Petra Gilmore

Identification and validation of novel cerebrospinal fluid biomarkers for staging early Alzheimer's disease.

Background Ideally, disease modifying therapies for Alzheimer disease (AD) will be applied during the ‘preclinical’ stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome. Methods and Findings CSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aβ42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85–0.94 95% confidence interval [CI]) and 0.88 (0.81–0.94 CI), respectively. Conclusions Four novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aβ42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions.

Quantitative Label-Free Proteomics for Discovery of Biomarkers in Cerebrospinal Fluid: Assessment of Technical and Inter-Individual Variation

Background Biomarkers are required for pre-symptomatic diagnosis, treatment, and monitoring of neurodegenerative diseases such as Alzheimers disease. Cerebrospinal fluid (CSF) is a favored source because its proteome reflects the composition of the brain. Ideal biomarkers have low technical and inter-individual variability (subject variance) among control subjects to minimize overlaps between clinical groups. This study evaluates a process of multi-affinity fractionation (MAF) and quantitative label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) for CSF biomarker discovery by (1) identifying reparable sources of technical variability, (2) assessing subject variance and residual technical variability for numerous CSF proteins, and (3) testing its ability to segregate samples on the basis of desired biomarker characteristics. Methods/Results Fourteen aliquots of pooled CSF and two aliquots from six cognitively normal individuals were randomized, enriched for low-abundance proteins by MAF, digested endoproteolytically, randomized again, and analyzed by nano-LC-MS. Nano-LC-MS data were time and m/z aligned across samples for relative peptide quantification. Among 11,433 aligned charge groups, 1360 relatively abundant ones were annotated by MS2, yielding 823 unique peptides. Analyses, including Pearson correlations of annotated LC-MS ion chromatograms, performed for all pairwise sample comparisons, identified several sources of technical variability: i) incomplete MAF and keratins; ii) globally- or segmentally-decreased ion current in isolated LC-MS analyses; and iii) oxidized methionine-containing peptides. Exclusion of these sources yielded 609 peptides representing 81 proteins. Most of these proteins showed very low coefficients of variation (CV<5%) whether they were quantified from the mean of all or only the 2 most-abundant peptides. Unsupervised clustering, using only 24 proteins selected for high subject variance, yielded perfect segregation of pooled and individual samples. Conclusions Quantitative label-free LC-MS/MS can measure scores of CSF proteins with low technical variability and can segregate samples according to desired criteria. Thus, this technique shows potential for biomarker discovery for neurological diseases.

Serum markers may distinguish biliary atresia from other forms of neonatal cholestasis

Background: Biliary atresia (BA) is the most serious liver disease in infants. Diagnosis currently depends on surgical exploration of the biliary tree. Noninvasive tests that distinguish BA from other types of neonatal liver disease are not available. Patients and Methods: To identify potential serum biomarkers that classify children with neonatal cholestasis, we performed 2-dimensional difference gel electrophoresis, statistical analysis, and tandem mass spectrometry using serum samples from 19 infants with BA and 19 infants with non-BA neonatal cholestasis. Results: Eleven potential serum biomarkers were found that could in combination classify children with neonatal cholestasis. Conclusions: Although no single biomarker or imaging test adequately distinguishes BA from other types of neonatal cholestasis, combinations of biomarkers, imaging tests, and noninvasive clinical criteria should be further explored as potential tests for rapid and accurate diagnosis of BA.

A unified sample preparation protocol for proteomic and genomic profiling of cervical swabs to identify biomarkers for cervical cancer screening

Cervical cancer screening is ideally suited for the development of biomarkers due to the ease of tissue acquisition and the well‐established histological transitions. Furthermore, cell and biologic fluid obtained from cervix samples undergo specific molecular changes that can be profiled. However, the ideal manner and techniques for preparing cervical samples remains to be determined. To address this critical issue a patient screening protein and nucleic acid collection protocol was established. RNAlater was used to collect the samples followed by proteomic methods to identify proteins that were differentially expressed in normal cervical epithelial versus cervical cancer cells. Three hundred ninety spots were identified via 2‐D DIGE that were expressed at either higher or lower levels (>three‐fold) in cervical cancer samples. These proteomic results were compared to genes in a cDNA microarray analysis of microdissected neoplastic cervical specimens to identify overlapping patterns of expression. The most frequent pathways represented by the combined dataset were: cell cycle: G2/M DNA damage checkpoint regulation; aryl hydrocarbon receptor signaling; p53 signaling; cell cycle: G1/S checkpoint regulation; and the ER stress pathway. HNRPA2B1 was identified as a biomarker candidate with increased expression in cancer compared to normal cervix and validated by Western blot.

Human Experimental Challenge With Enterotoxigenic Escherichia coli Elicits Immune Responses to Canonical and Novel Antigens Relevant to Vaccine Development

Background Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in the developing world. Enterotoxigenic E coli vaccinology has been challenged by genetic diversity and heterogeneity of canonical antigens. Examination of the antigenic breadth of immune responses associated with protective immunity could afford new avenues for vaccine development. Methods Antibody lymphocyte supernatants (ALS) and sera from 20 naive human volunteers challenged with ETEC strain H10407 and from 10 volunteers rechallenged 4-6 weeks later with the same strain (9 of whom were completely protected on rechallenge) were tested against ETEC proteome microarrays containing 957 antigens. Results Enterotoxigenic E coli challenge stimulated robust serum and mucosal (ALS) responses to canonical vaccine antigens (CFA/I, and the B subunit of LT) as well as a small number of antigens not presently targeted in ETEC vaccines. These included pathovar-specific secreted proteins (EtpA, EatA) as well as highly conserved E coli antigens including YghJ, flagellin, and pertactin-like autotransporter proteins, all of which have previously afforded protection against ETEC infection in preclinical studies. Conclusions Taken together, studies reported here suggest that immune responses after ETEC infection involve traditional vaccine targets as well as a select number of more recently identified protein antigens that could offer additional avenues for vaccine development for these pathogens.

Identification and validation of novel cerebrospinal fluid protein biomarkers for early Alzheimer's disease using proteomics and ELISA: a multi-institutional collaborative study

Disease Research Center, University of Washington Medical Center, Seattle, WA, USA; Choju Medical Institute, Fukushimura Hospital, Toyohashi, Japan; Immuno-Biological Laboratories Co., Ltd. (IBL), Fujioka, Japan; Department of Neurology, Emory Alzheimer’s Disease Research Center, Emory University, Atlanta, GA, USA; Department of Neurology, Oregon Health & Science University, Portland, OR, USA; Departments of Neurology and Neuropathology, University of Washington, Seattle, WA, USA; Department of Psychiatry, Alzheimer’s Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA; Departments of Neurology and Psychiatry, Mount Sinai School of Medicine, Alzheimer’s Disease Research Center, New York, NY, USA. Contact e-mail: sahata07@ hotmail.com