Edmond Coen

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.

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.

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.

Presynaptic serotonin receptors regulate the release of 3H-serotonin in hypothalamic slices of the rabbit.

SummaryHypothalamic slices of the rabbit brain were incubated with 10−7 M of 3H-serotonin (3H-5HT). After the incubation and an initial washout period, a nearly constant basal efflux of tritium was detected. This basal efflux was not significantly altered by Ca2+-free solution or by the 5HT-antagonist metitepin (10−5 M), but was augmented by chlorimipramine (10−5 M) and by unlabelled 5HT (10−6 M); the acceleration caused by unlabelled 5HT was absent in presence of chlorimipramine (10−5 M). Both electrical stimulation (4 Hz, 50 mA, 2 min) and high K+ (50 mM) induced an overflow of 3H. This overflow was nearly abolished in Ca2+-free solution. In presence of chlorimipramine (10−5 M) both the tritium overflow evoked by electrical stimulation and that evoked by high K+ were augmented by metitepin (10−5 M) and decreased in a concentration dependent manner by unlabelled serotonin (10−8–10−6 M); the latter effect was antagonized by metitepin (10−6 M and 10−5 M). These experiments suggest that in rabbit hypothalamic slices, the release of 3H-5HT is controlled by a negative feedback mechanism acting via presynaptic serotonin receptors.

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.

Phenotype plasticity and immunocytochemical evidence for ChAT and DβH co-localization in fetal pig superior cervical ganglion cells

The early expression of the cholinergic phenotype in sympathetic neurons was already studied in superior cervical ganglion cells derived from rat, quail and chicken embryo. In the present work, we set up a neuron culture derived from the superior cervical ganglia of fetal pigs. The yield is 1000 times of that of a neonatal rat [17], 100 times of a 10- to 13-day-old chick embryo [26] and 20 times of a 10-day-old quail embryo [3]. This high yield will greatly facilitate further biochemical studies concerning neuronal differentiation. Using these cells as a model, the phenotype plasticity was studied by both biochemical and immunocytochemical methods in normal physiological medium, in a high KCl (30 mM) medium and in a splenocyte co-culture. The phenotype shift occurs in the normal physiological medium and in the splenocyte co-culture, but not in the high KCl medium. Taking into account the species difference, the fetal pig superior cervical ganglion neurons behave in a comparable manner as reported in earlier studies for other animal models. Moreover, for the first time, using immunocytochemical methods, direct evidence for a co-localization of choline-acetyl-transferase and dopamine-beta-hydroxylase in mammalian fetal sympathetic neurons, at least during a certain period, is given.

Relationship between external calcium concentration and noradrenaline- and neuropeptide Y-evoked release from perfused dog spleen

Several neuropeptides have been demonstrated to coexist with classical neurotransmitters in the central and peripheral nervous systems and have been proposed as neurotransmitter or neuromodulator candidates. In this report, we investigated the relationship between the external calcium concentration and the electrically induced overflow of noradrenaline (NA) and neuropeptide Y (NPY) in dog perfused spleen. Perfusion solutions with calcium concentrations ranging from 0 to 10 mM were applied. The splenic nerves were electrically stimulated at 16 Hz. For NA analysis high-pressure liquid chromatography with electrochemical detection was used. NPY was determined by radioimmunoassay. The perfusion pressure and the overflow of NA and NPY increased in a calcium concentration dependent manner. The calcium concentration dependency of the overflow of NA and NPY was comparable, indicating a co-release of the two substances. The molar ratio of NA/NPY remained unchanged over the calcium concentration range applied and the half maximal saturation values for release of NA (5.55 mM) and NPY (6.66 mM) were similar. These results indicate that the preferential release of NPY at high frequency stimulation as previously shown in the pig spleen, if present in the dog, is not the result of a difference in calcium dependency of the evoked release of NA and NPY.

Presence and release of SR-17 (chromogranin B586-602) in the porcine splenic nerve and its enzymatic degradation by CD26/dipeptidyl peptidase IV

Using the pig splenic nerve as a model, we investigated the proteolytic processing of porcine chromogranin B (CgB) during its axonal transport. An ELISA was developed for SR-17 (CgB(586-602)), a novel CgB-derived peptide, originally found in the adrenal medulla. The results demonstrate that CgB is processed in an early stage during its axonal transport. Immunohistochemical data, based on a rabbit anti-SR-17 antiserum, show that the spleen CgB/SR-17 is exclusively present in the nerve endings. No SR-17 immunoreactivity (IR) was found in splenocytes. We also provide evidence that SR-17 is co-released with noradrenaline (NA) upon electrical stimulation of the splenic nerve. Its release is frequency-dependent and strongly enhanced in the presence of the alpha-blocking agent phentolamine. In addition, we show that the new CgB-peptide can serve as a substrate for the lymphocyte surface glycoprotein CD26, also known as dipeptidyl peptidase IV (DPP IV), generating a new peptide ER-15 (CgB(588-602)).

The processing of secretogranin II in the peripheral nervous system: release of secretoneurin from porcine sympathetic nerve terminals.

The distribution of secretoneurin (SN), a peptide derived from secretogranin II (SgII), in the coeliac ganglion, the splenic nerve and the spleen was examined by immunohistochemistry. In the ganglion, SN immunoreactivity (IR) was unevenly distributed. Positive nerve terminals densely surrounded some postganglionic perikarya in which also intense SN-IR was present. In the crushed splenic nerves, intense immunoreactivities appeared proximal (but to a less extent also distal) to the crush of the nerve. Analysis by cytofluorimetric scanning (CFS) demonstrated that SN-IR and neuropeptide Y immunoreactivity (NPY-IR) were predominant in the axons proximal to the crush representing anterogradely transported components. Using radioimmunoassay (RIA) we demonstrated that upon electrical stimulation (10 Hz, 1 min) of the splenic nerve, significant amounts of SN-IR (64.2+/-2.3 fmol) were released together with NA (4. 1x106+/-0.2 fmol) and NPY (330.0+/-7.2 fmol) from the isolated perfused porcine spleen. To evaluate the processing of SgII in sympathetic neurons, boiled tissue extracts (coeliac ganglia and splenic nerve) and boiled spleen perfusate (used as a suitable source for vesicle derived peptides) were analysed by gel filtration chromatography followed by SN-RIA. In all cases immunoreactivity was present solely as SN, indicating that SgII was fully processed to the free peptide. The evidence that SN is transported to the nerve terminals and is released from the porcine spleen upon nerve stimulation, suggests that it may modulate adrenergic neurotransmission and may also play a role in the neuroimmune communication.

Inhibition of evoked neurotransmitter release from rat hippocampus by a polypeptide toxin isolated from the marine snail Conus distans

The active fraction, isolated and partially purified from the crude venom of the marine snail Conus distans, with a molecular mass of about 25 kDa, inhibits neurotransmitter release in rat hippocampus. This toxin (distans Toxin) inhibits the electrically evoked tritium labelled noradrenaline release from rat hippocampal slices in a dose and time dependent manner. The neurotransmitter release is mainly regulated by N-type of voltage sensitive Ca(2+)-channels. The distans toxin behaves as a partial antagonist of calcium in the buffer, possibly by competing with calcium for this type of voltage sensitive Ca(2+)-channels.