Immune Activation in the Female Genital Tract: Expression Profiles of Soluble Proteins in Women at High Risk for HIV Infection.

Soluble cervicovaginal biomarkers of inflammation, immune activation and risk of HIV acquisition are needed to reliably assess the safety of new biomedical prevention strategies including vaccines and microbicides. However, a fuller understanding of expression profiles in women at high risk for HIV infection is crucial to the effective use of these potential biomarkers in Phase 3 trial settings. We have measured 45 soluble proteins and peptides in cervicovaginal lavage samples from 100 HIV negative women at high risk for HIV infection. Women were followed over one menstrual cycle to investigate modulation by hormonal contraception, menstrual cycle phase, recent sexual exposure and intravaginal practices. Women using injectable DMPA had increased concentration of several soluble proteins of the innate and adaptive immune system, including IL-1α, IL-1ß, IL-2, MIP-1ß, IP-10, IL-8, TGF-ß, HBD4, IgA, IgG1, and IgG2. Women using combined oral contraceptives had a similar signature. There were differences in concentrations among samples from post-ovulation compared to pre-ovulation, notably increased immunoglobulins. Increased prostate-specific antigen, indicative of recent sexual exposure, was correlated with increased IL-6, MCP-1, and SLPI, and decreased GM-CSF and HBD3. The identified signature profiles may prove critical in evaluating the potential safety and impact on risk of HIV acquisition of different biomedical intervention strategies.

Annexin A3 is a mammary marker and a potential neoplastic breast cell therapeutic target.

Breast cancers are the most common cancer-affecting women; critically the identification of novel biomarkers for improving early detection, stratification and differentiation from benign tumours is important for the reduction of morbidity and mortality.To identify and functionally characterise potential biomarkers, we used mass spectrometry (MS) to analyse serum samples representing control, benign breast disease (BBD) and invasive breast cancer (IDC) patients. Complementary and multidimensional proteomic approaches were used to identify and validate novel serum markers.Annexin A3 (ANX A3) was found to be differentially expressed amongst different breast pathologies. The diagnostic value of serum ANX A3 was subsequently validated by ELISA in an independent serum set representing the three groups. Here, ANX A3 was significantly upregulated in the benign disease group sera compared with other groups (P < 0.0005).In addition, paired breast tissue immunostaining confirmed that ANX A3 was abundantly expressed in benign and to a lesser extent malignant neoplastic epithelium. Finally, we illustrated ANX A3 expression in cell culture lysates and conditioned media from neoplastic breast cell lines, and its role in neoplastic breast cell migration in vitro.This study confirms the novel role of ANX A3 as a mammary biomarker, regulator and therapeutic target.

Intraocular biomarker identification in uveitis associated with juvenile idiopathic arthritis.

PURPOSE: To investigate the presence of biomarkers in aqueous humor (AH) from patients with uveitis associated with juvenile idiopathic arthritis (JIA). METHODS: AH (N = 73) AND SERUM (N = 105) SAMPLES FROM 116 CHILDREN WERE ANALYZED USING SURFACE ENHANCED LASER DESORPTION/IONIZATION TIME OF FLIGHT MASS SPECTROMETRY (SELDI-TOF MS). THE SAMPLES WERE DIVIDED INTO THE FOLLOWING GROUPS: JIA, silent chronic anterior uveitis (AU), other uveitis entities, and noninflammatory controls. Statistical biomarker identification was performed using the SELDI-ToF Biomarker Analysis Cluster Wizard followed by multivariate statistical analysis. Biochemical identification of biomarkers was performed by polyacrylamide gel protein separation, followed by liquid chromatography tandem mass spectrometry. ELISA was performed in a number of AH samples representing all four study groups. RESULTS: In the JIA group, one AH protein peak at mass/charge (m/z) 13,762 had qualitative and quantitative differences in expression compared with the other uveitis entities and the controls, but not to the group of silent chronic AU. Its quantitative expression in AH of patients with JIA and other silent chronic AU was positively associated with uveitis activity. The protein at m/z 13,762 in AH was identified as transthyretin (TTR). The TTR concentration in AH differed significantly between the study groups (P = 0.006) with considerably higher TTR concentrations in JIA and silent chronic AU samples positive for m/z 13,762 than those of the other uveitis and control groups. CONCLUSIONS: TTR is a potential intraocular biomarker of JIA- associated uveitis. Its role in the pathogenesis of silent chronic AU with and without arthritis needs further investigation.

Evaluation of mass spectrometry of urinary proteins and peptides as biomarkers for cats at risk of developing azotemia.

OBJECTIVE: To evaluate proteomic delineation of feline urine by mass spectrometry as a method for identifying biomarkers in cats at risk of developing azotemia. SAMPLES: Urine samples from geriatric cats (> 9 years old) with chronic kidney disease and nonazotemic cats that either remained nonazotemic (n = 10) or developed azotemia (10) within 1 year. PROCEDURES: Optimization studies with pooled urine were performed to facilitate the use of surface enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF-MS) for analysis of the urinary proteome of cats. Urine samples from nonazotemic cats at entry to the study were analyzed via SELDI-TOF-MS with weak cation exchange and strong anion exchange arrays. Spectral data were compared to identify biomarkers for development of azotemia. RESULTS: Low protein concentration in feline urine precluded direct application to array surfaces, and a buffer exchange and concentration step was required prior to SELDI-TOF-MS analysis. Three preparation conditions by use of weak cation and strong anion exchange arrays were selected on the basis of optimization studies for detection of biomarkers. Eight potential biomarkers with an m/z of 2,822, 9,886, 10,033, 10,151, 10,234, 11,653, 4,421, and 9,505 were delineated. CONCLUSIONS AND CLINICAL RELEVANCE: SELDI-TOF-MS can be used to detect urinary low-molecular weight peptides and proteins that may represent biomarkers for early detection of renal damage. Further study is required to purify and identify potential biomarkers before their use in a clinical setting.

In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts.

Efficient muscle regeneration requires cross talk between multiple cell types via secreted signaling molecules. However, as yet there has been no comprehensive analysis of this secreted signaling network in order to understand how it regulates myogenesis in humans. Using integrated proteomic and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling proteins, including 43 previously implicated in myogenesis, while others are known to modulate various cell types thus implying a much broader role for myoblasts in muscle remodeling. We also isolated and characterized two types of secreted membrane-derived vesicles: nanovesicles harboring typical exosomal features and larger, morphologically distinct, microvesicles. While they share some common features, their distinct protein and RNA cargos suggest independent functions in myogenesis. We further demonstrate that both types of microvesicles can dock and fuse with adjacent muscle cells but also deliver functional protein cargo. Thus, the intercellular signaling networks invoked during muscle differentiation and regeneration may employ conventional soluble signaling molecules acting in concert with muscle derived microvesicles delivering their cargos directly into target cells.

Proteomic analysis of archival breast cancer serum.

Large cohorts of archival samples are stored in tissue banks worldwide yet their contribution to biomarker discovery is limited. Proteomic profiling technologies have potential for early screening and diagnosis of cancer, and data from such samples can be the answer for many clinical questions. Here we introduce the notion of archival samples proteomics. Using SELDI-TOF MS analysis, we compared 30-year-old archival serum samples of healthy volunteers and patients diagnosed with non metastatic breast cancer. To validate the reproducibility of our results, analysis of the same samples was repeated in a different centre under standardised settings. Plausible differentially expressed protein peaks between the breast cancer and control groups were repeatedly detected. Our pilot study showed highly reproducible and concordant results between two independent analyses conducted in different centres. The feasibility and reliability of profiling serum archives of women with breast cancer was tested in this pilot study. Our results imply that proteomic profiling of serum may have an important role in biomarkers discovery regardless of the storage period. Clearly, multicentre validation of larger archival cohorts is vital.

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS.

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.

Proteomic changes in hearts of kyphoscoliosis (ky) mutant mice in the absence of structural pathology: implication for the analysis of early human heart disease.

Complex molecular changes associated with early stage human heart disease are poorly understood and prevent the development of effective treatments of human cardiac disease. Relatively minor structural changes in early disease may accompany some conditions such as arrhythmias. Our objective was to determine if significant proteomic changes occur in heart tissues in the absence of structural pathology. We used a proteomic "pipeline" based on Ciphergen SELDI-TOF/MS, gel electrophoresis and MALDI-TOF/MS. The kyphoscoliosis (ky) mouse carries a mutation in a putative transglutaminase causing a primary skeletal muscle disease. The ky protein is expressed usually in skeletal and cardiac muscle but its absence from the ky heart causes no structural pathology making it a good model of "occult" heart disease. We discovered 20 statistically validated biomarkers discriminating ky from normal hearts, one cardiac troponin-I was reduced by 40% in ky hearts. A 17% deficit was confirmed subsequently by Western blot. Thus, the proteome of ky hearts was abnormal, giving support to our contention that this SELDI-based analytical approach is capable of making a significant contribution to the analysis of complex proteomic changes in early stage human heart disease.

Evaluation of an integrated strategy for proteomic profiling of skeletal muscle.

Proteomic analysis of skeletal muscle presents particular challenges when trying to identify valid biomarkers of phenotypic change in small biopsies from genetically diverse human subjects. Currently, two-dimensional (2-D) gel electrophoresis and mass spectrometry are the chosen analytical strategies but 2-D gels are not appropriate for analyzing proteins less than 11 kDa, they can suffer from problems of reproducibility and in routine use are not a viable high-throughput technique. We have evaluated an integrated proteomic strategy employing Ciphergen ProteinChip arrays, one-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Protein fingerprints characteristic of fast and slow contracting muscles from normal and kyphoscoliosis (ky) mutant mice were obtained from Ciphergen protein arrays. Eight statistically validated protein biomarkers have so far been identified capable of discriminating fast from slow muscle. Five of these showed further differential expression in ky versus normal BDL soleus muscles. Several biomarkers have been formally identified, and were myosin light chain isoforms shown previously to be expressed differentially by fast versus slow skeletal muscles. This integrated experimental approach using a model mouse muscle system shows the potential of Ciphergen protein array technology for proteomic analysis of small proteins in small muscle samples and its applicability for phenotypic characterization of skeletal muscle in general.

Effect of NT-4 and BDNF delivery to damaged sciatic nerves on phenotypic recovery of fast and slow muscles fibres.

We investigated whether neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF) affected the reinnervation of slow and fast motor units. Neurotrophin-impregnated or plain fibronectin (FN) conduits were inserted into a sciatic nerve gap. Fast extensor digitorum longus (EDL) and slow soleus muscles were collected 4 months postsurgery. Muscles were weighed and fibre type proportion and mean fibre diameters were derived from muscle cross-sections. All fibre types in muscles from FN animals were severely atrophied and this correlated well with type 1 fibre loss and atrophy in soleus and type 2b loss and atrophy in EDL. Treatment with NT-4 reversed soleus but not EDL mass loss above the FN group by significantly restoring type 1 muscle fibre proportion and diameters towards those of normal unoperated animals. BDNF did not increase muscle mass but did have minor effects on fibre type and diameter. Thus, NT-4 significantly improved slow motor unit recovery, and provides a basis for therapies intended to aid the functional recovery of muscles after denervating injury.