Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4709—Amyloid plaque core protein
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event

Definitions

• the invention relates to an ex vivo method of differentially diagnosing dementias comprising the features of the preamble of independent claim 1.
• the present invention relates to such a method which is suitable for discriminating vascular dementias and/or frontotemporal lobe degenerations from differential diagnostic relevant dementias, such as Alzheimer's disease, and dementive disorders, such as depression.
• the invention also relates to differentiating between vascular dementias and frontotemporal lobe degenerations.
• the invention relates to a kit for executing such a method of differentially diagnosing dementias according to the preamble of independent claim 15.
• a ⁇ peptides amyloid-beta peptides
• a ⁇ -SDS-PAGE/immunoblot amyloid-beta-sodium-dodecyl-sulphate-polyacrylamide-gel- electrophoresis with western immunoblot
• AD Alzheimer's disease
• APP beta-amyloid precursor protein
• CCD-camera charge coupled device camera
• CSF cerebrospinal fluid
• DC depressive control
• DGN Deutsche Deutschen Deutschen f ⁇ r Neurologie
• FTD frontotemporal dementia
• FTLD frontotemporal lobe degeneration
• MMSE Mini-Mental-Status Examination
• NINCDS-ADRDA National Institute Neurological and Communicative Disorders and Stroke- Alzheimer's Disease and Related Disorders Association
• SDS sodium dodecyl sulphate
• %T percentage of acrylamide
• VAD vascular dementia.
• CSF-NDD multiparametric CSF-based neurochemical dementia diagnostics
• a quintet of A ⁇ peptide species i.e. A ⁇ 1-37/38/39/40/42, has been characterised as regular constituents of human CSF (Wiltfang et al., 2002; Lewczuk et al., 2004).
• FTLD Alzheimer's disease
• AD Alzheimer's disease
• This most common degenerative brain disease constitutes 70% of all cases of dementia in humans. Onset is usually in late middle life, and death typically ensues in 5 to 10 years.
• the heterogeneous group of FTLD and AD share neurochemical and clinical features, although temporal appearance of symptoms during the course of disease and neuropathological findings differ remarkably between these two dementia syndromes.
• a ⁇ aggregated amyloid beta
• a ⁇ 1-42 levels in body fluids such as cerebrospinal fluid (CSF) are significantly reduced in AD and to a lesser degree in FTLD (Hulstaert et al., 1999).
• a ⁇ peptides are derived from a transmembrane amyloid precursor protein (APP), after cleavage by two enzymes, ⁇ - and ⁇ -secretase (Haas and Selkoe, 1993).
• APP transmembrane amyloid precursor protein
• a ⁇ consists predominantly of A ⁇ 1-38, A ⁇ 1-40 and A ⁇ 1-42. Distinct ⁇ -secretase activities are hypothesized to be responsible for generation of either carboxy-terminal truncated (Ct-truncated) or elongated (Ct-elongated)
• a ⁇ peptides as referenced to A ⁇ 1-40 (Citron et al., 1984).
• the measurement of the different A ⁇ peptide species in body fluids can be assumed to more adequately represent disease-specific changes of APP metabolism rather than the simple absolute A ⁇ 1-42 levels (Wiltfang et al., 2001 , Bibl et al., 2006).
• a ⁇ forms can be used in conjunction with clinical diagnosis to support a differential diagnosis.
• Biomarkers to aid clinical diagnosis such as beta amyloid 1-42 tau, and phosphorylated tau have been systematically investigated in FTD, but not in PPA.
• a ⁇ peptide species i.e. A ⁇ 1-37/38/39/40/42, which have been characterised as regular constituents of human CSF before (Wiltfang et al., 2002; Lewczuk et al., 2004), in plasma.
• plasma A ⁇ peptides were characterised by the additional aminoterminally truncated species A ⁇ 2-40. The latter peptide species cannot be differentiated from A ⁇ 1-40 by currently available ELISA methods.
• the inventors found that the plasma concentrations of the individual A ⁇ peptide species show characteristic variations in case of vascular dementia and frontotemporal lobe degenerations.
• vascular dementias in addition to an increase of A ⁇ 1-40 the concentrations of A ⁇ 1-37, A ⁇ 1-38, A ⁇ 1-39, A ⁇ 1-42 and A ⁇ 2-40 are decreased.
• the decrease of these A ⁇ peptide species is characteristic for vascular dementias in that it is not displayed with other differential diagnostic relevant dementive disorders, such as Alzheimer's disease and depression. However, this decrease, except of the decrease in A ⁇ 1-42, and the increase in A ⁇ 1-40 are also displayed with frontotemporal lobe degenerations.
• a ⁇ peptide species which display particularly high decreases in their plasma concentration with vascular and frontotemporal dementias are A ⁇ 1-37, A ⁇ 1-38, A ⁇ 1-42 (not with frontotemporal dementia), and A ⁇ 2-40, the most characteristic decrease being displayed by A ⁇ 1-38.
• the present invention provides an ex vivo method of differentially diagnosing dementias using at least one carboxy-terminally truncated amyloid ⁇ peptide selected from the group consisting of A ⁇ 1-38, A ⁇ 1-37 and A ⁇ 1-39, preferably from the group consisting of A ⁇ 1-38 and A ⁇ 1-37, and even more preferably from the group consisting of A ⁇ 1-38.
• the method of the present invention makes use of a combination of the carboxy-terminally truncated A ⁇ species A ⁇ 1-37 and A ⁇ 1-38 as the biomarker.
• the body fluid used in the present invention is CSF (cerebrospinal fluid).
• a concentration of the at least one carboxy-terminally truncated amyloid ⁇ peptide species is determined in a blood sample taken from the patient, and, typically, the concentration of the at least one carboxy-terminally truncated amyloid ⁇ peptide species and all other amyloid ⁇ peptide species which will be determined in the new method are plasma concentrations, i.e. the concentrations will be determined in a blood plasma sample.
• plasma is a component of blood
• a blood plasma sample is a fraction of a blood sample taken from a patient
• the concentrations of the A ⁇ peptide species determined in the new method may also be related to the total blood sample, or they may even be directly determined in the blood sample.
• the A ⁇ peptides are preferably enriched using an antibody specific to A ⁇ peptides, and then further analysed with regard to the concentration of the individual A ⁇ peptide species.
• the use of an N-terminal specific antibody for enriching the A ⁇ peptides proved as being particularly successful.
• the absolute concentrations of the A ⁇ peptide species may show a considerable variation which is not due to the occurrence of vascular dementias or frontotemporal lobe degenerations.
• the relative concentrations of the A ⁇ peptide species display a much smaller variation without the occurrence of these diseases.
• the relative concentration of the A ⁇ peptide species can be determined as a fraction of the total A ⁇ peptide concentration, which will here be denoted as A ⁇ 1-X%, or as a ratio of the absolute concentration of the at least one carboxy-terminally truncated amyloid ⁇ peptide species and the absolute concentration of the further A ⁇ peptide A ⁇ 1-40 or A ⁇ 1-42, which will here be denoted as A ⁇ 1-X/1-40 and A ⁇ 1-X/1-42, respectively.
• the concentration of the at least one carboxy- terminally truncated amyloid ⁇ peptide species is compared to a threshold value for the respective one carboxy-terminally truncated amyloid ⁇ peptide species.
• the actual threshold value for the respective one carboxy-terminally truncated amyloid ⁇ peptide species is obtainable by comparing the levels of the biomarker(s) in the patient to those in non-demented controls (like for example in non-demented depressive controls) and/or in patients with other dementias (like for example Alzheimer's disease).
• a suitable threshold value for diagnosing vascular dementias is in a range of 7.6 % to 8.6 %.
• this threshold value is in a range of 8.29 % to 8.43 %.
• a threshold value within the latter range is most suited to discriminate VAD from AD.
• a suitable threshold value for diagnosing vascular dementias is in a range of 0.116 to 0.156. Preferably, this threshold value is about 0.136.
• the said mammal is suffering from FTLD provided that the concentration of the at least one carboxy-terminally truncated amyloid ⁇ peptide species is lower than a threshold value for the respective one carboxy-terminally truncated amyloid ⁇ peptide species which, quantified as a percentage, is at least 20% less and preferably ranges from 20% to 40% less as compared to the concentration of the respective carboxy-terminally truncated amyloid ⁇ peptide species in patients with Alzheimer's disease and in non-demented controls.
• the method of the present invention includes obtaining body fluid (for example, blood or CSF) samples from a mammal (for example, human) and determining the ratio of carboxy-terminal truncated A ⁇ peptides A ⁇ 1-37 to A ⁇ 1-40 wherein the ratio less than 0.125 indicates that the mammal is susceptible to, or has, FTLD.
• the method of the present invention includes obtaining body fluid samples (for example, blood or CSF) from a mammal (for example, human) and determining the ratio of carboxy-terminal truncated A ⁇ peptides Ap 1-38 to A ⁇ 1-40 wherein the ratio less than 0.22 indicates that the mammal is susceptible to, or has, FTLD.
• a concentration of the at least one carboxy-terminally truncated amyloid ⁇ peptide species selected from the group of concentrations consisting of the absolute concentration of the at least one carboxy-terminally truncated amyloid ⁇ peptide species, - the relative concentration of the at least one carboxy-terminally truncated amyloid ⁇ peptide species determined as a fraction of the total amyloid ⁇ peptide concentration, and the relative concentration of the at least one carboxy-terminally truncated amyloid ⁇ peptide species determined as a ratio of the absolute concentration of the at least one carboxy- terminally truncated amyloid ⁇ peptide species and the absolute concentration of the further carboxy-terminally truncated amyloid ⁇ peptide species A ⁇ 1-40, below the threshold value for the respective one carboxy-terminally truncated amyloid ⁇ peptide species is taken as an indication of a vascular dementia or a
• the relative concentration of the carboxy-terminally truncated amyloid ⁇ peptide species A ⁇ 1-38 determined as a ratio of the at least one carboxy- terminally truncated amyloid ⁇ peptide species and the absolute concentration of the further amyloid ⁇ peptide species A ⁇ 1-42 can be used to differentiate between a vascular dementia and a frontotemporal lobe degeneration in the patient, a relative concentration below the threshold value for the respective one carboxy-terminally truncated amyloid ⁇ peptide species indicating a frontotemporal lobe degeneration in the patient.
• this threshold value will be in the range of 1.2 to 1.4, preferably about 1.3, if the body fluid is blood plasma, for example.
• a relative concentration of the at least one carboxy-terminally truncated amyloid ⁇ peptide species A ⁇ 1-38 determined as a fraction of the total amyloid ⁇ peptide concentration below the threshold value for the respective one carboxy-terminally truncated amyloid ⁇ peptide species A ⁇ 1-38 may be taken as an indication of a frontotemporal dementia in the patient to distinguish such a dementia from, for example, a primary progressive aphasia (PPA) also belonging to the group of frontotemporal lobe degenerations.
• PPA primary progressive aphasia
• the patient from which the body fluid to be examined in the new method is obtained may be any mammal; in a particular embodiment of the present invention the mammal is a human.
• the methodology used for the quantification of the biomarker is a measure of immunological interaction of the biomarker with antibody(s) or antibody mimicking molecule(s), including specifically monoclonal antibodies specific to A ⁇ peptide biomarker.
• One such example of the methodology used for the quantification of the biomarker is gel electrophoresis or A ⁇ -SDS-PAGE/immunoblot.
• the biomarker may be comprised of a modified form of the carboxy- terminally truncated amyloid ⁇ peptide species of interest: the said A ⁇ peptide may be present in an oxidized form, in a phosphorylated form, or an N(or amino)-terminally truncated form.
• the new kit for differentially diagnosing dementias which include vascular dementias and/or frontotemporal lobe degenerations according to the new comprises all necessary components for executing the new method so far as these components are no standard components available in any well equipped laboratory.
• the new kit comprises at least components for determining a concentration of at least one carboxy-terminally truncated amyloid ⁇ peptide selected from the group consisting of A ⁇ 1-38, A ⁇ 1 -37 and A ⁇ 1-39 in a body fluid obtained from a patient.
• FIg. 1 is a graph showing the mean and the 95% confidence interval (Cl) of the ratio
• Example I A ⁇ 1 -38/1-40 for three diagnostic groups of Example I consisting of DC, AD and VAD patients.
• Fig. 2 shows operator curves of the ratios A ⁇ 1-38/1 -40, A ⁇ 1 -42/1 -40 and A ⁇ 2-40/1 -40, respectively, for the detection of VAD among all other patients in Example I.
• Fig. 3 belongs to Example Il and shows (a) an urea-based A ⁇ -SDS-PAGE/immunoblot, and (b) conventional SDS-PAGE of CSF (lane 1-3) and synthetic A ⁇ 1-37, A ⁇ 1- 38, A ⁇ 1-39, A ⁇ 1-40 and A ⁇ 1-42 peptides (lane 4). Ten microliters of an unconcentrated CSF were applied to each lane.
• the figure shows a blot of CSF of patients with FTLD (lane 2), Non-demented disease controls (NDC) (lane 3), and AD (lane 4), respectively.
• Fig. 4 is a graph showing the mean and the 95% confidence interval (Cl) of A ⁇ 1-38/
• Fig. 5 is a scatter plot of FTLD, AD, and NDC patients of Example Il divided by the ratio of and A ⁇ i. 42 [ng/mL]. The best cut off for differentiating FTLD from
• AD and NDC using A ⁇ ,. 38 / A ⁇ 1-40 is 0.215.
• Fig. 6 is a scatter plot of FTD, AD, PPA and NDC patients of Example III divided by their percentage abundance of A ⁇ and A ⁇ i. 38 , respectively, relative to the sum of all investigated A ⁇ peptides.
• Fig. 7 is a graph of A ⁇ 1-38% in CSF of non-demented disease controls (NDC),
• AD Alzheimer's disease
• FDD Frontoremporal dementias
• PPA Primary progressive aphasia
• Fig. 8 is a graph showing the mean and the 95% confidence interval (Cl) of the ratio plasma A ⁇ 1-38/1-40 for each diagnostic group of Example IV.
• Fig. 9 is a graph showing the mean and the 95% confidence interval (Cl) of the ratio plasma A ⁇ 1-38/1-42 for each diagnostic group of Example IV.
• Fig. 10 is a graph showing the mean and the 95% confidence interval (Cl) of the ratio plasma A ⁇ 1-40/1 -42 for each diagnostic group of Example IV.
• Table 1 represents plasma A ⁇ peptide patterns, CSF A ⁇ 1-42 and tau and demographic data of the diagnostic groups of Example I.
• Table 2 represents ratios of A ⁇ i -38 / A ⁇ 1-40 for the following diagnostic groups of Example
• Table 3 represents ratio of A ⁇ i -37 / Ap 1-40 for the following diagnostic groups of Example II: FTLD versus NDC and AD patients (Table 3a); FTLD versus AD (Table 3b);
• Table 4 represents ratio of APi -42 / Ap 1-38 for the following diagnostic groups of Example II:
• Table 5 represents ratio of Ap 1-42 / Ap 1-37 for the following diagnostic groups of Example II: FTLD versus NDC and AD patients (Table 4a); FTLD versus AD (Table 4b); FTLD versus NDC (Table 4c); AD versus NDC (Table 4d).
• Table 6 represents Ap peptide patterns, Ap 1-42 and tau in the CSF of the diagnostic groups NDC, AD and FTLD of Example II.
• Table 8 represents diagnostic accuracies of AU peptide patterns in FTLD, AD and NDC of
• Table 9 represents diagnostic accuracies of A ⁇ peptide patterns in AD, FTD, PPA among other dementias and NDC as described in Example III.
• Table 10 represents absolute and percentage abundances of plasma Ap peptide species, CSF Ap1-42, tau, and demographic data of the diagnostic groups of Example IV. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
• vascular dementia and Alzheimer's disease is based on the NINCDS-AIREN and NINCDS-ARDA criteria, respectively.
• the application of other criteria, like for example, the ADDTC criteria is also possible and leads to comparable results.
• biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathological processes, or pharmacological responses to a therapeutic intervention.
• the biomarker can for example describe a substance whose detection indicates a particular disease state.
• the biomarker may be a peptide that causes disease or is associated with susceptibility to disease.
• body fluids includes any fluids which can be obtained from a mammalian body.
• body fluids also includes homogenates of any tissues and other body matter. More particularly, however, the term “body fluids” includes fluids that are normally or abnormally secreted by or excreted from the body.
• the respective fluids may include, but are not limited to: blood, plasma, lymph, urine, and cerebrospinal fluid, blood, plasma, and cerebrospinal fluid being of most interest here.
• the term "quantifying the level” or “quantification of may relate to absolute or relative measurement of the biomarker. Any method may be used to quantify the biomarker(s) level in the bodily fluid. For example, quantitative analysis may involve techniques such as ELISA, SDS-PAGE/immunoblot, or immunocapture-based techniques (Lewczuk et al., 2004b). Other techniques, such as immunoassays, using enzyme labeling, fluorescent labeling, and electrochemiluminescence may also be used to validate the measurement of A ⁇ peptides. Such techniques may involve use of polyclonal or monoclonal antibodies specific to the biomarker of interest.
• quantification may represent the ratio of carboxy-terminal truncated A ⁇ forms to the total A ⁇ iJt2 - In another method, quantification may represent the ratio of carboxy- terminal truncated A ⁇ forms to the carboxy-terminal truncated
• the truncated A ⁇ forms may also include, but are not restricted to, modified forms such as the oxidized helical form of A ⁇ 1-4 o ox ).
• the two notations A ⁇ n . x and A ⁇ n-X used in this description are not intended to indicate different but the same amyloid ⁇ peptide species. Likewise, there is no difference between amyloid ⁇ peptide, beta amyloid peptide, and A ⁇ .
• antibody-mimicking molecule refers to any molecule capable of binding to the epitope of A ⁇ peptide biomarker.
• the molecule may be a synthetic form comprising all the characteristics of a monoclonal antibody.
• sensitivity refers to the capacity of a biomarker to identify a substantial percentage of mammals (for example humans) with the disease. A sensitivity of 100% would mean that a marker can identify 100% of mammals with the disease.
• the mathematical expression of sensitivity is the number of true positive cases of disease divided by the number of true positive cases plus the number of false negative cases.
• specificity refers to the capacity of a biomarker to identify a substantial percentage of mammals (for example humans) without the disease. A specificity of 100% would mean that a mammal is healthy.
• the mathematical expression of specificity is number of true negative cases divided by number of true negative cases plus number of false positive cases.
• control relates to healthy mammals or mammals (for example humans) suffering from mental illness that may include, but is not limited to, affective or emotional instability, behavioral dysregulation, and/or cognitive dysfunction or impairment.
• control refers to a psychiatric cognitive complainer (PCC) and includes mammals suffering from any psychiatric disorders that may lead to cognitive deficiencies complained of by the diseased individual. This may not necessarily have to be verified cognitive testing and includes many psychiatric disorders, such as schizophrenia, depression, or sleep disorders of any kind.
• PCC psychiatric cognitive complainer
• NDC non- demented disease controls
• controls are meant to have no clinical signs of any form of dementia.
• Psychiatric cognitive complainers patients suffering from any psychiatric disorder and complain about cognitive impairment during course of disease
• NDC Non-demented disease controls
• DCC Depressive cognitive complainers
• VAD cerebral spastic syndrome
• AD cerebral spastic syndrome
• AD patients were characterized by typical signs of cerebrovascular disease in brain imaging (CT or MRI) 1 whereas AD patients displayed mild white matter lesions at maximum.
• Patients with relevant cerebrovascular disease or clear focal atrophy in brain imaging, MMSE score below 26 or anamnesis of persistent cognitive decline for at least six month were excluded from the depressive control group.
• Plasma samples were taken from all patients between 2000 and 2004. Handling of the samples followed a standardized protocol according to previously published data, except for time span until freezing which ranged up to 48 hours (Lewczuk et al., 2004).
• a ⁇ peptides were enriched from plasma using the N(or amino)-terminal specific immunoprecipitation by the monoclonal antibody 1 E8, and the A ⁇ -SDS-PAGE/immunoblot served for analysis as published elsewhere (Wiltfang et al., 2002; Bibl et al., 2004; Lewczuk et al., 2004).
• Plasma peptide values are indicated as absolute (pg/ml) or percentage (amount of each peptide relative to the sum of all single investigated A ⁇ peptides) and ratio (amount of each peptide relative to A ⁇ 1-40) values, respectively.
• Significant group differences were evaluated in the Mann-Whitney U-test. The two-sided level of significance was taken as p ⁇ 0.05.
• Receiver operating characteristic curve analysis was used to determine cut-off points at the maximum Youden index. The kappa coefficient was used for measurement of agreement between clinical diagnosis and neurochemical testing. Fisher's z-transformation served for comparison of kappa- values.
• the statistical software package SPSS, version 10.0 was used for computations.
• Fig. 1 visualizes the ratio A ⁇ 1-38/ A ⁇ 1-40 for all three diagnostic groups.
• ROC curve analysis as depicted in Fig. 2 revealed areas under the curve (AUC) as follows: 0.92, 0.87 and 0.89 for A ⁇ 1-38/ A ⁇ 1-40, A ⁇ 1-42/ A ⁇ 1-40 and A ⁇ 2-40/ A ⁇ 1-40, respectively.
• the ratio A ⁇ 1-38/ A ⁇ 1-40 exhibited a sensitivity of 92% for detection of VAD at a specificity of 87% in a combined group of AD and DC.
• the discriminative power of A ⁇ 1-38/ A ⁇ 1- 40 between VAD and AD was even higher, yielding a sensitivity and specificity of 92% and 90%, respectively, at the same cut off point.
• a ⁇ 1- 40 was interpreted as a potential cerebrovascular risk factor, and combined with our results, it may be a potential biomarker to distinguish pure AD from mixed ADA/AD dementia or pure VAD, respectively.
• the specific role of A ⁇ 1-40 in VAD is underlined by the fact that this peptide comprises the major component of vascular amyloid plaques, but is less abundant in senile AD plaques (Gravina et al., 1995). It remains an open question whether the reduction of plasma A ⁇ peptides other than A ⁇ 1-40 may also be closely related to the cerebrovascular amyloid pathology. However, the diagnostic power of this effect can be increased, when A ⁇ 1-40 is assessed in relation to other A ⁇ peptides in plasma.
• FTLD frontotemporal lobe degeneration
• AD Alzheimer's disease
• NDC non- demented disease controls
• the patients were selected from wards and the dementia outpatient clinic of the University of G ⁇ ttingen (G ⁇ ttingen, Germany).
• Five AD patients came from the dementia outpatient clinic of the University of Er Weg-Nuremberg (Er Weg, Germany). Both specialized centers followed a standardized protocol of sample handling.
• Ail neurochemical measurements and quantifications were performed in the laboratory of neurobiology of the University of G ⁇ ttingen by two very experienced technical assistants blinded to clinical diagnosis. Diagnosis was established by a neurologist, a psychiatrist, and a neuropsychologist, all highly experienced in clinical differential diagnosis of dementias on the basis of thorough anamnesis, clinical examination, results of neuropsychological assessment, clinical records of the patients, and the best clinical judgement.
• MMSE Mini- Mental-State-Evaluation
• SCT Short Cognitive Performance Test
• MMSE was available for 26 patients, the mean MMSE score was 20.7 ⁇ 8.9 (mean ⁇ SD (standard deviation)). Neuropsychological assessment was hindered in 4 patients due to severe linguistic or aphasic deficiencies. Age of this group was 61.6 ⁇ 11.5 years (mean ⁇ SD).
• CT computerized tomography
• MRI magnetic resonance imaging
• Functional imaging using either 99mTc-hexamethylpropyleneamine oxime single photon emission computerized tomography (SPECT) or [18F]fluorodeoxyglucose PET investigation of the regional cerebral blood flow was available for 22 patients. Included patients exhibited frontal, frontotemporal, or anterior temporal focal atrophy or marked hypometabolism. A ⁇ 1-42 ELISA results were available for 22 patients, and tau ELISA for 25 patients.
• NDC Non-demented disease controls
• This group consisted of 30 non-demented patients (10 men and 20 women), who underwent lumbar puncture for the differential diagnosis of dementia or other differential diagnostic reasons. Patients with persistent cognitive decline for more than six months, an MMSE score below 26, or clear focal atrophy in brain imaging (CT or MRI) were excluded from the study.
• the mean age of this group was 61.5 ⁇ 11.1 years (mean ⁇ SD).
• MMSE Mini-Mental State Examination
• the mean MMSE score was 28.5 ⁇ 1.5 (mean ⁇ SD).
• the cognitive impairment of all depressive patients improved after antidepressant medication.
• Brain imaging CT or MRI was available for 21 patients.
• Results of A ⁇ 1-42 ELISA and tau ELISA were available for 23 patients.
• a volume of 10 ⁇ L CSF was boiled in sample buffer before analysis of A ⁇ peptides by SDS- PAGE.
• a ⁇ -SDS-PAGE/immunoblot was performed as described elsewhere (Wiltfang et al., 2002, Bibl et al., 2004).
• the stock solution of synthetic A ⁇ peptides (0.5 mg/mL) was diluted to 24 ng/mL (A ⁇ o), 12 ng/mL (A ⁇ i- 38 and Ap 1-42 ), and 6 ng/mL (A ⁇ 1-37 and The peptides were mixed to create the first dilution step of synthetic A ⁇ peptide dilution series and the mixture was diluted 1 :2 three more times.
• amino-terminal-selective mouse monoclonal antibody 1 E8 (Schering, Berlin, Germany) was applied overnight at 4 0 C for detection of the antigen (N-terminus with the first two aminoacids of
• the detection sensitivity of the 1E8 in this optimized immunoblot procedure was 0.6 pg (A ⁇ i. 38l A ⁇ 1-40 ) and 1 pg (A ⁇ 1-37 , A ⁇ 1-39 , A ⁇ 1-42 ), respectively (Bibl et al., 2004).
• the inter- and intra-assay coefficients of variation were below 10% for 80 and 20 pg of the synthetic A ⁇ peptides (Wiltfang et al., 2002; Bibl et al., 2004).
• the novel electrochemiluminescence detection technology was applied to determine CSF A ⁇ s levels independently of the A ⁇ -SDS-PAGE/ immunoblot. It was conducted according to the manufacturer's recommendations (Meso Scale Discovery, Gaithersburg, Maryland, USA). In brief, Multi-Spot 4, 96-well plates (Meso Scale Discovery, Gaithersburg, Maryland, USA) , precoated with the N-terminal-specific anti-A ⁇ antibody 6E10 were blocked with solution A for one hour.
• the plates were then incubated for one hour each with peptide dilution series or 100 ⁇ L CSF samples, followed by c-terminal SULFO-TAG A ⁇ 1-38 detection antibody (Meso Scale Discovery, Gaithersburg, Maryland, USA) , and read buffer. Washing with 1xTris buffer was performed between all incubation steps. The emitted light was read at a wavelength of - 620nm.
• the commercially available assays INNOTEST ® hTAU Antigen and INNOTEST ® ⁇ -Amyloid O -42 ), lnnogenetics (Gent, Belgium) were used for the quantification of tau protein and A ⁇ 1-42 levels, respectively, in CSF.
• the assays were performed according to previously published standard methods (Hulstaert et al., 1999).
• the inter- and intra-assay variability of the A ⁇ 1-42-ELISA was below 10%, the detection sensitivity for A ⁇ 1-42 was 50 pg/mL (Hulstaert et al., 1999).
• a ⁇ peptide levels were expressed as absolute values (ng/mL). The data on peptide levels was obtained from individual blots of each patient sample. Patient groups were characterized by mean and standard deviation (SD).
• the Mann-Whitney U-test was applied to evaluate significant group differences.
• the Wilcoxon signed ranks test was used to calculate significant differences of two related samples. Multiple comparisons were not performed.
• the two-sided level of significance was taken as p ⁇ 0.05. A p-value of less than 0.01 was considered as highly significant.
• ROC Receiver operating characteristic
• the mean age of the diagnostic groups did not differ significantly (p> 0.05).
• the A ⁇ -SDS-PAGE/immunoblot allowed the electrophoretic separation and subsequent analysis of A ⁇ 1-37, A ⁇ 1-38, A ⁇ 1-39 and A ⁇ 1-40 in addition to A ⁇ 1-42. All A ⁇ peptides migrate as a single band, if urea is absent in otherwise unchanged separation gels (Fig. 3)
• the A ⁇ 1-42/ A ⁇ 1- 38 ratio showed no reasonable accuracy in the differentiation of FTLD from NDC or in a combined group of AD and NDC.
• the ratio of A ⁇ 1-38 to A ⁇ 1-40 improved the discrimination of FTLD and NDC to a sensitivity of 87 % and a specificity of 90%. Additionally, this ratio provided a sensitivity of 87% and a specificity of 87% for differentiation of FTLD from AD.
• the ratio of A ⁇ 1-38 to A ⁇ 1-40 achieved the best discrimination of FTLD among both NDC and AD, yielding a sensitivity and specificity of 87% and 88%, respectively.
• This assay reached a sensitivity of 91 % and a specificity of 81% for the discrimination of FTLD and AD.
• the sensitivity and specificity of FTLD detection among NDC was 50% and 61 %, respectively.
• the diagnostic power of the test for FTLD detection among AD was increased to a sensitivity of 86 % and a specificity of 100%.
• the sensitivity and specificity for detection of FTLD among NDC patients was 81 % and 61%, respectively.
• the detection of FTLD among both NDC and AD yielded a sensitivity of 100% and specificity of 45% .
• PD Parkinson's disease
• PDD Parkinson's disease dementia
• DLB DLB
• Diagnoses were rendered by a psychiatrist, a neurologist and a neuro-psychologist, who were all very experienced in clinical differential diagnosis of dementias, on the basis of thorough anamnesis, clinical examination, results of a neuropsychological assessment, clinical records, and best clinical judgement. All three investigators were blinded to the results of the neurochemical outcome measurements. Investigations were carried out with the informed consent of patients or their authorized caregiver. If possible, neuropsychological assessment (MMSE at minimum) was performed on patients suffering from cognitive impairments at the time of lumbar puncture. The study was conducted under the guidelines of the Declaration of Helsinki and approved by the ethics committees of the Universities of Goettingen, Er Weg- Nuremberg and Hessen.
• NDC Non-demented disease controls
• the NDC group consisted of three subgroups:
• the mean age of this subgroup was 63.0 ⁇ 10.3 years (mean ⁇ SD).
• Neurodegenerative diseases without dementia ND
• Depressive cognitive complainers Twenty-six depressive patients (8 men and 18 women) underwent lumbar puncture for differential diagnosis of cognitive complaints during the course of disease. The diagnosis of depression was made according to the DSM IV criteria and cognitive impairment was assessed by MMSE at minimum. The mean MMSE score was 28.6 ⁇ 1.4 (mean ⁇ SD). The mean age of this subgroup was 62.9 ⁇ 10.3 years (mean ⁇ SD).
• PPA primary progressive aphasia
• VAD vascular dementia
• Parkinson's disease dementia was diagnosed in 21 patients according to the UK Parkinson's Disease Society Brain Bank clinical diagnostic criteria for idiopathic Parkinson's disease and the consensus criteria. All patients presented with parkinsonism at least one year before onset of dementia.
• CJD Creutzfeld-Jakob's disease
• the novel electrochemiluminescence detection technology was applied to determine CSF A ⁇ 1-38 levels independently of the A ⁇ -SDS-PAGE/ immunoblot. It was conducted according to the manufacturer's recommendations (Meso Scale Discovery). In brief, Multi-Spot 4, 96 well plates, precoated with the N-terminal-specific anti-A ⁇ antibody 6E10 were blocked with solution A for one hour. The plate was then incubated with peptide dilution series or 100 ⁇ L CSF sample, followed by C-terminal SULFO-TAG A ⁇ i -38 detection antibody and Read Buffer, each for one hour. Washing with 1xTris buffer was performed between incubation steps. The measurement of emitted light was performed at - 620 nm.
• the mean age of FTD and NDC was significantly younger than all other patient groups.
• the mean MMSE score did not significantly differ between the dementia groups.
• the A ⁇ -SDS- PAGE/immunoblot revealed a regular abundant pattern of six peptides: A ⁇ o, A ⁇ 1-3 8, A ⁇ , A ⁇ 1-39 , A ⁇ 1-37 and A ⁇ 1-40 OX .
• AD versus other dementias A specific decrease of Ap 1-42 WaS evident in AD, whereas other dementias with low A ⁇ 1-42 levels displayed an overall decrease of all A ⁇ peptides.
• percentage terms the aforementioned alterations were only present for A ⁇ 1 . 38 %, A ⁇ L39 0 Zo and A ⁇ ⁇ 2 %, respectively.
• a ⁇ 1-38 % The pronounced percentage reduction of A ⁇ 1-38 % in FTD exhibited satisfactory accuracies of above 85% for discrimination among all other dementias and NDC.
• the loss of accuracy of the A ⁇ s/A ⁇ o ratio as compared to A ⁇ 1-38 % was mainly due to elevated A ⁇ 1-40 levels in individual AD, DLB, and PPA patients.
• This example aimed at obtaining detailed knowledge about the plasma A ⁇ peptide patterns in VAD and FTD.
• Plasma and CSF samples of AD, VAD, FTD and patients with depression came from the memory clinic of the Department of Psychiatry, University of Goettingen or from hospitalized patients. All patients, except patients with FTD and one with vascular dementia have been investigated in a previous study and the results have been published (Bibl et al., 2007a)
• Non-demented depressive controls The 7 patients with depression (2 men and 5 women) underwent lumbar puncture for differential diagnosis of cognitive complaints during the course of disease. The diagnosis of depression was made according to the criteria of DSM IV and cognitive impairment was assessed by MMSE at minimum. Patients with history of persistent cognitive decline for more than six months, MMSE score below 26, clear focal atrophy or with signs of relevant vascular disease in brain imaging were excluded.
• AD Alzheimer's disease
• AD patients displayed no signs of relevant cerebrovascular disease in either CT or MRI.
• VAD vascular dementias
• FTP Frontotemporal dementia
• CSF and plasma were drawn from patients by lumbar and venous puncture, respectively, on the same day.
• the preanalytical handling of samples followed a standardized protocol according to previously published data, except that the time span until freezing of plasma ranged up to 12 hours (Lewczuk et al. 2004).
• Magnetic sheep anti-mouse IgG Dynabeads M-280 (Dynal, Hamburg, Germany) were incubated overnight at +4°C with the monoclonal A ⁇ amino-terminally-selective antibody, 1 E8 (provided by Schering, Berlin, Germany) according to the manufacturer's protocol.
• Five hundred ⁇ L of plasma were added to 200 ⁇ l_ of five-fold concentrated RIPA detergent buffer (Wiltfang et al. 2002), 25 ⁇ L of the activated magnetic Dynabeads (1 ⁇ g mAb 1 E8/1.68 x 10 7 beads) and 300 ⁇ L of H 2 O.
• the amino-terminally-selective mouse monoclonal antibody 1 E8 (provided by Schering, Berlin, Germany; stock: 0.25 mg/ mL) was diluted 4000-fold and applied overnight at 4°C. Membranes were further incubated with a biotinylated anti-mouse polyclonal antibody (Vector Laboratories, Burlingame, CA, USA) and horseradish peroxidase coupled streptavidin (Amersham Pharmacia Biotech, Buckinghamshire, England) for 1 h each. Washing steps were performed in between and chemiluminescent visualization was done by ECL-Plus solution (Amersham Pharmacia).
• Plasma samples and peptide standards were run on the same gel as triplicates at minimum. Each gel carried a five point dilution series of synthetic A ⁇ peptides A ⁇ 1-37, A ⁇ 1-38, A ⁇ 1-39, A ⁇ 1-40 and A ⁇ 1-42. A ⁇ 2-40 was only applied in the two lower concentrations, because bands of A ⁇ 2-40 and A ⁇ 1-42 tended to diffuse into one single band when higher concentrations were applied. Thus, the three higher concentrations of dilution series of A ⁇ 1-42 were used to complement the standard row of A ⁇ 2-40.
• Synthetic peptides A ⁇ 1-38, A ⁇ 1-40, A ⁇ 1-42 and A ⁇ 2- 40 were obtained from Bachem (Bubendorf, Switzerland), A ⁇ 1-37 and A ⁇ 1-39 were synthesized automatically according to Janek et al. (Janek K et al 2001). Standard preparations of synthetic A ⁇ peptide mixture were created as described previously (Bibl M et al. 2004) and bands were quantified from individual blots of each patient relative to this dilution series using a charge coupled device camera (CCD-camera) and Quantity-ONE software (FluorSMax Multilmager, Bio-Rad)
• the detection sensitivity of the A ⁇ -SDS-PAGE/immunoblot was 0.6-1 pg (Wiltfang et al. 2002, Bibl et al. 2004). Coefficients of variation of the A ⁇ peptides' concentrations ranged between 14.0 and 18.5% (Lewczuk et al. 2004).
• CSF peptide levels are given in absolute values (pg/ml).
• Plasma peptide values are given in absolute (pg/ml) or percentage (amount of each peptide relative to the sum of all single investigated A ⁇ peptides) values, respectively.
• Significant group differences were evaluated with the Mann-Whitney litest. Comparisons of multiple groups were evaluated by the Kruskal-Wallis test. Spearman's rho was used for correlation analysis. We used the biserial correlation coefficient to determine the relationship between clinical diagnosis and neurochemical testing. Fisher's z-transformation served for comparison of biserial correlation coefficients. The two-sided level of significance was taken as p ⁇ 0.05.
• Plasma A ⁇ peptides in DC and AD did not significantly differ.
• FTD and VAD displayed similarities in elevated percentage levels of A ⁇ 1-40 and decreased percentage levels of A ⁇ 1-38 as compared to DC and AD. Conversely, percentage levels of A ⁇ 1-42 and A ⁇ 2-40 were diminished only in VAD, but not in FTD.
• Diagnosis of FTD The positive diagnosis of FTD among DC and AD could be most effectively supported by elevated levels of A ⁇ 1-40 accompanied by decreased levels of A ⁇ 1-38 and A ⁇ 1-37, respectively.
• a detailed characterization of A ⁇ peptide species in plasma of FTD in comparison to VAD, AD and depressive controls (DC) is presented.
• a ⁇ 1-40 and A ⁇ 1-42 in AD the results are in line with previous studies of others (Irizarry 2004, Mehta and Pirttila 2005).
• a prominent percentage increase of plasma A ⁇ 1-40 (A ⁇ 1-40%) in VAD as compared to AD and DC was found, which was paralleled by decreases of A ⁇ 1-38%, A ⁇ 1-42% and A ⁇ 2- 40%.
• FTD revealed alterations similar to VAD with regard to A ⁇ 1-40% and A ⁇ 1-38%.
• FTD displayed lower absolute levels of A ⁇ 1-37 and higher percentage levels of A ⁇ 1-42 than VAD.
• a ⁇ 1-40 in VAD is underlined by the fact that this peptide comprises the major component of vascular amyloid plaques, but is less abundant in senile AD plaques (Gravina et al. 1995). Nevertheless, it remains an open question whether the relative reduction of plasma A ⁇ peptides other than A ⁇ 1-40 (i.e. A ⁇ 1-38, A ⁇ 1-42 and A ⁇ 2-40 in comparison DC) may also be related to cerebrovascular amyloid pathology. However, from the above analysis, the diagnostic power of plasma A ⁇ 1-40 levels will be increased if assessed in relation to A ⁇ 1-38 or A ⁇ 1-42, respectively.
• plasma samples have to be investigated following a standardized pretreatment protocol.
• the focus should be particularly on the effect of sample storage under distinct conditions (e.g. storage time and temperature) prior to analyses.
• Selected carboxy-terminally truncated amyloid ⁇ peptides as parts of the overall plasma A ⁇ peptide pattern are a biomarker for the improved differential diagnosis of FTD and VAD.
• Both FTD and VAD may be differentiated from other non-demented patients and other dementias, such as AD 1 by plasma concentrations of A ⁇ 1-40, A ⁇ 1-38 and A ⁇ 1-37.
• discrimination between FTD and VAD should additionally consider plasma A ⁇ 1-42 levels.
• Citron M Vigo-Pelfrey C, Teplow DB, Miller C, Schenk D, Johnston J, Winblad B, Venizelos N, Lannfelt L, Selkoe DJ. Excessive production of amyloid beta-protein by peripheral cells of symptomatic and presymptomatic patients carrying the Swedish familial Alzheimer disease mutation. Proc Natl Acad Sci USA JID - 7505876 1994; 91 : 11993-11997.
• Alzheimer's disease initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 1984; 120: 885-890.
• Gurol ME Irizarry MC, Smith EE, Raju S, Diaz-Arrastia R, Bottiglieri T, Rosand J, Growdon JH, Greenberg SM. Plasma ⁇ -amyloid and white matter lesions in AD, MCI and cerebral amyloid angiopathy. Neurology 2006; 66:23-29.
• Irizarry MC Biomarkers of Alzheimer disease in plasma. NeuroRX 2004; 1 :226-234.
• AUC Area under curve
• Cl Confidence interval

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