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

<p>Abstract</p> <p>Background</p> <p>Cervical-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.</p> <p>Results</p> <p>In 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.</p> <p>Conclusion</p> <p>We 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.</p

Climate and colonialism

Recent years have seen a growth in scholarship on the intertwined histories of climate, science and European imperialism. Scholarship has focused both on how the material realities of climate shaped colonial enterprises, and on how ideas about climate informed imperial ideologies. Historians have shown how European expansion was justified by its protagonists with theories of racial superiority, which were often closely tied to ideas of climatic determinism. Meanwhile, the colonial spaces established by European powers offered novel ‘laboratories’ where ideas about acclimatisation and climatic improvement could be tested on the ground. While historical scholarship has focused on how powerful ideas of climate informed imperial projects, emerging scholarship in environmental history, history of science and historical geography focuses instead on the material and cognitive practices by which the climates of colonial spaces were made known and dealt with in fields such as forestry, agriculture and human health. These heretofore rather disparate areas of historical research carry great contemporary relevance of studies of how climates and their changes have been understood, debated and adapted to in the past

Identification of Protein Biomarkers for Cervical Cancer Using Human Cervicovaginal Fluid

<div><p>Objectives</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>The proteome analysis revealed 16 candidate biomarkers of which <i>alpha-actinin-4</i> (p = 0.001) and <i>pyruvate kinase isozyme M1/M2</i> (p = 0.014) were most promising. Verification of <i>alpha-actinin-4</i> 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 <i>alpha-actinin-4</i> levels correlated with virus persistence and clearing, with a discrimination of approximately 18 pg/ml.</p><p>Conclusions</p><p>Our results show that CVF is an excellent source of protein biomarkers for detection of lower female genital tract pathologies and that <i>alpha-actinin-4</i> 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.</p></div

Evolution of ACTN4 levels and HPV infection on longitudinal samples.

<p>Comparison of ACTN4 levels with genotype and viral load in longitudinal samples from 9 different patients. Similar genotypes are illustrated in the same colours. At the left side the viral load (in number of copies/cell) is shown on a logarithmical scale while at the right side the concentration of ACTN4 is expressed in pg/ml. The y-axe indicates the time (expressed in days) while the patients were followed. A black dotted line illustrates the ACTN4 threshold of 17.3 pg/ml. ACTN4 levels were determined on the time points when a CVF sample was available.</p

Patient information of samples used for the validation of ACTN4 by ELISA.

<p>HPV viral load was expressed as number of copies per cell. Colposcopy: Normal  =  no aberrant lesions detected, LSIL  =  low-grade squamous intra-epithelial lesions, HSIL  =  high-grade squamous intra-epithelial lesions, ASCUS  =  atypical squamous cells of undertimined significance. Cytology results were listed according to the Cervical Intraepithelial Neoplasia (CIN) nomenclature. The concentration of ACTN4 was described in pg/ml.</p><p>Patient information of samples used for the validation of ACTN4 by ELISA.</p

Proteins with a statistical significant difference in frequency between the healthy and precancerous group.

<p>Acc No represents the accession number of the identified proteins. Statistical significance is listed in column named p-value. Columns “# Healthy” and “# Precanc” show the number of samples that contain the corresponding protein.</p><p>Proteins with a statistical significant difference in frequency between the healthy and precancerous group.</p

ACTN4 levels between different study groups.

<p>Concentration of ACTN4 for the healthy individuals (n = 16), the HR-HPV infected patients (n = 8) and the LR-HPV infected patients (n = 4). Statistical testing of ACNT-4 levels between healthy and HR-HPV infected patients resulted in a p-value of 0.023 and between healthy versus LR-HPV in a p-value of 0.052. When the HR- and LR-HPV infected patients were grouped as HPV infected patients, a p-value of 0.009 was found between non-infected (healthy patients) and infected groups.</p

Proteins with a statistical significant difference in abundance between the healthy and precancerous group.

<p>Columns “Mean Healthy” and “Mean Precanc.” show the mean normalized spectral abundance factors (NSAF-value) while (x) indicates the number of samples that contain this ID. Ratio is expressed as precancerous/healthy. “Acc No” represents the accession number of the identified proteins and “p-value” shows the statistical significance.</p><p>Proteins with a statistical significant difference in abundance between the healthy and precancerous group.</p