Background: The 14-3-3 protein is a physiological cellular protein expressed in various tissues, and its release to CSF reflects extensive neuronal damage as in Creutzfeldt-Jakob disease (CJD), but also in other neurological diseases. 14-3-3 protein in CSF in the proper clinical context is a reliable diagnostic tool for sporadic CJD. However, the sensitivity varies across molecular CJD subtypes.Objective: We determined the level of the 14-3-3 protein in CSF from 70 sporadic CJD patients with distinct molecular subtypes using an improved enzyme-linked immunosorbent assay (ELISA) protocol technique.Results: The 14-3-3 levels varied markedly across various molecular subtypes. The most elevated levels of 14-3-3 protein were observed in the frequently occurring and classical subtypes, whereas the levels were significantly lower in the subtypes with long disease duration and atypical clinical presentation. PRNP codon 129 genotype, PrPsc isotype, disease stage and clinical subtype influenced the 14-3-3 level and the test sensitivity.Conclusions: The 14-3-3 protein levels differ across molecular subtypes and might be used for their early pre-mortem identification when the codon 129 genotype is known, especially for the less common molecular subtypes such as MV2 and MM2. (C) 2008 Elsevier Inc. All fights reserved.

We investigated cerebrospinal fluid (CSF) samples from patients with Creutzfeldt-Jakob disease (CJD) and other neurological diseases. Concentrations of pro- and anti-inflammatory cytokines IL-1beta, IL-6, IL-8, IL-12, TNF-alpha and TGF-beta 2 were determined in CSF using ELISA. Significant changes were found for IL-8 and TGF-beta 2. IL-8 levels were elevated in the CSF of CJD patients. Of interest, the increase was significant to other dementia and to controls. In contrast, TGF-beta 2 was significantly decreased in CSF of CJD compared to all groups. IL-1beta, IL-12 and TNF-alpha could not be detected in CSF or in case of IL-6 in only low concentrations without significant difference.

OBJECTIVES:To analyze the diagnostic sensitivity and specificity of various brain-derived proteins (14-3-3, Tau, neuron specific enolase [NSE], and S100b) in the CSF of patients with Creutzfeldt-Jakob disease (CJD) and to analyze biologic factors that modify these parameters.METHODS:CSF was tested for 14-3-3, Tau, NSE, and S100b in 1,859 patients with sporadic, genetic, iatrogenic, and variant CJD, and in 1,117 controls.RESULTS:The highest sensitivity was achieved for 14-3-3 and Tau in sporadic CJD (85% and 86%), and a combined determination of 14-3-3 and Tau, S100b, or NSE increased the sensitivity to over 93%. A multivariate analysis showed that the sensitivity of all tests was highest in patients with the shortest disease duration, age at onset >40 years, and homozygosity at codon 129 of the prion protein gene. In a group of patients with repeated lumbar punctures, a second test also increased the diagnostic sensitivity.CONCLUSIONS:The detection of elevated levels of brain-derived proteins in the CSF in patients with suspected Creutzfeldt-Jakob disease is a valuable diagnostic test. A second lumbar puncture may be of value in patients with atypical clinical course in whom the first test was negative.

Background In neurodegenerative diseases, increasing attention has been focused on inflammatory mediators such as pro-inflammatory and anti-inflammatory cytokines and their potential influence in the process of neurodegeneration. In prion diseases, much data has been gained on the cell culture and animal disease models level, but only limited information is available on humans affected by Creutzfeldt-Jakob disease (CJD). Objective To obtain data on anti-inflammatory cytokines interleukin 4 and interleukin 10 in the cerebrospinal fluid of patients with CJD, patients with other dementia, and nondemented neurological patients and controls. Design Cerebrospinal fluid samples were collected from CJD patients and control subjects, and concentrations of the anti-inflammatory cytokines interleukin 4 and interleukin 10 were determined using an enzyme-linked immunosorbent assay. Patients Cerebrospinal fluid samples from 61 patients were analyzed. The group was composed of patients with CJD (n = 20), patients with other forms of dementia (n = 10), patients with motoneuron disease (n = 6), patients with normal pressure hydrocephalus (n = 5), and control subjects (n = 20). Results Interleukin 10 levels were significantly elevated in the cerebrospinal fluid of CJD patients (median, 9.8 pg/mL). The elevation was significant to other dementia (median, 7.9 pg/mL,P<.05), motoneuron disease (median, 7.9 pg/mL,P<.05), normal pressure hydrocephalus (median, 7.0 pg/mL,P<.05), and controls (median, 1.3 pg/mL,P<.001). Levels of interleukin 4 were significantly elevated in cerebrospinal fluid of patients with CJD (median, 26.4 pg/mL) compared with control subjects (median, 6.2 pg/mL,P<.001) and patients with a motoneuron disease (median, 10.5 pg/mL,P<.001) Conclusions Elevated levels of the anti-inflammatory cytokines interleukin 4 and interleukin 10 in cerebrospinal fluid of patients with CJD are new findings. The data of the present study provide a clue toward the possible role of cytokines as immunological modifiers in the neurodegenerative process of CJD.

Zu den humanen Prionenerkrankungen gehört die Creutzfeldt-Jakob-Erkrankung mit einer rasch progredienten Demenz und weiteren neurologischen Defiziten. Nach der gegenwärtigen Vorstellung wird die Erkrankung durch eine Konformationsänderung des physiologischen Prionproteins (PrPc) verursacht. Allerdings ist die Ursache dafür noch weitgehend ungeklärt. Anhand vorliegender Daten (Zellkulturstudien, Tierversuche) werden dem PrPc neuroprotektive Eigenschaften zugeschrieben. In der vorliegenden Studie sollten anhand von Liquoruntersuchungen erste Anhaltspunkte über mögliche Alteration der Konzentrationen des PrPc bei Patienten mit Erkrankungen des zentralen Nervensystems im Verlauf gewonnen werden. In einem weiteren Schritt wurden die PrPc-Konzentrationen mit weiteren neuronalen Proteinen korreliert (Tau und 14–3-3).

Human plasminogen has been shown to interact with the abnormal disease-specific prion protein. Till now, no data are available for patients with Creutzfeldt-Jakob disease (CJD). Therefore, we compared plasminogen concentrations and plasminogen activities in patients with sporadic CJD and controls with other dementia, which were collected in the framework of the German CJD Surveillance study. Patients with CJD had significantly higher plasminogen concentrations than patients with other forms of dementia and plasminogen specific activities were lower in CJD patients. The reasons for these abnormalities are not clear at the moment. The results may reflect a disease-specific prion protein and plasminogen interaction in patients with CJD. Other possible explanations are plasminogen polymorphisms and genotypes with distinct plasminogen activity levels in CJD than in controls, which should be a subject for further studies.

BACKGROUND: CJD is usually diagnosed by clinical and neuropathological findings. A number of proteins regarded as markers for neuronal damage in plasma or serum have recently been described. Markers typical for tissue damage, although not usually associated with CJD, are another possibility. An evaluation of the relative usefulness of markers of neuronal and tissue damage in identifying CJD could be beneficial.STUDY DESIGN AND METHODS: Plasma samples were collected from 46 patients with sporadic CJD and from a control group of 42 healthy subjects. The samples were analyzed with tests that were specific for C-reactive protein (CRP), IL-6, neuron-specific enolase (NSE), S-100 proteins, and cellular prion protein (PrPc). The results were compared, and a cutoff level for each test used was defined as the 90th percentile from the control group.RESULTS: The assay specific for NSE identified only 13 percent of the sporadic CJD patients as positive. The identification rate of the other markers was significantly higher: S-100, 76.1 percent; PrP, 76.1 percent; CRP, 78.3 percent; and IL-6, 73.3 percent. Only three of the samples were positive in all five tests.CONCLUSION: The markers for tissue damage, CRP and IL-6, are as useful as the previously described markers for neuronal damage in the diagnosis of CJD in plasma. All the markers tested are, however, of only limited value in the diagnosis of CJD in plasma. A combination of all surrogate markers improves the specificity but still provides no definitive diagnosis of the disease.

BACKGROUND: Creutzfeldt-Jakob disease is thought to be caused by conversion of cellular prion protein (PrP) from its soluble form (PrPsen) to a pathologic form (PrPres). The occurrence of a new variant of CJD has increased the demand for a rapid assay capable of detecting a theoretical risk of transmission of the disease by blood or plasma.STUDY DESIGN AND METHODS: A quantitative sandwich ELISA for routine screening was developed for analysis of PrP levels in plasma. The time-resolved dissociation-enhanced fluorescence technology allowed a detection limit in plasma samples of approximately 50 pg/mL. Levels of PrPsen were tested in plasma from 31 patients with CJD, from 11 patients with other neurodegenerative diseases, and from a control group of 200 healthy subjects.RESULTS: The assay recognized both PrPsen and pathologic PrPres, but did not differentiate between the two isoforms. PrPsen levels were higher in plasma from both patient groups than in plasma from the control group: 27 of the 31 (87%) CJD patients and all patients with other neurodegenerative diseases had higher levels than the highest concentration found in the control group. No correlation was found between age and PrP level. No signal could be detected in the CJD samples after protease K digestion, indicating that all detected PrP was protease-sensitive and therefore not pathologic.CONCLUSION: These data suggest that soluble PrPsen in plasma samples might be useful as a surrogate marker for a broad spectrum of neurologic diseases as well as for CJD.

OBJECTIVE:To improve diagnostic criteria for sporadic Creutzfeldt-Jakob disease (CJD).METHODS:Pooled data on initial and final diagnostic classification of suspected CJD patients were accumulated, including results of investigations derived from a coordinated multinational study of CJD. Prospective analysis for a comparison of clinical and neuropathologic diagnoses and evaluation of the sensitivity and specificity of EEG and 14-3-3 CSF immunoassay were conducted.RESULTS:Data on 1,003 patients with suspected CJD were collected using a standard questionnaire. After follow-up was carried out, complete clinical data and neuropathologic diagnoses were available in 805 cases. In these patients, the sensitivity of the detection of periodic sharp wave complexes in the EEG was 66%, with a specificity of 74%. The detection of 14-3-3 proteins in the CSF correlated with the clinical diagnosis in 94% (sensitivity). The specificity (84%) was higher than that of EEG. A combination of both investigations further increased the sensitivity but decreased the specificity.CONCLUSIONS:Incorporation of CSF 14-3-3 analysis in the diagnostic criteria for CJD significantly increases the sensitivity of case definition. Amended diagnostic criteria for CJD are proposed.

According to the recently established molecular basis for phenotypic heterogeneity of sporadic Creutzfeldt-Jakob disease (CJD), six different phenotypes are characterized by the size of the protease-resistant fragment of the pathological prion protein (types 1 and 2) and homozygosity or heterozygosity for methionine or valine at codon 129 of the prion protein gene (designated by MM1, MM2, MV1, MV2, W1, and W2). In the present investigation, we analyzed the value of commonly used clinical tests (electroencephalogram [EEG], detection of 14-3-3 protein in cerebrospinal fluid [CSF], and hyperintensity of the basal ganglia in magnetic resonance imaging) for the clinical diagnosis in each CJD phenotype. The detection of periodic sharp and slow wave complexes in the EEG is reliable in the clinical diagnosis of MM1 and MV1 patients only. The CSF analysis for 14-3-3 protein showed high sensitivity in all analyzed subgroups with the exception of MV2 patients. Valine-homozygous patients had a negative EEG, but most had detectable levels of neuronal proteins in the CSF. The sensitivity of the magnetic resonance imaging was 70%, irrespective of the subgroup, but was particularly reliable in the clinical diagnosis of MV2 patients. The widening spectrum of diagnostic techniques in CJD is not only useful in the increased accuracy of the clinical diagnosis but should also lead to the identification of more atypical cases of sporadic CJD.

Creutzfeldt-Jakob disease (CJD) is a rare neurodegenerative human disorder with an incidence of one case per 1000000 per year. Recently new diagnostic tests such as neuron-specific enolase (NSE), S-100, tau-protein and protein 14-3-3 have been established as markers in prion diseases. NSE is elevated in case of rapid nerve cell loss so quantitative measurement of NSE in cerebrospinal fluid (CSF) might correlate with the disease progression. To further evaluate this hypothesis we analysed longitudinal CSF samples from 16 CJD patients. The first spinal tap was taken two weeks after the first clinical signs of a neurodegenerative disorder. This showed an elevation of NSE which continued during the course of the disease. Longitudinal examination of neuron-specific enolase in cerebrospinal fluid therefore may be useful for differentiation between CJD and other dementias.

Two-dimensional polyacrylamide gel electrophoresis of CSF has been used in the diagnosis of Creutzfeldt-Jakob disease (CJD). One of the two diagnostic protein spots was identified as isoform(s) of the 14-3-3 family of abundant brain proteins. This has led to the development of one-dimensional 14-3-3 sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot, which is currently used to support the diagnosis of CJD. In the present study employing western blot analysis, we have identified the panel of 14-3-3 isoforms that appear in the CSF of 10 patients with CJD compared with 10 patients with other dementias. The results clearly show that the 14-3-3 isoforms beta, gamma, epsilon, and eta are present in the CSF of patients with CJD and can be used to differentiate other dementias. 14-3-3eta also gave a baseline signal in all patients with other dementias, including six patients with Alzheimer's disease. The presence of 14-3-3eta in the CSF of a patient with herpes simplex encephalitis was particularly noteworthy. This study has determined that isoform-specific 14-3-3 antibodies against beta, gamma, and epsilon should be considered for the neurochemical differentiation of CJD from other neurodegenerative diseases.

Human prion diseases may be acquired as infectious diseases, they may be inherited in an autosomal dominant fashion or occur sporadically. Mutations and polymorphisms in the sequence of the coding region of the prion protein gene (PRNP) have been established as an important factor in all of these three types of prion diseases. Therefore, a total of 578 patients with suspect prion diseases referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit over a period of 4.5 years have been examined for mutations and polymorphisms in the coding region of PRNP. We found 40 cases with a missense mutation previously reported as pathogenic. Amongst these, the aspartate to asparagine change at codon 178 (D178N) was the most common mutation. All of these cases carried the D178N mutation in coupling with methionine at codon 129, resulting in the typical fatal familial insomnia (FFI) genotype. Most cases with pathogenic mutations were not found in the group of clinically "probable" cases according to established clinical criteria, supporting the notion that inherited prion diseases often exhibit atypical features. Two novel missense mutations (T188R and P238S) and several silent polymorphisms were found, demonstrating the quality of our screening procedure based on a modified version of the single-stranded conformational polymorphism technique. In "definite" CJD cases with no pathogenic mutation, the patients clinically classified as "probable" were mostly homozygous for methionine at the common polymorphism at codon 129, whereas there was a marked over-representation of patients homozygous for valine amongst those clinically classified as "possible". This large study on suspect cases of human prion diseases in Germany clearly shows that PRNP genetics is essential for a comprehensive analysis of prion diseases.

OBJECTIVE:To report the clinical and pathologic features of patients with the D178N-129M mutation living in Germany.METHODS:Patients with clinically suspected Creutzfeldt-Jakob disease (CJD) were seen in an ongoing, prospective epidemiologic study from June 1993 to August 1997 throughout Germany. Suspect patients were referred to the CJD unit by the participating hospitals or physicians. Patients were seen by a physician, and each patient underwent a detailed neurologic examination. Prion protein gene (PRNP) analysis was performed to distinguish patients with familial forms of CJD.RESULTS:The constellation D178N-129M was identified in eight individuals; in one patient, the diagnosis was made by neuropathologic examination. Four affected men and five women belong to eight unrelated families. A family history of a neurodegenerative disorder was recalled in only five patients. In contrast to the first reported fatal familial insomnia (FFI) patient, none of our patients complained of severe, untreatable insomnia in the early stages. Dysautonomia was observed in varying degrees in most patients. The clinical course of these patients resembled sporadic CJD. In six patients, brain tissue was available for neuropathologic study. In one patient, the neuropathologic examination showed changes that were more reminiscent of forms of sporadic CJD; in the remaining five, the histopathology was typical of FFI.CONCLUSIONS:The clinical presentation in patients with FFI may vary to a great extent. Genotyping of the patients was crucial in providing laboratory confirmation of the diagnosis of FFI, even when there was no family history of a prion disease.

The analysis of 14-3-3 protein in cerebrospinal fluid (CSF) was shown to be highly sensitive and specific for the diagnosis of Creutzfeldt-Jakob disease (CJD). However, the predictive value of this test in the clinical diagnosis of, and its relation to, sporadic, genetic, and iatrogenic CJD cases have yet to be established. CSF samples of suspect CJD cases seen in the prospective German surveillance study were tested for the presence of 14-3-3 protein by using a modified western blot (WB) technique. WB detected 14-3-3 protein in 95.4% of definite and 92.8% of probable cases. In two patients classified initially as not having CJD the test was positive, and both were later proved to have definite CJD. The positive predictive value is 94.7% and the negative predictive value is 92.4%. False-positive results in a single CSF analysis were seen in patients with herpes simplex encephalitis, hypoxic brain damage, atypical encephalitis, intracerebral metastases of a bronchial carcinoma, metabolic encephalopathy, and progressive dementia of unknown cause. WB analysis for 14-3-3 protein was positive in only 5 of 10 cases of familial forms of spongiform encephalopathies. CSF analysis for 14-3-3 protein should thus be performed in any case suspect for CJD.

Since its first description by H.G. Creutzfeldt and A. Jakob, six forms of human spongiform encephalopathies have been described. Besides Creutzfeldt-Jakob disease (CJD), a new variant CJD (nvCJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI) and potentially familial progressive subcortical gliosis have been reported. The most likely causative agent of these and at least six animal-transmissible spongiform encephalopathies (TSE) is a structurally altered form of a regular cellular protein, designated prion. The best known animal forms are bovine spongiform encephalopathy (BSE) and scrapie. The clinical spectrum of human spongiform encephalopathies has been expanded in recent years by the discovery of new, partially genetically determined forms. The currently available clinical, neurophysiological, neuroradiological, biochemical and molecular-biological methods permit only a probable diagnosis of CJD. A definite diagnosis can only be achieved by the neuropathological demonstration of the pathological prion protein (PrPSc). The transmission of BSE to humans has neither been shown nor definitely excluded.

The sensitivity and specificity of PCR of CSF for the diagnosis of progressive multifocal leuko encephalopathy is estimated at 75 and 98.5%, respectively. However, inter-laboratory and inter-technique variations have been shown to produce wide variations. A 10-fold dilution series of JC virus in cerebrospinal fluid was prepared and circulated for `blind' evaluation in laboratories participating in a European Union Concerted Action on Virus Meningitis and Encephalitis. Six of seven laboratories returned results with sensitivity of between 10 and 1 JCV DNA copy equivalents per 10 μl of CSF, one laboratory detected 105 copies per 10 μl of CSF. These results demonstrate the feasibility of using virus diluted in CSF for comparison of PCR techniques, and that the range of sensitivity of JCV PCR in proficient laboratories is between 10 and 1 copy equivalents per 10 μl of CSF.