B. Van Everbroeck

A prospective study of CSF markers in 250 patients with possible Creutzfeldt–Jakob disease

Objective: To investigate various cerebrospinal fluid (CSF) markers that could assist in the clinical diagnosis of Creutzfeldt–Jakob disease (CJD). Methods: CSF samples were analysed for the presence of 14-3-3 protein, microtubule associated protein tau, and β amyloid in 250 patients with possible CJD. Densitometric analysis was used to quantify the level of 14-3-3 in all patients. Results: Analysis of the clinical data showed that cerebellar signs or myoclonus combined with progressive dementia were the main features leading to a clinical suspicion of CJD. While 14-3-3 detection had a sensitivity of 100% and a specificity of 92%, tau determination using a threshold of 1300 pg/ml had a sensitivity of 87% and a specificity of 97%. If the protocol for the analysis of 14-3-3 was modified (using densitometric analysis) a higher specificity (97%) could be obtained, but with a lower sensitivity (96%). Maximum sensitivity, specificity, and positive predictive value were obtained with a combination of 14-3-3 and β amyloid determinations. The concentrations of 14-3-3 and tau in the CSF were reduced in CJD patients with a long duration of disease (more than one year; p < 0.05). The concentrations of 14-3-3 or tau were lowest at the onset or at the end stage of the disease, while the β amyloid concentration remained low throughout the course of the disease. Conclusions: Both 14-3-3 and tau protein are sensitive and specific biomarkers for CJD. The combination of 14-3-3 and β amyloid analysis resulted in the maximum sensitivity, specificity, and positive predictive value. When these biomarkers are used in the diagnosis of CJD, the phase of the disease in which the CSF sample was obtained should be taken into account. Disease duration, dependent on the PrP genotype, also has a significant influence on the level of 14-3-3 and tau in the CSF.

The role of cytokines, astrocytes, microglia and apoptosis in Creutzfeldt-Jakob disease

In order to investigate inflammation and apoptosis in Creutzfeldt-Jakob disease (CJD) patients, we analyzed astrocytes, microglia and apoptotic neurons in brain and IL-1beta in cerebrospinal fluid (CSF). Our results showed increased numbers of astrocytes in CJD and increased numbers of microglia and apoptotic neurons both in CJD and Alzheimers disease (AD) as compared to controls. All these markers correlated (P < 0.001) with the severity of the neuropathological lesions. An increased IL-1beta concentration was found in AD and CJD CSF that correlated with the number of microglia and which did not change in the disease course of CJD.In conclusion, apoptotic neurons in CJD correlates to the neuropathological lesions and are probably related to the presence of inflammatory cells and cytokines which are present during the whole CJD disease process.

Cerebrospinal fluid biomarkers in Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) is a rare neurodegenerative disorder. Since the emergence of variant CJD (vCJD) vigilance concerning the diseases incidence has increased and the interest in accurate in vivo diagnosis has augmented. So far, a large number of biomarkers has been investigated as aid in the differential diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) and vCJD. These include, among others, neuron-specific enolase (NSE), microtubuli associated protein Tau, S-100beta, amyloid-beta (Abeta(1-42)) and the 14-3-3 protein. Multiple studies have confirmed that CSF detection of 14-3-3 protein by Western blot was the best single biomarker for sCJD with an average sensitivity and specificity of 92%. Also, in genetic and iatrogenic CJD (iCJD) patients with an average disease duration of less than 1 year, 14-3-3 is the best differential biomarker. Unfortunately, the 14-3-3 protein has a lower sensitivity if the disease duration exceeds beyond 1 year in both sporadic CJD and other CJD types (vCJD, and specific genetic or iatrogenic CJD types).

Decreased Levels of Amyloid-β 1-42 in Cerebrospinal Fluid of Creutzfeldt-Jakob Disease Patients

Creutzfeldt–Jakob disease (CJD) is a rare neurodegenerative disease caused by the prion protein. In the search for biochemical markers for CJD, cerebrospinal fluid (CSF) of 101 patients was analysed for 14-3-3 protein, hTau-protein and amyloid-beta 1-42 (Abeta_1-42). The 14-3-3 test had a specificity of 91.5% and a sensitivity of 84%. The hTau test resulted in 95% specificity and 74% sensitivity, when a cut-off of 1530 pg/ml was used. Abeta_1-42 detection in CSF of 29 probable or definite CJD patients revealed significantly decreased values (p=0.01) compared to a group of 22 neurological controls. In the CJD patients a mean of 319+/-102 pg/ml was found. In the neurological control group a mean of 553+/-268 pg/ml was observed. In patients with a false positive 14-3-3 test (n=5) a mean of 716+/-441 pg/ml was found. We conclude that determination of Abeta_1-42 levels in CSF can be useful for identifying false positive 14-3-3 results in suspected CJD patients. We also compared the presence of senile plaques and the Abeta_1-42 levels in CSF of CJD patients. No clear correlation between them was found in this series. This signifies that the deceased Abeta_1-42 levels in CSF are not just due to plaque retention but that other mechanisms must also play a role.

Antigen Retrieval in Prion Protein Immunohistochemistry

Transmissible spongiform encephalopathies are a group of neurodegenerative diseases occurring in both humans and animals and are most likely caused by prions. Neuropathological confirmation of the clinical diagnosis has been a problem because of the difficulty in epitope retrieval from formalin-fixed, paraffin-embedded brain specimens. Many different protocols for the detection of prions in brain tissue have been used. Thus far, picric and/or formic acid, steam autoclaving at 121C of sections, microwave treatment, and 4 M guanidine thiocyanate treatment have been suggested. The objective of our experiment was to obtain the standard pretreatment(s) resulting in optimal immunostaining. In the experiment, successive tissue slides of brain specimens of several Creutzfeldt-Jakob disease and control patients were stained using different combinations of pretreatments. Using densitometric analysis, several well-defined locations per section were examined and prion immunostaining was quantified. The results showed that autoclaving is necessary for antigen retrieval and cannot be substituted by microwave treatment. The best results were obtained when the following combination was used in the specified order: 15 min saturated picric acid, 10 min steam autoclaving at 121C, 5 min 88% formic acid, and 2 hr 4 M guanidine thiocyanate at 4C.

Phosphorylated tau in cerebrospinal fluid as a marker for Creutzfeldt–Jakob disease

Objective: To determine the concentrations of microtubule associated protein tau and multiple phosphorylated tau epitopes in the cerebrospinal fluid of patients with sporadic Creutzfeldt–Jakob disease (sCJD), dementias, and controls, in order to evaluate their diagnostic use and clinical relevance. Methods: The CSF concentrations of total tau and phosphorylated tau at epitope 181 were determined by enzyme linked immunosorbent assay in 66 definite and nine probable sCJD patients, and in 97 controls. Other phosphorylated tau epitopes were investigated by western blot. Results: In the sCJD population, determination of 14-3-3 protein and total tau protein concentrations was of the highest diagnostic value, with a sensitivity of 96% and 92%, respectively, and a specificity of 94% and 97%. Two distinct subgroups could be identified among the 75 sCJD patients based on the detection of phosphorylated tau at threonine 181 and serines 199, 202, and 404. A high phosphorylated tau concentration was clinically correlated with a significantly shorter disease duration, early onset of akinetic mutism, and a higher rate of typical EEGs (p < 0.05). Conclusions: Although the determination of phosphorylated tau levels cannot be used as a diagnostic biomarker, it may prove useful for estimating the prognosis of an sCJD patient. These experiments reconfirm that sCJD is a disease with a complex pathology.

14-3-3 γ-isoform detection distinguishes sporadic Creutzfeldt–Jakob disease from other dementias

We developed a polyclonal antiserum directed to the γ-isoform of the human 14-3-3 protein and compared the immunoreactivity with a commercially available antibody (CG31). We analysed 14-3-3 in 253 cerebrospinal fluid samples blinded for the diagnosis by western blot and ELISA, with a commonly used polyclonal antiserum (Sc-731) and the γ specific antibodies. Our patient population consisted of 52 patients with definite sporadic Creutzfeldt–Jakob disease (sCJD) and 201 patients with a different final diagnosis. We obtained similar sensitivity, ranging from 96% to 98% with all antibodies. Of all the samples that were false positive with Sc-731, 50% were negative with both γ-isoform specific antibodies resulting in a significantly higher specificity (85% v 93%, respectively). If only sCJD and patients with dementia differing from sCJD were analysed we found that 64% of false positives were negative which also resulted in significantly increased specificity and positive predictive value. The γ-isoform specific antibodies strongly improve the specificity of the immunoblot and might improve worldwide acceptance of the use of the 14-3-3 assay in the differential diagnosis of sCJD.

Retrospective study of Creutzfeldt-Jakob disease in Belgium: neuropathological findings

Abstract Creutzfeldt-Jakob disease (CJD) is a spongiform encephalopathy that affects about 1 in 106 inhabitants in most countries. Recently, a new variant of CJD has been linked to the epidemic of bovine spongiform encephalopathy. Therefore, vigilance concerning the disease’s incidence has been increased. We conducted a comprehensive, nation-wide and retrospective study. In 79 Belgian autopsies, we found the characteristic triad of spongiosis, neuronal loss and reactive gliosis. The occipital cortex was most affected, while the cerebellum was mostly spared. Immunohistochemistry was performed using hydrated autoclave pretreatment and several monoclonal antibodies directed against the prion protein. We identified prion-immunoreactive patterns and locations reflecting the important heterogeneity, independently of the antibody that was used. Granular prion immunoreactivity was observed in astrocytes. We studied the regional intensity of the prion immunostaining and determined that the frontal cortex with 95% positive immunoreactivity was best suited for a biopsy. We studied the disease duration in sporadic CJD patients who showed neuropathological lesions of other neurodegenerative disorders (such as Alzheimer’s disease). The study shapes the framework in which a prospective neuropathological registry will be able to function.

A retrospective study of Creutzfeldt–Jakob disease in Belgium

Using data from Belgian neuropathological archives, completed with the results of a comprehensive study of available medical records, we found 100 patients who fulfilled diagnostic criteria for probable or definite Creutzfeldt–Jakob disease (CJD). Mean age at death was 63 years. The median disease duration was 9 months. Progressive mental deterioration was present in all cases, whereas signs of cerebellar dysfunction and myoclonus were found in approximately 80% of the patients. In 50% of the population, the EEG revealed characteristic abnormalities. Ninety-six patients suffered from the sporadic type of CJD, while 4 suffered from a hereditary form. In our series, we could find no evidence for the new variant, neither for an iatrogenic cause.