Xaveer Van Ostade

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples.

BackgroundCervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C4(RP)-LC protein separation) followed by C18(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.ResultsIn total, we were able to identify 339 proteins in human CVF of which 151 proteins were not identified in any other proteomics study on human CVF so far. Those included antimicrobial peptides, such as human beta-defensin 2 and cathelicidin, which were known to be present in CVF, and endometrial proteins such as glycodelin and ribonucleoprotein A. Comparison of our results with previously published data led to the identification of a common protein set of 136 proteins. This overlapping protein set shows increased fractions of immunological and extracellular proteins, confirming the extracellular immunological role of CVF.ConclusionWe demonstrated here that CVF colposcopy samples can be used in proteomics experiments and hence are applicable for biomarker discovery experiments. The delineation of an overlapping set of proteins that is identified in most proteomics studies on CVF may help in the description of a reference proteome when performing proteomics studies on human CVF.

Use of cervicovaginal fluid for the identification of biomarkers for pathologies of the female genital tract.

Cervicovaginal fluid has an important function in the homeostasis and immunity of the lower female genital tract. Analysis of the cervicovaginal fluid proteome may therefore yield important information about the pathogenesis of numerous gynecological pathologies. Additionally, cervicovaginal fluid has great potential as a source of biomarkers for these conditions.This review provides a detailed discussion about the human cervicovaginal proteome and the proteomics studies performed to characterize this biological fluid. Furthermore, infection-correlated pathological conditions of the female genital tract are discussed for which cervicovaginal fluid has been used in order to identify potential biomarkers. Recent years, numerous studies have analyzed cervicovaginal fluid samples utilizing antibody-based technologies, such as ELISA or Western blotting, to identify biomarkers for preterm birth, premature preterm rupture of membranes, bacterial vaginosis and cervical cancer. The present article will discuss the importance of proteomic technologies as alternative techniques to gain additional meaningful information about these conditions. In addition, the review focuses on recent proteomic studies on cervicovaginal fluid samples for the identification of potential biomarkers. We conclude that the use of proteomic technology for analysis of human cervicovaginal fluid samples is promising and may lead to the discovery of new biomarkers which can improve disease prevention and therapy development.

The proteome of the human neuroblastoma cell line SH-SY5Y: an enlarged proteome

The human neuroblastoma cell line SH-SY5Y (ATCC: CRL-2266) is widely used as a neural cellular model system. The hitherto existing proteome data (115 proteins) are here extended. A total of 1103 unique proteins of this cell line were identified using 2D-LC combined with MALDI-TOF/TOF-MS, SDS-PAGE with nano-LC-MS/MS, N-terminal COFRADIC analysis with nano-LC-MS/MS and 2D-PAGE with MALDI-TOF/TOF-MS peptide mass fingerprinting. The obtained proteome profile of this cell line is discussed.

The expression of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (E-NPP1) is correlated with astrocytic tumor grade

OBJECTIVE Astrocytic brain tumors are subdivided in four grades. The most aggressive and invasive one is grade IV or glioblastoma multiforme (GBM). Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (E-NPP1), a membrane-bound enzyme, is involved in many cellular processes such as modulation of purinergic signalling, nucleotide recycling, regulation of extracellular pyrophosphate levels and stimulation of cell motility. In this study, the use of anti-NPP1 antibody in the determination of astrocytic tumor grade is evaluated. MATERIALS AND METHODS Paraffin-embedded surgical specimens from 41 primary human astrocytic brain tumors (grade I=2; grade II=10; grade III=9; grade IV=20) and 5 control samples are immunostained against NPP1 and glial fibrillary acid protein an astrocytic marker. RESULTS In this communication, we report the expression of NPP1 in human astrocytic brain tumors. No expression could be detected in control tissue. We observed a remarkable up regulated expression of NPP1 in GBM. Taking the latter as 100%, grade I has a relative NPP1 staining of 7%, whereas grade II and III have a similar NPP1 expression level of 53% and 47% respectively. CONCLUSION A correlation is found between the up-regulated expression of NPP1 and the grade of the astrocytic tumor. Further investigation of NPP1 expression, especially in GBM, is necessary to determine the role of NPP1 in astrocytic brain tumor progression.

Expression Analysis of LEDGF/p75, APOBEC3G, TRIM5alpha, and Tetherin in a Senegalese Cohort of HIV-1-Exposed Seronegative Individuals

Background HIV-1 replication depends on a delicate balance between cellular co-factors and antiviral restriction factors. Lens epithelium-derived growth factor (LEDGF/p75) benefits HIV, whereas apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and tetherin exert anti-HIV activity. Expression levels of these proteins possibly contribute to HIV-1 resistance in HIV-1-exposed populations. Methodology/Principal Findings We used real-time PCR and flow cytometry to study mRNA and protein levels respectively in PBMC and PBMC subsets. We observed significantly reduced LEDGF/p75 protein levels in CD4+ lymphocytes of HIV-1-exposed seronegative subjects relative to healthy controls, whereas we found no differences in APOBEC3G, TRIM5α, or tetherin expression. Untreated HIV-1-infected patients generally expressed higher mRNA and protein levels than healthy controls. Increased tetherin levels, in particular, correlated with markers of disease progression: directly with the viral load and T cell activation and inversely with the CD4 count. Conclusions/Significance Our data suggest that reduced LEDGF/p75 levels may play a role in resistance to HIV-1 infection, while increased tetherin levels could be a marker of advanced HIV disease. Host factors that influence HIV-1 infection and disease could be important targets for new antiviral therapies.

Identification of protein biomarkers for cervical cancer using human cervicovaginal fluid.

Objectives Cervicovaginal fluid (CVF) can be considered as a potential source of biomarkers for diseases of the lower female reproductive tract. The fluid can easily be collected, thereby offering new opportunities such as the development of self tests. Our objective was to identify a CVF protein biomarker for cervical cancer or its precancerous state. Methods A differential proteomics study was set up using CVF samples from healthy and precancerous women. Label-free spectral counting was applied to quantify protein abundances. Results The proteome analysis revealed 16 candidate biomarkers of which alpha-actinin-4 (p = 0.001) and pyruvate kinase isozyme M1/M2 (p = 0.014) were most promising. Verification of alpha-actinin-4 by ELISA (n = 28) showed that this candidate biomarker discriminated between samples from healthy and both low-risk and high-risk HPV-infected women (p = 0.009). Additional analysis of longitudinal samples (n = 29) showed that alpha-actinin-4 levels correlated with virus persistence and clearing, with a discrimination of approximately 18 pg/ml. Conclusions Our results show that CVF is an excellent source of protein biomarkers for detection of lower female genital tract pathologies and that alpha-actinin-4 derived from CVF is a promising candidate biomarker for the precancerous state of cervical cancer. Further studies regarding sensitivity and specificity of this biomarker will demonstrate its utility for improving current screening programs and/or its use for a cervical cancer self-diagnosis test.

Comparative proteomics of copper exposure and toxicity in rainbow trout, common carp and gibel carp.

Species specific differences in transporters, chaperones, metal binding proteins and other targets are important in metal toxicity. Therefore, we have studied the effects of copper exposure on the proteome of gill tissue from Oncorhynchus mykiss, Cyprinus carpio and Carassius auratus gibelio, which have different sensitivities toward copper. Fish were exposed to the Flemish water quality standard for surface waters, being 50μg/L, for 3 days. Sampled gill tissue was subjected to a 2D-Dige and an iTRAQ analysis. While gibel carp showed more positive responses such as increased apolipoprotein A-I, transferrin and heat shock protein 70, common carps gill tissue on the other hand displayed a changed actin cytoskeleton, and indications of a changed metabolism. These last two traits were evident in rainbow trout as well, together with decreased expressions of transferrin and albumin. urthermore, the Weighted Gene Co-Expression Network Analysis of rainbow trout data revealed a network of 98 proteins related to Cu accumulation in gill, of which the occurrence of proteins related to oxidative stress, such as superoxide dismutase and cytochrome c were promising. Additionally, the outcome of the different proteomics techniques demonstrates the usefulness of iTRAQ analysis compared to 2D-Dige and the need for fully annotated genomes.

Increased Serpin A5 levels in the cervicovaginal fluid of HIV-1 exposed seronegatives suggest that a subtle balance between serine proteases and their inhibitors may determine susceptibility to HIV-1 infection

HIV-exposed seronegative individuals (HESNs) are persons who remain seronegative despite repeated exposure to HIV, suggesting an in vivo resistance mechanism to HIV. Elucidation of endogenous factors responsible for this phenomenon may aid in the development of new classes of microbicides and therapeutics. We compared cervicovaginal protein abundance profiles between high-risk HESN and two control groups: low-risk HESN and HIV-positives. Four iTRAQ-based quantitative experiments were performed using samples classified based on presence/absence of particular gynaecological conditions. After statistical analysis, two proteins were shown to be differentially abundant between high-risk HESNs and control groups. Serpin A5, a serine proteinase inhibitor and Myeloblastin, a serine protease, were up- and downregulated, respectively. Commercially available ELISA assays were used to confirm differential Serpin A5 levels. These results suggest that HIV resistance in CVF of HESNs is the result of a delicate balance between two complementary mechanisms: downregulation of serine proteinases and upregulation of their inhibitors.

Intracellular detection of differential APOBEC3G, TRIM5alpha, and LEDGF/p75 protein expression in peripheral blood by flow cytometry

Expression studies on specific host proteins predominantly use quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based assay to study intracellular expression levels of three important host proteins involved in HIV-1 replication: apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and lens epithelium-derived growth factor (LEDGF/p75). An indirect intracellular staining (ICS) method was optimized using antibodies designed for other applications like enzyme-linked immunosorbent assay (ELISA), confocal imaging, and western blotting. The median fluorescence intensity (MFI) value--a measure for the protein expression level--increased upon higher antibody concentration and longer incubation time, and was reduced following preincubation with recombinant proteins. Staining of stably transfected or knock-down cell lines supported the methods specificity. Moreover, confocal microscopy analysis of peripheral blood mononuclear cells (PBMC), when stained according to the ICS method, confirmed the localization of APOBEC3G and TRIM5α in the cytoplasm, and of LEDGF/p75 in the nucleus. Also, stimulation with mitogen, interferon-alpha, or interferon-beta resulted in detectable, albeit weak, increases in intracellular expression of APOBEC3G and TRIM5α. After optimization, the method was applied to healthy control and HIV-1 infected subjects. For all subjects studied, the memory subset of CD4+ T cells showed significantly higher expression levels of APOBEC3G, TRIM5α, and LEDGF/p75, while the CD16+ subset of monocytes was characterized by higher expression levels of LEDGF/p75. In addition, we observed that therapy-naïve HIV-1 patients tended to have lower expression levels of APOBEC3G and TRIM5α than HIV-1 negative controls. In summary, our data provide proof-of-principle for the detection of specific host factors at the level of a single cell, which may prove useful for our further understanding of their role in virus-host interactions.

Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass Spectrometry.

Background The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. Methodology/Principal Findings To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. Conclusions This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.