Seminal plasma as a source of prostate cancer peptide biomarker candidates for detection of indolent and advanced disease

Background:Extensive prostate specific antigen screening for prostate cancer generates a high number of unnecessary biopsies and over-treatment due to insufficient differentiation between indolent and aggressive tumours. We hypothesized that seminal plasma is a robust source of novel prostate cancer (PCa) biomarkers with the potential to improve primary diagnosis of and to distinguish advanced from indolent disease. <br>Methodology/Principal Findings: In an open-label case/control study 125 patients (70 PCa, 21 benign prostate hyperplasia, 25 chronic prostatitis, 9 healthy controls) were enrolled in 3 centres. Biomarker panels a) for PCa diagnosis (comparison of PCa patients versus benign controls) and b) for advanced disease (comparison of patients with post surgery Gleason score <7 versus Gleason score >>7) were sought. Independent cohorts were used for proteomic biomarker discovery and testing the performance of the identified biomarker profiles. Seminal plasma was profiled using capillary electrophoresis mass spectrometry. Pre-analytical stability and analytical precision of the proteome analysis were determined. Support vector machine learning was used for classification. Stepwise application of two biomarker signatures with 21 and 5 biomarkers provided 83% sensitivity and 67% specificity for PCa detection in a test set of samples. A panel of 11 biomarkers for advanced disease discriminated between patients with Gleason score 7 and organ-confined (<pT3a) or advanced (≥pT3a) disease with 80% sensitivity and 82% specificity in a preliminary validation setting. Seminal profiles showed excellent pre-analytical stability. Eight biomarkers were identified as fragments of N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase​,prostatic acid phosphatase, stabilin-2, GTPase IMAP family member 6, semenogelin-1 and -2. Restricted sample size was the major limitation of the study.</br> <br>Conclusions/Significance: Seminal plasma represents a robust source of potential peptide makers for primary PCa diagnosis. Our findings warrant further prospective validation to confirm the diagnostic potential of identified seminal biomarker candidates.</br&gt

Multicentric validation of proteomic biomarkers in urine specific for diabetic nephropathy

Background: Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration >= 5 years, cases of DN were defined as albuminuria >300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82). Methodology/Principal Findings: Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In <10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patient's subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients. Conclusions: These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin

Proteomics as a quality control tool of pharmaceutical probiotic bacterial lysate products

Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots

Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of Chlamydophila pneumoniae IgA antibodies

<p>Abstract</p> <p>Background</p> <p>Serologic diagnosis of <it>Chlamydophila pneumoniae </it>(Cpn) infection routinely involves assays for the presence of IgG and IgM antibodies to Cpn. Although IgA antibodies to Cpn have been found to be of interest in the diagnosis of chronic infections, their significance in serological diagnosis remains unclear. The microimmunofluorescence (MIF) test is the current method for the measurement of Cpn antibodies. While commercial enzyme linked immunosorbent assays (ELISA) have been developed, they have not been fully validated. We therefore evaluated and optimized a commercial ELISA kit, the SeroCP IgA test, for the detection of Cpn IgA antibodies.</p> <p>Methods</p> <p>Serum samples from 94 patients with anti-Cpn IgG titers ≥ 256 (study group) and from 100 healthy blood donors (control group) were tested for the presence of IgA antibodies to Cpn, using our in-house MIF test and the SeroCP IgA test. Two graph receiver operating characteristic (TG-ROC) curves were created to optimize the cut off given by the manufacturer.</p> <p>Results</p> <p>The MIF and SeroCP IgA tests detected Cpn IgA antibodies in 72% and 89%, respectively, of sera from the study group, and in 9% and 35%, respectively, of sera from the control group. Using the MIF test as the reference method and the cut-off value of the ELISA test specified by the manufacturer for seropositivity and negativity, the two tests correlated in 76% of the samples, with an agreement of Ƙ = 0.54. When we applied the optimized cut-off value using TG-ROC analysis, 1.65, we observed better concordance (86%) and agreement (0.72) between the MIF and SeroCP IgA tests.</p> <p>Conclusion</p> <p>Use of TG-ROC analysis may help standardize and optimize ELISAs, which are simpler, more objective and less time consuming than the MIF test. Standardization and optimization of commercial ELISA kits may result in better performance.</p

Non-detection of Chlamydia species in carotid atheroma using generic primers by nested PCR in a population with a high prevalence of Chlamydia pneumoniae antibody

BACKGROUND: The association of Chlamydia pneumoniae with atherosclerosis is controversial. We investigated the presence of C. pneumoniae and other Chlamydia spp. in atheromatous carotid artery tissue. METHODS: Forty elective carotid endarterectomy patients were recruited (27 males, mean age 65 and 13 females mean age 68), 4 had bilateral carotid endarterectomies (n= 44 endarterectomy specimens). Control specimens were taken from macroscopically normal carotid artery adjacent to the atheromatous lesions (internal controls), except in 8 cases where normal carotid arteries from post mortem (external controls) were used. Three case-control pairs were excluded when the HLA DRB gene failed to amplify from the DNA. Genus specific primers to the major outer membrane protein (MOMP) gene were used in a nested polymerase chain reaction (nPCR) in 41 atheromatous carotid specimens and paired controls. PCR inhibition was monitored by spiking with target C. trachomatis. Atheroma severity was graded histologically. Plasma samples were tested by microimmunofluorescence (MIF) for antibodies to C. pneumoniae, C. trachomatis and C. psittaci and the corresponding white cells were tested for Chlamydia spp. by nPCR. RESULTS: C. pneumoniae was not detected in any carotid specimen. Twenty-five of 38 (66%) plasma specimens were positive for C. pneumoniae IgG, 2/38 (5%) for C. trachomatis IgG and 1/38 (3%) for C. psittaci IgG. CONCLUSIONS: We were unable to show an association between the presence of Chlamydia spp. and atheroma in carotid arteries in the presence of a high seroprevalence of C. pneumoniae antibodies in Northern Ireland

Use of a nested PCR-enzyme immunoassay with an internal control to detect Chlamydophila psittaci in turkeys

BACKGROUND: Laboratory diagnosis of Chlamydophila psittaci, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay (PCR-EIA) was developed to detect the Cp. psittaci outer membrane protein A (ompA) gene in pharyngeal swabs. METHODS: The fluorescein-biotin labelled PCR products were immobilized on streptavidin-coated microtiter plates and detected with anti-fluorescein peroxidase conjugate and a colorimetric substrate. An internal inhibition control was included to rule out the presence of inhibitors of DNA amplification. The diagnostic value of the ompA nested PCR-EIA in comparison to cell culture and a 16S-rRNA based nested PCR was assessed in pharyngeal turkey swabs from 10 different farms experiencing respiratory disease. RESULTS: The sensitivity of the nested PCR-EIA was established at 0.1 infection forming units (IFU). Specificity was 100%. The ompA nested PCR-EIA was more sensitive than the 16S-rRNA based nested PCR and isolation, revealing 105 out of 200 (52.5%) positives against 13 and 74 for the latter two tests, respectively. Twenty-nine (23.8%) out of 122 ompA PCR-EIA negatives showed the presence of inhibitors of DNA amplification, although 27 of them became positive after diluting (1/10) the specimens in PCR buffer or after phenol-chloroform extraction and subsequent ethanol precipitation. CONCLUSION: The present study stresses the need for an internal control to confirm PCR true-negatives and demonstrates the high prevalence of chlamydiosis in Belgian turkeys and its potential zoonotic risk. The ompA nested PCR-EIA described here is a rapid, highly sensitive and specific diagnostic assay and will help to facilitate the diagnosis of Cp. psittaci infections in both poultry and man

Proteomic Candidate Biomarkers of Drug-Induced Nephrotoxicity in the Rat

Improved biomarkers of acute nephrotoxicity are coveted by the drug development industry, regulatory agencies, and clinicians. In an effort to identify such biomarkers, urinary peptide profiles of rats treated with two different nephrotoxins were investigated. 493 marker candidates were defined that showed a significant response to cis-platin comparing a cis-platin treated cohort to controls. Next, urine samples from rats that received three consecutive daily doses of 150 or 300 mg/kg gentamicin were examined. 557 potential biomarkers were initially identified; 108 of these gentamicin-response markers showed a clear temporal response to treatment. 39 of the cisplatin-response markers also displayed a clear response to gentamicin. Of the combined 147 peptides, 101 were similarly regulated by gentamicin or cis-platin and 54 could be identified by tandem mass spectrometry. Most were collagen type I and type III fragments up-regulated in response to gentamicin treatment. Based on these peptides, classification models were generated and validated in a longitudinal study. In agreement with histopathology, the observed changes in classification scores were transient, initiated after the first dose, and generally persistent over a period of 10–20 days before returning to control levels. The data support the hypothesis that gentamicin-induced renal toxicity up-regulates protease activity, resulting in an increase in several specific urinary collagen fragments. Urinary proteomic biomarkers identified here, especially those common to both nephrotoxins, may serve as a valuable tool to investigate potential new drug candidates for the risk of nephrotoxicity

A Distinct Urinary Biomarker Pattern Characteristic of Female Fabry Patients That Mirrors Response to Enzyme Replacement Therapy

Female patients affected by Fabry disease, an X-linked lysosomal storage disorder, exhibit a wide spectrum of symptoms, which renders diagnosis, and treatment decisions challenging. No diagnostic test, other than sequencing of the alpha-galactosidase A gene, is available and no biomarker has been proven useful to screen for the disease, predict disease course and monitor response to enzyme replacement therapy. Here, we used urine proteomic analysis based on capillary electrophoresis coupled to mass spectrometry and identified a biomarker profile in adult female Fabry patients. Urine samples were taken from 35 treatment-naive female Fabry patients and were compared to 89 age-matched healthy controls. We found a diagnostic biomarker pattern that exhibited 88.2% sensitivity and 97.8% specificity when tested in an independent validation cohort consisting of 17 treatment-naive Fabry patients and 45 controls. The model remained highly specific when applied to additional control patients with a variety of other renal, metabolic and cardiovascular diseases. Several of the 64 identified diagnostic biomarkers showed correlations with measures of disease severity. Notably, most biomarkers responded to enzyme replacement therapy, and 8 of 11 treated patients scored negative for Fabry disease in the diagnostic model. In conclusion, we defined a urinary biomarker model that seems to be of diagnostic use for Fabry disease in female patients and may be used to monitor response to enzyme replacement therapy

Functional Analysis of the Cytoskeleton Protein MreB from Chlamydophila pneumoniae

In rod-shaped bacteria, the bacterial actin ortholog MreB is considered to organize the incorporation of cell wall precursors into the side-wall, whereas the tubulin homologue FtsZ is known to tether incorporation of cell wall building blocks at the developing septum. For intracellular bacteria, there is no need to compensate osmotic pressure by means of a cell wall, and peptidoglycan has not been reliably detected in Chlamydiaceae. Surprisingly, a nearly complete pathway for the biosynthesis of the cell wall building block lipid II has been found in the genomes of Chlamydiaceae. In a previous study, we discussed the hypothesis that conservation of lipid II biosynthesis in cell wall-lacking bacteria may reflect the intimate molecular linkage of cell wall biosynthesis and cell division and thus an essential role of the precursor in cell division. Here, we investigate why spherical-shaped chlamydiae harbor MreB which is almost exclusively found in elongated bacteria (i.e. rods, vibrios, spirilla) whereas they lack the otherwise essential division protein FtsZ. We demonstrate that chlamydial MreB polymerizes in vitro and that polymerization is not inhibited by the blocking agent A22. As observed for MreB from Bacillus subtilis, chlamydial MreB does not require ATP for polymerization but is capable of ATP hydrolysis in phosphate release assays. Co-pelleting and bacterial two-hybrid experiments indicate that MreB from Chlamydophila (Chlamydia) pneumoniae interacts with MurF, MraY and MurG, three key components in lipid II biosynthesis. In addition, MreB polymerization is improved in the presence of MurF. Our findings suggest that MreB is involved in tethering biosynthesis of lipid II and as such may be necessary for maintaining a functional divisome machinery in Chlamydiaceae