Human CSF Research Articles

Detection of West Nile virus in six mosquito species in synchrony with seroconversion among sentinel chickens in India

BackgroundWest Nile virus (WNV) is a zoonotic flavivirus maintained in mosquito-bird transmission cycle. Although humans are accidental hosts, fatal outcomes following WNV infection have been reported from India. Studies have identified WNV as an important etiological agent causing acute encephalitis syndrome in Assam, Northeast India. While circulation of WNV is evident, the role of vectors and avian hosts involved in the transmission remains unclear. In this study we identified local mosquito species for evidence of WNV infection along with seroconversion among sentinel chickens.MethodsMosquitoes were collected and pooled species wise from June 2014 through December 2015. Virus was screened using reverse transcriptase PCR followed by sequencing and phylogenetic analysis. Sentinel chicken blood was screened for WNV antibody to assess their role in WNV transmission.ResultsA total of 52,882 mosquitoes belonging to 16 species were collected. WNV was detected in 18 pools of Culex vishnui, Culex tritaeniorhynchus, Culex quinquefasciatus, Culex whitmorei, Culex pseudovishnui and Mansonia uniformis. Phylogenetic analysis revealed that all mosquito derived sequences belonged to Lineage 5 and were 99–100% similar to the Assam strain of WNV isolated from human CSF sample in 2007. All sentinel chickens had seroconverted by the month of July that happens to be the peak WNV transmission month among humans as well.ConclusionTo the best of our knowledge, this is the first report of WNV identification from field-collected Cx. pseudovishnui and Mansonia uniformis in India. Our study demonstrates potential vectors which may play a crucial role in WNV transmission and should be considered in the vector control strategies. Additionally, our study highlights the role of sentinel chickens for WNV surveillance.

Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System.

Sirtuin proteins have a variety of intracellular targets, thereby regulating multiple biological pathways including neurodegeneration. However, relatively little is currently known about the role or expression of the 7 mammalian sirtuins in the central nervous system. Western blotting, PCR and ELISA are the main techniques currently used to measure sirtuin levels. To achieve sufficient sensitivity and selectivity in a multiplex-format, a targeted mass spectrometric assay was developed and validated for the quantification of all seven mammalian sirtuins (SIRT1-7). Quantification of all peptides was by multiple reaction monitoring (MRM) using three mass transitions per protein-specific peptide, two specific peptides for each sirtuin and a stable isotope labelled internal standard. The assay was applied to a variety of samples including cultured brain cells, mammalian brain tissue, CSF and plasma. All sirtuin peptides were detected in the human brain, with SIRT2 being the most abundant. Sirtuins were also detected in human CSF and plasma, and guinea pig and mouse tissues. In conclusion, we have successfully applied MRM mass spectrometry for the detection and quantification of sirtuin proteins in the central nervous system, paving the way for more quantitative and functional studies.

P.5.a.007 - Altered expression of dystrophin protein in the brain of an animal model of Alzheimer’s disease

Truncated forms and full-length forms of the amyloid-beta 40 (Aβ40) are key molecules in the pathogenesis of dementia, and are detectable in CSF. Reliable methods to detect these biomarkers in CSF are of great importance for understanding the disease mechanisms and for diagnostic purposes.VU-α-Aβ40, a monoclonal antibody (mAb) specifically detecting Aβ40, was generated and characterized by solid and fluid phase ELISA, surface plasmon resonance spectroscopy (SPRS), immunoprecipitation (IP), immunohistochemical and Western blot (WB) analysis. In addition, an ELISA with VU-α-Aβ40 as catching and 6E10 as detecting mAbs was set up and validated. This ELISA was used to measure Aβ40 in CSF of controls (N = 27), patients with Alzheimer's disease (AD; N = 20), frontotemporal lobe dementia (FTLD; N = 14), noninflammatory (N = 15) and inflammatory (N = 15) neurological conditions.VU-α-Aβ40 specifically recognizes Aβ40 with high affinity (KA = 1.3 × 109 M− 1) and detects Aβ40 in AD brain specimens. The developed sandwich ELISA has a detection limit of 0.21 ng/mL, a mean recovery of 90%, and an intra- and inter-assay CV of 1.4% and 7.3%. FTLD patients had a lower mean level of Aβ40 (8.8 (1.9) ng/mL) than controls (12.0 (1.7) ng/mL); p < 0.01).VU-α-Aβ40 was successfully implemented in an ELISA which enables us to measure Aβ40 accurately in human CSF. Clinical validation revealed lower levels of Aβ40 in FTLD patients. This finding opens new possibilities for early and differential diagnosis of dementia.

Beneficial effects of increased lysozyme levels in Alzheimer's disease modelled in Drosophila melanogaster.

Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer's disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome‐wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aβ1‐42 or AβPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aβ1‐42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aβ1‐42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aβ1‐42 and caused a beneficial effect by binding of lysozyme to toxic species of Aβ1‐42, which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.

Differential Neuroproteomic and Systems Biology Analysis of Spinal Cord Injury

Acute spinal cord injury (SCI) is a devastating condition with many consequences and no known effective treatment. Although it is quite easy to diagnose traumatic SCI, the assessment of injury severity and projection of disease progression or recovery are often challenging, as no consensus biomarkers have been clearly identified. Here rats were subjected to experimental moderate or severe thoracic SCI. At 24h and 7d postinjury, spinal cord segment caudal to injury center versus sham samples was harvested and subjected to differential proteomic analysis. Cationic/anionic-exchange chromatography, followed by 1D polyacrylamide gel electrophoresis, was used to reduce protein complexity. A reverse phase liquid chromatography-tandem mass spectrometry proteomic platform was then utilized to identify proteome changes associated with SCI. Twenty-two and 22 proteins were up-regulated at 24 h and 7 day after SCI, respectively; whereas 19 and 16 proteins are down-regulated at 24 h and 7 day after SCI, respectively, when compared with sham control. A subset of 12 proteins were identified as candidate SCI biomarkers - TF (Transferrin), FASN (Fatty acid synthase), NME1 (Nucleoside diphosphate kinase 1), STMN1 (Stathmin 1), EEF2 (Eukaryotic translation elongation factor 2), CTSD (Cathepsin D), ANXA1 (Annexin A1), ANXA2 (Annexin A2), PGM1 (Phosphoglucomutase 1), PEA15 (Phosphoprotein enriched in astrocytes 15), GOT2 (Glutamic-oxaloacetic transaminase 2), and TPI-1 (Triosephosphate isomerase 1), data are available via ProteomeXchange with identifier PXD003473. In addition, Transferrin, Cathepsin D, and TPI-1 and PEA15 were further verified in rat spinal cord tissue and/or CSF samples after SCI and in human CSF samples from moderate/severe SCI patients. Lastly, a systems biology approach was utilized to determine the critical biochemical pathways and interactome in the pathogenesis of SCI. Thus, SCI candidate biomarkers identified can be used to correlate with disease progression or to identify potential SCI therapeutic targets.

The Lyme Disease Spirochete Borrelia Burgdorferi Induces Increased Expression of Multiple Neuroinflammatory Factors in Human Microglial Cells In Vitro

Many microorganisms can cause inflammation in the central nervous system (CNS) by damaging or penetrating the protective blood‐brain and blood‐cerebral spinal fluid barriers that sequester the brain from the periphery. Breakdown of the blood‐brain barrier (BBB) or blood‐cerebral spinal fluid (blood‐CSF) barrier could facilitate entry of both B. burgdorferi and activated immune cells into the CNS leading to multiple neurological complications, collectively termed neuroborreliosis. Indeed, B. burgdorferi has an affinity for the CNS and can be isolated from the CSF of humans as early as 18 days after the bite of an infected tick. However, the mechanism by which the bacterium initiates neuroinflammation leading to neural disfunction remains unknown. The ensuing neurological symptoms are likely a result of a glial‐driven, host inflammatory response to the bacterium. However, the specific contributions of human microglial cells to the pathogenesis of neuroborreliosis have not yet been elucidated.We hypothesize that dead B. burgdorferi and debris persist in the CNS after antibiotic treatment and contribute to an ongoing inflammatory response in the CNS. To begin characterization of this response, highly pure cultures of primary human microglia were incubated with live virulent B. burgdorferi and the membrane lipoprotein Pam3Cys to elucidate the cell‐ and lipoprotein‐specific responses to the bacterium. Cultures were analyzed by immuncytochemistry, real time PCR array and ELISA analysis. Array results were confirmed with individual sets of PCR primers. Our results demonstrate a robust increase in numerous inflammatory chemokines, cytokines and related genes in all treatment conditions examined. Most prominent was an increase in the neurophil chemoattractants IL‐8, IL‐6, CXCL1, CXCL10 and LIF. These data suggest that, in addition to B. burgdorferi, infiltrating phagocytic cells, particularly potentially neurotoxic neutrophils, may be important additional contributors to the early inflammatory events associated with the onset of neuroborreliosis.Our continuing studies are now focused on elucidating microglial phagocytosis of B. burgdorferi using real time PCR arrays and culture monolayers coupled with confocal microscopy and trans emission microscopy. Interestingly, initial data has demonstrated that although no there is no phagocytosis of live bugs in vitro, primary human microglia highly up regulate numerous genes associated with phagocytosis and associated cell signaling when exposed to dead and/or dead‐sonicated forms of B. burgdorferi. More specifically, in microglia exposed to dead and/or dead‐sonicated B. burgdorferi, there was a significant increase in expression of genes known to be involved in recognition of Bb (TLRs, NOD1, CD14); Fc receptors; kinases (LYN, MAPK); and things involved in uptake/phagolysosomes (GULP‐1, members of the Rac/Rho family, phospholipases, VAMP, syntaxin). Taken together, these results suggest that primary human microglia continue to mount a pro‐inflammatory response in the absence of viable Borrelia burgdorferi.Support or Funding InformationThis study was funded by a grant from the University of North Dakota Office of Research Development and Compliance (2014).

Human cerebrospinal fluid microRNA: temporal changes following subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating form of hemorrhagic stroke with 30-day mortality between 33 and 45%. Delayed cerebral ischemia (DCI) is the chief cause of morbidity and mortality in patients who survive the initial aSAH. DCI accounts for almost 50% of deaths in patients surviving to treatment of the ruptured aneurysm. The mechanisms for brain injury after aSAH and the brain's response to this injury are not fully understood in humans. MicroRNAs (miRs) are 22- to 25-nucleotide single-stranded RNA molecules that inhibit the expression of specific messenger RNA targets. In this work, miR profiling of human cerebrospinal fluid from eight patients after aSAH was performed daily for 10 days with the goal of identifying changes in miR abundance. Using the nanoString nCounter Expression Assay, we identified two specific clusters of miR that were differentially regulated over time. Quantitative RT-PCR was performed on select miRs from each cluster. The first cluster contained miRs known to be present in blood and decreased in abundance over time. miRs in this group include miR-92a and let-7b. The second cluster contained several poorly characterized miRs that increased in abundance over time. miRs in this group included miR-491. This second cluster of miRs may be released into the CSF by the brain itself as a result of the initial SAH. Temporal changes in the abundance of specific miRs in human CSF after aSAH may provide novel insight into the role of miRs in brain injury and the brain's response.

Pharmacological BACE1 and BACE2 inhibition induces hair depigmentation by inhibiting PMEL17 processing in mice.

Melanocytes of the hair follicle produce melanin and are essential in determining the differences in hair color. Pigment cell-specific MELanocyte Protein (PMEL17) plays a crucial role in melanogenesis. One of the critical steps is the amyloid-like functional oligomerization of PMEL17. Beta Site APP Cleaving Enzyme-2 (BACE2) and γ-secretase have been shown to be key players in generating the proteolytic fragments of PMEL17. The β-secretase (BACE1) is responsible for the generation of amyloid-β (Aβ) fragments in the brain and is therefore proposed as a therapeutic target for Alzheimer’s disease (AD). Currently BACE1 inhibitors, most of which lack selectivity over BACE2, have demonstrated efficacious reduction of amyloid-β peptides in animals and the CSF of humans. BACE2 knock-out mice have a deficiency in PMEL17 proteolytic processing leading to impaired melanin storage and hair depigmentation. Here, we confirm BACE2-mediated inhibition of PMEL17 proteolytic processing in vitro in mouse and human melanocytes. Furthermore, we show that wildtype as well as bace2+/− and bace2−/− mice treated with a potent dual BACE1/BACE2 inhibitor NB-360 display dose-dependent appearance of irreversibly depigmented hair. Retinal pigmented epithelium showed no morphological changes. Our data demonstrates that BACE2 as well as additional BACE1 inhibition affects melanosome maturation and induces hair depigmentation in mice.

A comparative evaluation of ELISA test employing antigen 85C with ZN staining, culture and PCR in the diagnosis of Tuberculosis in children

Objectives: Childhood tuberculosis is difficult to diagnose. A rapid, simple and relatively inexpensive diagnostic test will be crucial to future control efforts. Therefore, diagnostic potential of secretory antigen 85C in human sera specimen, CSF and other body fluids of Mycobacterium tuberculosis in childhood tuberculosis cases were evaluated using ELISA technique and reactivity was compared with the gold standards (Ziehl Neelsen staining, BACTEC culture) and IS6110 targeted PCR. Methods: In the present study, 73 fresh, untreated childhood tuberculosis (TB) cases under 18 years of age out of these 27 had pulmonary tuberculosis and 46 patient had extra pulmonary tuberculosis. Twenty healthy children were included. Results: ELISA with Antigen 85C in sera was positive in 88.89% and 80.43% of pulmonary and extrapulmonary cases, respectively. Overall sensitivity and specificity were 83.56% and 65%, (p 0.05). PCR targeting IS6110 was positive in 96.30% and 65.22% of pulmonary and extrapulmonary cases, respectively. Overall sensitivity and specificity were 76.71% and 90%, respectively. Conclusions: Our findings demonstrates the potential of Ag 85C in sera in the detection of antibody in childhood TB cases and this antigen showed good concordance with PCR positivity.

Proteolytic degradation of neuropeptide Y (NPY) from head to toe: Identification of novel NPY-cleaving peptidases and potential drug interactions in CNS and Periphery.

The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed exvivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application. The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed exvivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application.

Advances and new insights in the neuropathogenesis of dengue infection.

Dengue virus (DENV) infects approximately 390 million persons every year in more than 100 countries. Reports of neurological complications are more frequently. The objective of this narrative review is to bring up the advances in the dengue neuropathogenesis. DENV can access the nervous system through blood-brain barrier disturbance mediated by cytokine. The blood-cerebrospinal fluid (CSF) barrier seems to be also involved, considering the presence of the virus in the CSF of patients with neurological manifestations. As for neurotropism, several studies showed the presence of RNA and viral antigens in brain tissue and CSF in humans. In murine model, different virus mutations were associated to neurovirulence. Despite the advances in the dengue neuropathogenesis, it is still necessary to determine a more appropriate animal model and increase the number of cases of autopsy. The detection of neurovirulence markers may contribute to establish a prognosis, the disease control and vaccine development.

A candidate reference method for analysis of amyloid beta 1-42 (Aß1-42) peptide in human CSF using 2D-HPLC with a xevo t q-s mass spectrometer

A candidate reference method for analysis of amyloid beta 1-42 (Aß1-42) peptide in human CSF using 2D-HPLC with a xevo t q-s mass spectrometer

New insights into the human brain proteome: Protein expression profiling of deep brain stimulation target areas

Deep brain stimulation (DBS) is a neurosurgical procedure that provides therapeutic benefits for movement and affective disorders. The nucleus basalis of Meynert (NBM) and substantia nigra (SN) are considered target areas to apply DBS. Even though the degeneration of NBM and SN underlies the cognitive decline observed in neurological diseases, the protein knowledge derived from both areas is scarce. We have characterized the proteome present in both subcortical brain areas using the Triple TOF 5600 mass spectrometer, identifying 2775 and 3469 proteoforms in NBM and SN respectively. Data mining of MS-generated proteomic data have revealed that: i) 675 proteins tend to localize to synaptic ending, ii) 61% of the global dataset is also present in human CSF and/or plasma, and iii) 894 proteins have not been previously identified in healthy brain by MS. The correlation of NBM and SN proteomic expression profiles with human brain transcriptome data available at Allen Brain Atlas has revealed protein evidence for 250 genes considered with brain-wide expression and 112 genes with region-specific expression in human brain. In addition, protein datasets have been classified according to their chromosomal origin, increasing the current proteome coverage in healthy human brain. Biological significanceThe nucleus basalis of Meynert and substantia nigra are brain areas of clinical interest to apply the deep brain stimulation (DBS) technology in neurosurgery. Our proteomic characterization has revealed 675 proteins involved in the regulation of synaptic transmission, electrical machinery, and neurotransmitter release in both DBS target areas. Moreover, 2599 identified proteins present capacity to be secreted to the CSF and plasma. Our data contribute to a further step towards the characterization of the anatomical atlas of the human brain proteome, detecting 652 proteins that are common between different basal ganglia structures.This article is part of a Special Issue entitled: HUPO 2014.

Inspiration is the major regulator of human CSF flow.

The mechanisms behind CSF flow in humans are still not fully known. CSF circulates from its primary production sites at the choroid plexus through the brain ventricles to reach the outer surface of the brain in the subarachnoid spaces from where it drains into venous bloodstream and cervical lymphatics. According to a recent concept of brain fluid transport, established in rodents, CSF from the brain surface also enters the brain tissue along para-arterial routes and exits through paravenous spaces again into subarachnoid compartments. This unidirectional flow is mainly driven by arterial pulsation. To investigate how CSF flow is regulated in humans, we applied a novel real-time magnetic resonance imaging technique at high spatial (0.75 mm) and temporal (50 ms) resolution in healthy human subjects. We observed significant CSF flow exclusively with inspiration. In particular, during forced breathing, high CSF flow was elicited during every inspiration, whereas breath holding suppressed it. Only a minor flow component could be ascribed to cardiac pulsation. The present results unambiguously identify inspiration as the most important driving force for CSF flow in humans. Inspiratory thoracic pressure reduction is expected to directly modulate the hydrostatic pressure conditions for the low-resistance paravenous, venous, and lymphatic clearance routes of CSF. Furthermore, the experimental approach opens new clinical opportunities to study the pathophysiology of various forms of hydrocephalus and to design therapeutic strategies in relation to CSF flow alterations.

A novel, ultrasensitive assay for tau: potential for assessing traumatic brain injury in tissues and biofluids.

Traumatic brain injury (TBI) is a cause of death and disability and can lead to tauopathy-related dementia at an early age. Pathologically, TBI results in axonal injury that is coupled to tau hyperphosphorylation, leading to microtubule instability and tau-mediated neurodegeneration. This suggests that the forms of this protein might serve as neuroinjury-related biomarkers for diagnosis of injury severity and prognosis of the neurological damage prior to clinical expression. We initially determined whether we could detect tau in body fluids using a highly sensitive assay. We used a novel immunoassay, enhanced immunoassay using multi-arrayed fiberoptics (EIMAF) either alone or in combination with rolling circle amplification (a-EIMAF) for the detection of total (T) and phosphorylated (P) tau proteins from brains and biofluids (blood, CSF) of rodents following controlled cortical impact (CCI) and human patients post severe TBI (sTBI). This assay technology for tau is the most sensitive to date with a detection limit of approximately 100 ag/mL for either T-tau and P-tau. In the rodent models, T-tau and P-tau levels in brain and blood increased following CCI during the acute phase and remained high during the chronic phase (30 d). In human CSF samples, T-tau and P-tau increased during the sampling period (5-6 d). T-tau and P-tau in human serum rose during the acute phase and decreased during the chronic stage but was still detectable beyond six months post sTBI. Thus, EIMAF has the potential for assessing both the severity of the proximal injury and the prognosis using easily accessible samples.

The remarkable properties of amyloid-β derived from human Alzheimer's disease brain: swinging the streetlight.

This scientific commentary refers to ‘Highly potent soluble amyloid-β seeds in human Alzheimer brain but not cerebrospinal fluid’ by Fritschi et al. (doi: 10.1093/brain/awu255). Despite many years of intensive study, the specific amyloid-β species in the human brain responsible for the pathophysiological processes underlying Alzheimer’s disease have yet to be identified. In part, this may be because we have been ‘searching under the streetlight’: examination of other sources of amyloid-β such as synthetic preparations, material derived from the brains of transgenic animals, and amyloid-β recovered from human CSF after lumbar puncture has been much more convenient than direct evaluation of human brain-derived material. However, telltale signs over the last several years have indicated that amyloid-β derived from human brain may have properties or constituents that are qualitatively and quantitatively different from those of amyloid-β from other sources. The paper by Fritschi et al. (2014) from Mathias Jucker’s group in this issue of Brain is a major contribution to this line of investigation. Notably, the paper provides perhaps the most definitive evidence yet that human brain-derived amyloid-β has fundamentally dissimilar properties to human CSF-derived material. Specifically, Fritschi et al. demonstrate that <1 …

Abstract 078: Angiotensin Converting Enzyme type 2 shedding in the central nervous system of hypertensive patients

ACE2 (Angiotensin Converting Enzyme type 2) is involved in the conversion of the vasoconstrictor peptide Angiotensin (Ang)-II into its vasodilatory metabolite Ang-(1-7), thereby offering a new perspective for the treatment of cardiovascular diseases associated with over-activity of the renin-angiotensin system. Our group recently reported that ACE2 shedding, a process by which ACE2 is cleaved from the plasma membrane and secreted into the surrounding milieu, contributes to the loss of the enzyme’s compensatory activity in the central nervous system (CNS) during the development of experimental hypertension. The objective of this study was to determine whether ACE2 shedding is taking place in the CNS of patients during hypertension. Unused cerebrospinal fluid, collected from patients (17 males and 27 females, aged 22-66) for diagnostic purposes, was divided in 3 groups: normotensive (n=23), hypertensive (n=9), and hypertensive controlled with medication (n=12). In addition, blood pressure values and current medications were recorded. While ELISA lacked sensitivity, soluble levels of hACE2 (sACE2) could be detected by Mass spectrometry-based RAS-Fingerprint™ in all patients. Validation experiments (n=16) indicated that pre-treatment with MLN4760 (ACE2 inhibitor) abolished the conversion of Ang-II to Ang-(1-7) by 76 ±3 %, suggesting that ACE2 is the main enzyme converting Ang-II to Ang-(1-7) in human CSF. In hypertensive patients not taking blood pressure medications, (Systolic: 157 ±10, Diastolic: 95 ±7 mmHg; n=6) CSF sACE2 activity was significantly higher (21.7 ±8.3 AU; P&lt;0.05) than in normotensive (Systolic: 114 ±4, Diastolic: 75 ±4 mmHg; n=15) patients (6.6 ±0.9 AU). However, in patients with controlled hypertension (Systolic: 129 ±6, Diastolic: 86 ±5 mmHg; n=5), CSF sACE2 levels were normalized (6.9 ±1.9 AU). Comparison of systolic or diastolic blood pressure vs. sACE2 activity levels showed no significant correlation. These data suggest that ACE2 shedding is taking place in the CNS of hypertensive patients. Soluble ACE2 activity in the CSF is correlated with high blood pressure and could be used as a biomarker of neurogenic hypertension. AU: arbitrary unit.

Methodological aspects of ELISA analysis of thioredoxin 1 in human plasma and cerebrospinal fluid.

Thioredoxin-1 (Trx1) is a protein antioxidant involved in major cellular processes. Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8–87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases.

Mass spectrometry-based candidate reference measurement procedure for quantification of amyloid-β in cerebrospinal fluid.

Cerebrospinal fluid (CSF) amyloid-β (Aβ42) is a well-established biomarker for Alzheimer disease. Several immunoassays for Aβ42 exist but differ in absolute concentrations and may suffer from matrix interference, thereby hampering interlaboratory comparisons and the use of general cutoff levels. Together with the IFCC Working Group on CSF Proteins, we developed a candidate reference measurement procedure (RMP) for Aβ42. The antibody-independent candidate RMP was based on solid-phase extraction and isotope-dilution LC-MS/MS. The candidate RMP used 2 differently stable isotope-labeled Aβ42 peptides for calibration in human CSF, an important aspect since there was no analyte-free matrix available. Because no CSF certified reference material (CRM) exists, we used a nonlabeled Aβ42 standard, the concentration of which was determined by amino acid analysis. We performed measurements on a high-resolution quadrupole-Orbitrap hybrid instrument. The results were compared with a method run in a second laboratory with triple quadrupole instrumentation. The candidate RMP allowed quantification of CSF Aβ42 from 150 to 4000 pg/mL. Validation of the method showed a recovery of 100% (15%), intraassay and interassay imprecision of 5.0% and 6.4%, respectively, and an expanded uncertainty of 15.7%. No analytical interferences or carryover were detected. This method will help set the value of CSF Aβ42 in a CRM, which could be used to harmonize Aβ42 assays and facilitate the introduction of general cutoff concentrations for CSF Aβ42 in clinical trials and practice.

ACCURACY OF CSF BIOMARKERS FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE IN MCI PATIENTS: A SYSTEMATIC REVIEW AND META-ANALYSIS

Background: Blood biomarkers have the advantage of being readily available and less expensive and therefore offer the perfect method for enhancing utility of neuroimaging biomarkers. We are working towards developing a diagnostic blood test based on the changes in specific protein profiles that occur during the development of Alzheimer’s disease (AD). The specific aim is to differentiate between healthy controls (HC) and AD individuals, predict conversion to MCI (Mild cognitively impaired) and AD. Validation of these candidate proteins in the well-characterised longitudinal Australian Imaging Biomarkers and Lifestyle (AIBL) Study of Aging cohort will allow us to identify biomarkers which demonstrate sensitivity and specificity to AD for potential use in an early diagnostic clinical test. Methods: To test this hypothesis soluble AD brain extracts were serially diluted. In vivo seeding capacity was assessed by intracerebral inoculations of young APP23 transgenic mice. We then examined the seeding efficacy of human CSF from subjects with and without AD from 50 to 80 years of age. Results: Eight months after inoculation seeded Ab deposition was evident even when the hippocampus received subattomolar amounts of brain-derived Ab. In contrast, CSF from AD patients, which contained more than 10-fold higher levels of Ab than the most concentrated soluble brain extracts, yielded no apparent seeding activity. Similarly, CSF from aged APP transgenic donor mice failed to induce cerebral Ab deposition. When soluble, brain-derived seeds were mixed with CSF, the seeding potency of the brain seeds was maintained, indicating that the CSF per se does not inhibit seeding. Conclusions: These results suggest that highly potent, seeding-competent soluble Ab species are present in the AD brain but are absent or very rare in CSF. It is also possible that the seeding-competent Ab species adhere to structures in brain such as cellular membranes, which prevent their passive transport to the CSF. Finally, it cannot be excluded that the Ab seeds are rapidly cleared from the CSF or that the b-amyloid-inducing activity is dependent on a co-factor that only occurs in the brain.