULTS: The method offers good performance allowing an automatic peak detection and compound identification. The limit of identification is equivalent to 50 µg/L for the majority of the studied compounds. The process efficiency (PE) is higher than 70% for 65% of the evaluated compounds. Thus, a sufficient detection capability in terms of limit of detection for identification and PE satisfied the expected performance. CONCLUSION: The described methodology allows the identification of the main drugs incriminated in intoxications within a quite short analysis time. The separation of most of the analytes is performed in 15 min. The procedure is sufficiently sensitive and selective.

The effects of CYP2D6 and CYP3A activities on the pharmacokinetics of immediate release oxycodone.

BACKGROUND AND PURPOSE: There is high interindividual variability in the activity of drug-metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug-drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored. EXPERIMENTAL APPROACH: A randomized crossover double-blind placebo-controlled study was performed with 10 healthy volunteers genotyped for CYP2D6 [six extensive (EM), two deficient (PM/IM) and two ultrarapid metabolizers (UM)]. The volunteers randomly received on five different occasions: oxycodone 0.2 mg x kg(-1) and placebo; oxycodone and quinidine (CYP2D6 inhibitor); oxycodone and ketoconazole (CYP3A inhibitor); oxycodone and quinidine+ketoconazole; placebo. Blood samples for plasma concentrations of oxycodone and metabolites (oxymorphone, noroxycodone and noroxymorphone) were collected for 24 h after dosing. Phenotyping for CYP2D6 (with dextromethorphan) and CYP3A (with midazolam) were assessed at each session. KEY RESULTS: CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs and C(max) (-0.71 < Spearman correlation coefficient rhos < -0.92). Oxymorphone C(max) was 62% and 75% lower in PM than EM and UM. Noroxymorphone C(max) reduction was even more pronounced (90%). In UM, oxymorphone and noroxymorphone concentrations increased whereas noroxycodone exposure was halved. Blocking CYP2D6 (with quinidine) reduced oxymorphone and noroxymorphone C(max) by 40% and 80%, and increased noroxycodone AUC(infinity) by 70%. Blocking CYP3A4 (with ketoconazole) tripled oxymorphone AUC(infinity) and reduced noroxycodone and noroxymorphone AUCs by 80%. Shunting to CYP2D6 pathway was observed after CYP3A4 inhibition. CONCLUSIONS AND IMPLICATIONS: Drug-drug interactions via CYP2D6 and CYP3A affected oxycodone pharmacokinetics and its magnitude depended on CYP2D6 genotype.

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety.

BACKGROUND AND PURPOSE: The major drug-metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug-drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A-mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored. EXPERIMENTAL APPROACH: We conducted a randomized crossover (five arms) double-blind placebo-controlled study in 10 healthy volunteers genotyped for CYP2D6. Oral oxycodone (0.2 mg x kg(-1)) was given alone or after inhibition of CYP2D6 (with quinidine) and/or of CYP3A (with ketoconazole). Experimental pain (cold pressor test, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed. KEY RESULTS: CYP2D6 activity was correlated with oxycodone experimental pain assessment. CYP2D6 ultra-rapid metabolizers experienced increased pharmacodynamic effects, whereas cold pressor test and pupil size were unchanged in CYP2D6 poor metabolizers, relative to extensive metabolizers. CYP2D6 blockade reduced subjective pain threshold (SPT) for oxycodone by 30% and the response was similar to placebo. CYP3A4 blockade had a major effect on all pharmacodynamic assessments and SPT increased by 15%. Oxymorphone C(max) was correlated with SPT assessment (rho(S)= 0.7) and the only independent positive predictor of SPT. Side-effects were observed after CYP3A4 blockade and/or in CYP2D6 ultra-rapid metabolizers. CONCLUSIONS AND IMPLICATIONS: The modulation of CYP2D6 and CYP3A activities had clear effects on oxycodone pharmacodynamics and these effects were dependent on CYP2D6 genetic polymorphism.

The AmpliChip CYP450 test: cytochrome P450 2D6 genotype assessment and phenotype prediction.

Polymorphisms of the cytochrome P450 2D6 (CYP2D6) gene affecting enzyme activity are involved in interindividual variability in drug efficiency/toxicity. Four phenotypic groups are found in the general population: ultra rapid (UM), extensive (EM), intermediate (IM) and poor (PM) metabolizers. The AmpliChip CYP450 test is the first genotyping array allowing simultaneous analysis of 33 CYP2D6 alleles. The main aim of this study was to evaluate the performance of this test in CYP2D6 phenotype prediction. We first verified the AmpliChip CYP450 test genotyping accuracy for five CYP2D6 alleles routinely analysed in our laboratory (alleles 3,4,5,6, x N; n=100). Results confirmed those obtained by real-time PCR. Major improvements using the array are the detection of CYP2D6 intermediate alleles and identification of the duplicated alleles. CYP2D6 phenotype was determined by assessing urinary elimination of dextromethorphan and its metabolite dextrorphan and compared to the array prediction (n=165). Although a low sensitivity of UM prediction by genotyping was observed, phenotype prediction was optimal for PM and satisfying for EM and IM.

Cardiac biomarkers for risk stratification in non-massive pulmonary embolism: a multicenter prospective study.

BACKGROUND: Troponins (cTnI and cTnT), N-terminal pro-Brain Natriuretic Peptide (NT-proBNP), myoglobin, heart-type fatty acid-binding protein (H-FABP) and fibrin D-Dimer are emergent candidates for risk stratification in pulmonary embolism (PE). OBJECTIVE: To compare the respective prognostic values of biomarker with non-massive PE to predict an adverse outcome at 3 months. PATIENTS/METHODS: One hundred and forty-six consecutive patients with non-massive PE were included in this multicenter prospective study. The combined outcome consisted of intensive care monitoring on admission, death or hospitalization attributable to either a PE-related complication [defined by PE/deep vein thrombosis (DVT) relapse or major bleeding under anticoagulation] or to dyspnoea with or without chest pain during follow-up. RESULTS: The outcome was met in 12% of patients. In univariate analysis, a NT-proBNP level above 300 pg/ml was the strongest predictor of unfavorable outcome with an odds ratio (OR) of 15.8 [95% confidence interval (CI): 2.05-122). ORs for the other variables were: 8.0 for D-dimer >2000 ng/ml (95% CI: 1.1-64), 4.7 for H-FABP >6 ng/ml (95% CI:1.5-14.8), 3.5 for cTnI >0.09 ng/ml (95% CI:1.2-9.7), 3.4 for myoglobin >70 ng/ml (95% CI:0.9-12.2). Receiver operating curve (ROC) analysis indicated that NT-proBNP was the best predictor [area under the curve (AUC) 0.84; 95%CI: 0.76-0.92; P < 0.0001] with a negative predictive value of 100% (95% CI: 91-100) at 300 pg/ml. At that cut-off, the true negative rate for NT-proBNP was 40%. In multivariate analysis, NT-proBNP was the only significant independent predictors. CONCLUSIONS: NT-proBNP appears to be a good risk stratification marker in identifying low-risk patients with non-massive PE who could be treated in an outpatient setting.

Early legume responses to inoculation with Rhizobium sp. NGR234.

Interactions between legumes and rhizobia are controlled by the sequential exchange of symbiotic signals. Two different techniques, 2D-PAGE electrophoresis and differential display were used to study the effects of rhizobial signals on legume development. Application of variously substituted lipo-oligo-saccharidic Nod-factors to roots of Vigna unguiculata resulted in changes in the phosphorylation patterns of microsomal proteins. Reliable amino-acid sequences were obtained for one Nod-factor enhanced protein which was highly homologous to the 57-kDa subunit from Arabidopsis thaliana vacuolar membrane H(+)-ATPase. Immuno-blotting techniques demonstrated that Nod-factors cause rapid and massive increases of this enzyme in treated roots, suggesting that H(+)-ATPases play symbiotic roles. Concomitantly, we used differential display (DD) techniques on mRNA isolated from root-hairs to analyse early root responses to NGR234. Significant matches of several DD clones to known sequences were found. Clone D2.62 was homologous to a multitude of receptor kinases including S receptor-like kinases of A. thaliana and clone D4.1 showed similarities to Lotus japonicus phosphatidylinositol transfer-like protein III and late nodulin 16. Independent confirmatory analyses of these differentially expressed clones indicated expression at very low levels.

Changes of the cortex proteome and Apolipoprotein E in transgenic mouse models of Alzheimer's Disease.

Transgenic mice carrying human Amyloid Precursor Protein mutations present amyloid plaque deposition in the brain upon aging. In this study, we characterized the changes of cortex proteome and endogenous Apolipoprotein E in these mice. Differential analysis of two-dimensional electrophoresis images revealed spots altered upon aging, transgene addition and plaque deposition. Alpha-synuclein and cytochrome oxidase polypeptide Va were up-regulated in transgenic mice. Upon aging, expression of ATP synthase alpha, alpha enolase, UMP-CMP kinase, and dihydropyrimidinase like-2 protein was modified. These proteins and their modification probably play a role in the amyloid aggregate formation in these mice.

A humanist's legacy in medical informatics: visions and accomplishments of Professor Jean-Raoul Scherrer.

OBJECTIVE: To report about the work of Prof. Jean-Raoul Scherrer, and show how his humanist vision, his medical skills and his scientific background have enabled and shaped the development of medical informatics over the last 30 years. RESULTS: Starting with the mainframe-based patient-centered hospital information system DIOGENE in the 70s, Prof. Scherrer developed, implemented and evolved innovative concepts of man-machine interfaces, distributed and federated environments, leading the way with information systems that obstinately focused on the support of care providers and patients. Through a rigorous design of terminologies and ontologies, the DIOGENE data would then serve as a basis for the development of clinical research, data mining, and lead to innovative natural language processing techniques. In parallel, Prof. Scherrer supported the development of medical image management, ranging from a distributed picture archiving and communication systems (PACS) to molecular imaging of protein electrophoreses. Recognizing the need for improving the quality and trustworthiness of medical information on the Web, Prof. Scherrer created the Health-On-the-Net (HON) foundation. CONCLUSIONS: These achievements, made possible thanks to his visionary mind, deep humanism, creativity, generosity and determination, have made of Prof. Scherrer a true pioneer and leader of the human-centered, patient-oriented application of information technology for improving healthcare.

Contribution of proteomics to the molecular analysis of renal cell carcinoma with an emphasis on manganese superoxide dismutase.

Renal cell carcinoma (RCC) originates in the renal cortex. It accounts for 2-3 percent of all cancers occurring in adults and it is characterised by lack of early clinical manifestations, unpredictable outcome, and absence of effective treatment modalities except early surgery. RCC comprises a heterogeneous group of tumours with various molecular and cytogenetic abnormalities and different histological features as cell types and tumour architecture. Molecular genetic and proteomic tools led to the discovery of potential diagnostic prognostic and therapeutic biomarkers of RCC. In this review we discuss recent developments in understanding genotype-phenotype relationships, with attention to manganese superoxide dismutase, a mitochondrial enzyme related to the redox cycle which affects various regulatory functions of cells. The expression of this protein has been evaluated in numerous human tumour types including RCC, and post-translational modifications are being investigated.

New perspectives in the Escherichia coli proteome investigation.

Escherichia coli is a model organism for biochemical and biological studies as it is one of the best characterised prokaryote. Two-dimensional polyacrylamide gel electrophoresis, computer image analysis and different protein identification techniques gave rise, in 1995, to the Escherichia coli SWISS-2D PAGE database (http://www.expasy.ch/ch2d/). In the E. coli 3.5-10 SWISS-2D PAGE map, 40% of the E. coli proteome was displayed. The present study demonstrated that the use of narrow range pH gradients is able to potentially display up to a few copies of protein per E. coli cell. Moreover, the six new E. coli SWISS-2D PAGE maps (pH 4-5, 4.5-5.5, 5-6, 5.5-6.7, 6-9 and 6-11) presented here displayed altogether more than 70% of the entire E. coli proteome.

The mouse SWISS-2D PAGE database: a tool for proteomics study of diabetes and obesity.

A number of two-dimensional electrophoresis (2-DE) reference maps from mouse samples have been established and could be accessed through the internet. An up-to-date list can be found in WORLD-2D PAGE (http://www.expasy.ch/ch2d/2d- index.html), an index of 2-DE databases and services. None of them were established from mouse white and brown adipose tissues, pancreatic islets, liver nuclei and skeletal muscle. This publication describes the mouse SWISS-2D PAGE database. Proteins present in samples of mouse (C57BI/6J) liver, liver nuclei, muscle, white and brown adipose tissue and pancreatic islets are assembled and described in an accessible uniform format. SWISS-2D PAGE can be accessed through the World Wide Web (WWW) network on the ExPASy molecular biology server (http://www.expasy.ch/ ch2d/).

Identification of ribosome-associated viral and cellular basic proteins during the course of infection with herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) infection induces severe alterations of the translational apparatus, including the phosphorylation of a few ribosomal proteins, and the progressive association of several nonribosomal proteins to ribosomes. Therefore, we hypothesized that ribosomes themselves could contribute to the HSV-1-induced translational control of host and viral gene expression. As a prerequisite to test this hypothesis, we undertook the identification of the nonribosomal proteins associated to the ribosomes during the course of HSV-1 infection. After separation by two-dimensional polyacrylamide gel electrophoresis of basic proteins extracted from the ribosomal fraction, the identification of unknown protein spots was carried out by N-terminal sequencing and peptide mass determination by mass spectrometry. This allowed us to identify HSV-1 VP19C and VP26 that associated to ribosomes with different kinetics. Another nonribosomal protein turned out to be the poly(A)-binding protein 1 (PAB1P). Newly synthesized PAB1P continued to associate to ribosomes all along infection.

Cellular distribution of translationally controlled tumor protein in rat and human testes.

In a recent proteomic study we identified 53 spermatogonial proteins among which was the translationally controlled tumor protein (TCTP). This is a protein previously reported as being implicated in proliferation events in normal and tumoral tissues that had never previously been seen in the testis. The present study was aimed at establishing the complete cellular distribution of TCTP and its transcript and the ontogenetic expression of this gene within the testis. Using an immunohistochemistry technique, an intense TCTP signal was detected in gonocytes (the prespermatogonia) in the fetal rat testis and in spermatogonia within adult human and neonatal and adult rat testes. Meiotic spermatocytes and postmeiotic haploid spermatids were also strongly immunostained in a stage-dependent manner in human and rat testes. In addition, different levels of TCTP expression were also observed in the testicular somatic cells, with strong expression in Leydig cells and peritubular cells, and weak expression in Sertoli cells. Western and Northern blot analyses confirmed the presence of TCTP at all ages studied, with higher levels of RNA expression at 9 and 20 d postpartum, when spermatogonia and primary spermatocytes represent the highest proportion of germ cells: it was also confirmed that TCTP is present in all populations of isolated testicular cells. A transcript of 0.85 kb corresponding to TCTP, was expressed at all ages studied. This transcript was found to be expressed strongly in spermatogonia, somewhat less in isolated Leydig, resident macrophage, peritubular and Sertoli cells, weakly in the primary spermatocytes but not at all in spermatids. Interestingly, in the latter, a different transcript of 1.1 kb was present. The same 1.1 kb transcript appeared in testis extracts from 35 days postpartum onwards, corresponding to an age when spermatids accumulate within the tubules. Of note is that resident macrophages were found to express both the 0.85 and the 1.1 kb transcripts. We conclude that the strong expression of TCTP in spermatogonia makes it highly likely that the protein plays a significant role in spermatogenesis.

Assessing cerebrospinal fluid rhinorrhea: a two-dimensional electrophoresis approach.

Assessment of nasal cerebrospinal fluid (CSF) fistula commonly relies on the determination of CSF markers in an aqueous rhinorrhea, such as the beta2-transferrin immunofixation assay. While generally reliable, false positive and false negative results have been reported for most of the laboratory tests yet available. Based on the hypothesis that the simultaneous assessment of several CSF markers may yield an increased sensitivity and specificity, we used a proteomics, two-dimensional electrophoresis 2-DE based approach to study samples of nasal secretions obtained from 18 patients suspected of CSF rhinorrhea. Since CSF, nasal mucus and plasma may coexist in the nasal cavities, we first defined five specific markers for each of these biological fluids (transferrin, prostaglandin-D synthase, transthyretin, and two unknown trains of spots for CSF, immunoglobulin A (IgA) S-chain, lipocortin-1, lipocalin-1, prolactine-inducible protein and palatal lung nasal epithelium clone protein for mucus, haptoglobin alpha1/2- and beta-chains, fibrinogen alpha-, beta- and gamma-chains for plasma). Gels from the rhinorrhea patients were then compared to these 2-DE reference maps to determine the presence or absence of the defined markers, and clinical data were independently compared to the results of the 2-DE study. In all cases, the biological fluid(s) anticipated to be present in the nasal secretions based on clinical data were correctly identified by 2-DE. Moreover, an excellent correlation was found in nine patients who underwent extensive workup for suspected CSF rhinorrhea, since CSF was found by the 2-DE method in four patients in whom a CSF fistula was confirmed, whereas the test was negative in five patients in whom a CSF fistula was excluded. In the remaining patients, mucus, sometimes contamined with blood, was found to be the major component of the nasal secretions, confirming that clear mucus may mimick CSF rhinorrhea. These preliminary results suggest that a 2-DE-based multimarker approach is a valid, sensitive, and specific method to assess the presence of CSF in occult rhinorrhea.

CSF detection of the 14-3-3 protein in unselected patients with dementia.

OBJECTIVE: To determine the usefulness of the 14-3-3 test in patients with dementia of various causes. BACKGROUND: Recent reports have suggested that the detection of the 14-3-3 protein in the CSF of patients with Creutzfeldt--Jakob disease is a highly sensitive and specific marker of the disease that might be used as a diagnostic criterion. We examined the validity of this test when applied to a cohort of unselected patients prospectively examined for an ongoing dementing process. METHODS: One hundred patients underwent an extensive neurologic examination for dementia, including a CSF 14-3-3 protein immunoblotting assay. Final clinical diagnoses were compared with the qualitative results of the test, and statistical measures of test validity were carried out. RESULTS: We found a positive test in 14 of 100 patients, only two of whom had definite Creutzfeldt--Jakob disease. Positive results were found in patients with various degenerative dementias, including AD (4), frontotemporal dementia (2), and dementia with Lewy body (1), and in patients with vascular dementia (1), carcinomatous meningitis (1), and anoxic encephalopathy (1). In two other positive patients, the dementia could not be confidently classified. Sensitivity, specificity, and negative predictive value were fairly good, but positive predictive value was poor. Similar results were found independently of the disease duration. There was no correlation between intensity nor pattern of the 14-3-3 protein expression and diagnosis. CONCLUSIONS: The 14-3-3 test is not valid for discriminating between Creutzfeldt--Jakob disease and non-Creutzfeldt--Jakob disease in unselected patients with dementia. Positive results are found in various degenerative and secondary, prion-unrelated dementias.

Proteomic dissection of dome formation in a mammary cell line: role of tropomyosin-5b and maspin.

In this work we extended the study of genes controlling the formation of specific differentiation structures called "domes" formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the beta-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC beta-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC beta-subunit and the rat8 proteins, and is under the negative control of maspin.