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1.  Th1/Th2 Cytokine Ratio in Tissue Transudates from Patients with Oral Lichen Planus 
Mediators of Inflammation  2007;2007:19854.
Objective. The characteristics of oral lichen planus (OLP) provoke investigators to explore possible biomarkers by which to monitor disease activity and therapeutic efficacy. Oral fluids may provide an accessible medium for analysis of such biomarkers. Previous studies have shown that activation of nuclear factor-kappa B (NF-κ B) plays an important role in the pathogenesis of oral lichen planus (OLP), which is a chronic inflammatory disorder mediated by T cells. Prior to the present investigation, reports of the levels of NF-κ B and its dependent cytokines in oral fluids have not been forthcoming. The purpose of this study was to detect the level of NF-κ B dependent cytokines, TNF-alpha, IL-1-alpha, IL-6, and IL-8 in tissue transudates directly from lesions of OLP, and explore the feasibility of the data for clinical application. Study design. Thirteen definitively diagnosed OLP subjects were enrolled in the study as were 13 age-sex matched controls. In each subject, lesion tissue transudates (TTs) were collected by a novel collection technique with a filter paper. The level of cytokines, TNF-alpha, IL-1-alpha, IL-6, and IL-8 in three types of oral fluids were determined by ELISA. Results. In the tissue transudate(TT), there were significantly higher level of cytokines TNF-alpha, IL-1-alpha, IL-6, and IL-8 detected in OLP patients than in controls: (TT: 40.0 ± 9.8 versus 4.5 ± 0.7, 710 ± 114 versus 305 ± 78, 150 ± 25 versus 1.7 ± 0.5, 2800 ± 260 versus 1450 ± 130, P < .0001; unit: pg/mL). Conclusions. These results indicate that NF-κ B dependent inflammatory cytokines may be detected at increased levels in oral lesion tissue transudates which may have diagnostic and prognostic potentials for monitoring disease activity and making therapeutic decisions in patients with OLP.
PMCID: PMC1847474  PMID: 17497029
2.  Genetics and Genomics of Sjogren's Syndrome: Research provides Clues to Pathogenesis and Novel Therapies 
While the key inciting events that drive the progression from autoantibodies to clinical disease remain to be clarified, new light has been shed on the factors contributing to disease susceptibility and the role of genetic factors in determining Sjogren's syndrome (SS) disease phenotypes. The purpose of this review is to provide an update on the role of genetic markers in the susceptibility to and pathogenesis of Sjogren's syndrome. This paper also discusses how genomic and proteomic technology can help in the design of specific therapeutics.
Key Findings
Recent evidence suggests that inflammatory genes associated with interferon pathways, and specific regulatory genes that control the maturation and proliferation of B cells, contribute to the pathogenesis of Sjogren's syndrome. Both gene expression profiling technology and gene association studies have been used to identify these key biologic pathways. Molecularly defined subsets of pSS patients are also being revealed by these studies. Previously identified gene loci which predispose to multiple autoimmune disorders have been confirmed supporting the paradigm of “general” autoimmune disease genes. Association of SS with many additional susceptibility loci are likely to be established through ongoing genome-wide association scans (GWAS). Clues from genetic studies suggest that targeting B cells will prove to be an effective way of reducing the systemic manifestations of pSS and are supported by early clinical trials.
Genome-wide technologies are likely to identify new genes and molecular pathways in the pathogenesis of SS that will be useful not only to identify patients at risk for SS, but also to identify subsets of patients at risk for variable levels of disease severity. In the future, these studies could identify novel biomarkers that will lead to significant advances in management by providing the means to tailor therapeutic strategies to individual patients.
PMCID: PMC3095716  PMID: 21497524
Sjogren's; genetics; genomics; autoimmune; inflammatory; Sjögren's Syndrome; Gene Expression Profiling; Interferon; Genetics
3.  Variants at multiple loci implicated in both innate and adaptive immune responses are associated with Sjögren’s syndrome 
Nature genetics  2013;45(11):10.1038/ng.2792.
Sjögren’s syndrome is a common autoimmune disease (~0.7% of European Americans) typically presenting as keratoconjunctivitis sicca and xerostomia. In addition to strong association within the HLA region at 6p21 (Pmeta=7.65×10−114), we establish associations with IRF5-TNPO3 (Pmeta=2.73×10−19), STAT4 (Pmeta=6.80×10−15), IL12A (Pmeta =1.17×10−10), FAM167A-BLK (Pmeta=4.97×10−10), DDX6-CXCR5 (Pmeta=1.10×10−8), and TNIP1 (Pmeta=3.30×10−8). Suggestive associations with Pmeta<5×10−5 were observed with 29 regions including TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2, and PHIP amongst others. These results highlight the importance of genes involved in both innate and adaptive immunity in Sjögren’s syndrome.
PMCID: PMC3867192  PMID: 24097067
4.  Quantitative Proteomic Analysis of Oral Brush Biopsies Identifies Secretory Leukocyte Protease Inhibitor as a Promising, Mechanism-Based Oral Cancer Biomarker 
PLoS ONE  2014;9(4):e95389.
A decrease in the almost fifty percent mortality rate from oral cancer is needed urgently. Improvements in early diagnosis and more effective preventive treatments could affect such a decrease. Towards this end, we undertook for the first time an in-depth mass spectrometry-based quantitative shotgun proteomics study of non-invasively collected oral brush biopsies. Proteins isolated from brush biopsies from healthy normal tissue, oral premalignant lesion tissue (OPMLs), oral squamous cell carcinoma (OSCC) and matched control tissue were compared. In replicated proteomic datasets, the secretory leukocyte protease inhibitor (SLPI) protein stood out based on its decrease in abundance in both OPML and OSCC lesion tissues compared to healthy normal tissue. Western blotting in additional brushed biopsy samples confirmed a trend of gradual decreasing SLPI abundance between healthy normal and OPML tissue, with a larger decrease in OSCC lesion tissue. A similar SLPI decrease was observed in-vitro comparing model OPML and OSCC cell lines. In addition, exfoliated oral cells in patients’ whole saliva showed a loss of SLPI correlated with oral cancer progression. These results, combined with proteomics data indicating a decrease in SLPI in matched healthy control tissue from OSCC patients compared to tissue from healthy normal tissue, suggested a systemic decrease of SLPI in oral cells correlated with oral cancer development. Finally, in-vitro experiments showed that treatment with SLPI significantly decreased NF-kB activity in an OPML cell line. The findings indicate anti-inflammatory activity in OPML, supporting a mechanistic role of SLPI in OSCC progression and suggesting its potential for preventative treatment of at-risk oral lesions. Collectively, our results show for the first time the potential for SLPI as a mechanism-based, non-invasive biomarker of oral cancer progression with potential in preventive treatment.
PMCID: PMC3991667  PMID: 24748380
5.  Comparison of the American-European Consensus Group Sjögren's syndrome classification criteria to newly proposed American College of Rheumatology criteria in a large, carefully characterized sicca cohort 
Annals of the rheumatic diseases  2013;73(1):10.1136/annrheumdis-2013-203845.
To compare the performance of the American-European Consensus Group (AECG) and the newly proposed American College of Rheumatology (ACR) classification criteria for Sjögren's syndrome in a well-characterized sicca cohort, given ongoing efforts to resolve discrepancies and weaknesses in the systems.
In a multidisciplinary clinic for the evaluation of sicca, we assessed features of salivary and lacrimal gland dysfunction and autoimmunity as defined by tests of both AECG and ACR criteria in 646 participants. Global gene expression profiles were compared in a subset of 180 participants.
Application of the AECG and ACR criteria resulted in classification of 279 and 268 participants with SS, respectively. Both criteria were met by 244 participants (81%). In 26 of the 35 AECG+/ACR- participants, the minor salivary gland biopsy focal score was ≥1 (74%), while 9 had positive anti-Ro/La (26%). There were 24 AECG-/ACR+ who met ACR criteria mainly due to differences in the scoring of corneal staining. All patients with SS, regardless of classification, had similar gene expression profiles, which were distinct from the healthy controls.
The two sets of classification criteria yield concordant results in the majority of cases and gene expression profiling suggests that patients meeting either set of criteria are more similar to other SS participants than to healthy controls. Thus, there is no clear evidence for increased value of the new ACR criteria over the old AECG criteria from the clinical or biological perspective. It is our contention, supported by this report, that improvements in diagnostic acumen will require a more fundamental understanding of the pathogenic mechanisms than is at present available.
PMCID: PMC3855629  PMID: 23968620
Sjögren's syndrome; Classification; Diagnosis
6.  A comparison of screening methods in two early phase oral leukoplakia clinical trials 
Oral diseases  2012;18(7):720-723.
Clinical trial accrual for oral dysplasia is difficult in the U.S. and elsewhere. Patients with dysplastic oral leukoplakia progress to frank invasive carcinoma at a rate of 5-37% over 5 years. We compared two clinical trial screening efforts to hopefully devise better accrual strategies to these types of clinical trials.
For the first trial we identified 244 patients with dysplastic oral leukoplakia in our university database and a media campaign. Patients were notified, screened by examination and biopsy. For the second clinical trial we established a preneoplastic lesions clinic and teaching and communications network with regional oral health care professionals.
Only one of 244 patients accrued to the first clinical trial through an organized screening effort based on database/medical records review. The second clinical trial accrued 16/30 screened patients through redirected efforts in teaching, communications and a preneoplastic lesions clinic.
We conclude that significant difficulties resulted from medical record /database review of leukoplakia patients as a screening method for leukoplakia clinical trial entry. We feel that persistent direct contact and education of health care professionals who are likely to examine leukoplakia patients improved accrual to the second clinical trial.
PMCID: PMC3440539  PMID: 22524155
Oral leukoplakia; clinical trial; preneoplasia; clinical trial; barrier; cancer; oral carcinoma; referral clinic; dysplasia; health education
7.  Large-Scale Phosphoproteomics Analysis of Whole Saliva Reveals a Distinct Phosphorylation Pattern 
Journal of proteome research  2011;10(4):1728-1736.
In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in “extracellular” and “plasma membrane” categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation.
PMCID: PMC3070063  PMID: 21299198
Phosphoproteomics; Whole Saliva; Dynamic Range Compression; Mass Spectrometry
8.  Hexapeptide libraries for enhanced protein PTM identification and relative abundance profiling in whole human saliva 
Journal of proteome research  2011;10(3):1052-1061.
Dynamic range compression (DRC) by hexapeptide libraries increases MS/MS-based identification of lower-abundance proteins in complex mixtures. However, two unanswered questions impede fully realizing DRC’s potential in shotgun proteomics. First, does DRC enhance identification of post-translationally modified proteins? Second, can DRC be incorporated into a workflow enabling relative protein abundance profiling? We sought to answer both questions analyzing human whole saliva. Addressing question one, we coupled DRC with covalent glycopeptide enrichment and MS/MS. With DRC we identified ~2 times more N-linked glycoproteins and their glycosylation sites than without DRC, dramatically increasing the known salivary glycoprotein catalog. Addressing question two, we compared differentially stable isotope-labeled saliva samples pooled from healthy and metastatic breast cancer women using a multidimensional peptide fractionation-based workflow, analyzing in parallel one sample portion with DRC and one portion without. Our workflow categorizes proteins with higher absolute abundance, whose relative abundance ratios are altered by DRC, from proteins of lower absolute abundance detected only after DRC. Within each of these salivary protein categories we identified novel abundance changes putatively associated with breast cancer, demonstrating feasibility and benefits of DRC for relative abundance profiling. Collectively, our results bring us closer to realizing the full potential of DRC for proteomic studies.
PMCID: PMC3049962  PMID: 21142092
9.  Peripheral blood gene expression profiling in Sjögren’s syndrome 
Genes and Immunity  2009;10(4):285-296.
Sjögren’s syndrome (SS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands. Affected cases commonly present with oral and ocular dryness, thought to be the result of inflammatory cell-mediated gland dysfunction. To identify important molecular pathways involved in SS, we used high-density microarrays to define global gene expression profiles in peripheral blood. We first analyzed 21 SS cases and 23 controls and identified a prominent pattern of overexpressed genes that are inducible by interferons (IFNs). These results were confirmed by evaluation of a second independent dataset of 17 SS cases and 22 controls. Additional inflammatory and immune-related pathways with altered expression patterns in SS cases included B and T cell receptor, IGF-1, GM-CSF, PPARα/RXRα, and PI3/AKT signaling. Exploration of these data for relationships to clinical features of disease revealed that expression levels for most IFN-inducible genes were positively correlated with titers of anti-Ro/SSA (P<0.001) and anti-La/SSB (P<0.001) autoantibodies. Diagnostic and therapeutic approaches targeting IFN signaling pathway may prove most effective in the subset of SS cases who produce anti-Ro/SSA and anti-La/SSB autoantibodies. Our results strongly support innate and adaptive immune processes in the pathogenesis of SS and provide numerous candidate disease markers for further study.
PMCID: PMC3273959  PMID: 19404300
10.  Evaluating the potential of a novel oral lesion exudate collection method coupled with mass spectrometry-based proteomics for oral cancer biomarker discovery 
Clinical proteomics  2011;8(1):13.
Early diagnosis of Oral Squamous Cell Carcinoma (OSCC) increases the survival rate of oral cancer. For early diagnosis, molecular biomarkers contained in samples collected non-invasively and directly from at-risk oral premalignant lesions (OPMLs) would be ideal.
In this pilot study we evaluated the potential of a novel method using commercial PerioPaper absorbent strips for non-invasive collection of oral lesion exudate material coupled with mass spectrometry-based proteomics for oral cancer biomarker discovery.
Our evaluation focused on three core issues. First, using an "on-strip" processing method, we found that protein can be isolated from exudate samples in amounts compatible with large-scale mass spectrometry-based proteomic analysis. Second, we found that the OPML exudate proteome was distinct from that of whole saliva, while being similar to the OPML epithelial cell proteome, demonstrating the fidelity of our exudate collection method. Third, in a proof-of-principle study, we identified numerous, inflammation-associated proteins showing an expected increase in abundance in OPML exudates compared to healthy oral tissue exudates. These results demonstrate the feasibility of identifying differentially abundant proteins from exudate samples, which is essential for biomarker discovery studies.
Collectively, our findings demonstrate that our exudate collection method coupled with mass spectrometry-based proteomics has great potential for transforming OSCC biomarker discovery and clinical diagnostics assay development.
PMCID: PMC3200993  PMID: 21914210
Oral Pre-Malignant Lesion (OPML); Oral Squamous Cell Carcinoma (OSCC); exudate; mass spectrometry-based proteomics; biomarker
11.  Novel In Situ Collection of Tumor Interstitial Fluid from a Head and Neck Squamous Carcinoma Reveals a Unique Proteome with Diagnostic Potential 
Clinical Proteomics  2010;6(3):75-82.
Tumors lack normal drainage of secreted fluids and consequently build up tumor interstitial fluid (TIF). Unlike other bodily fluids, TIF likely contains a high proportion of tumor-specific proteins with potential as biomarkers.
Here, we evaluated a novel technique using a unique ultrafiltration catheter for in situ collection of TIF and used it to generate the first catalog of TIF proteins from a head and neck squamous cell carcinoma (HNSCC). To maximize proteomic coverage, TIF was immunodepleted for high abundance proteins and digested with trypsin, and peptides were fractionated in three dimensions prior to mass spectrometry.
We identified 525 proteins with high confidence. The HNSCC TIF proteome was distinct compared to proteomes of other bodily fluids. It contained a relatively high proportion of proteins annotated by Gene Ontology as “extracellular” compared to other secreted fluid and cellular proteomes, indicating minimal cell lysis from our in situ collection technique. Several proteins identified are putative biomarkers of HNSCC, supporting our catalog’s value as a source of potential biomarkers.
In all, we demonstrate a reliable new technique for in situ TIF collection and provide the first HNSCC TIF protein catalog with value as a guide for others seeking to develop tumor biomarkers.
Electronic supplementary material
The online version of this article (doi:10.1007/s12014-010-9050-3) contains supplementary material, which is available to authorized users.
PMCID: PMC2937136  PMID: 20930922
Biomarker discovery; Head and neck squamous cell carcinoma; In situ collection; Tumor interstitial fluid
12.  Quantitative Proteomics Reveals Myosin and Actin as Promising Saliva Biomarkers for Distinguishing Pre-Malignant and Malignant Oral Lesions 
PLoS ONE  2010;5(6):e11148.
Oral cancer survival rates increase significantly when it is detected and treated early. Unfortunately, clinicians now lack tests which easily and reliably distinguish pre-malignant oral lesions from those already transitioned to malignancy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need.
Methodology/Principal Findings
To discover such proteins, in a first-of-its-kind study we used advanced mass spectrometry-based quantitative proteomics analysis of the pooled soluble fraction of whole saliva from four subjects with pre-malignant lesions and four with malignant lesions. We prioritized candidate biomarkers via bioinformatics and validated selected proteins by western blotting. Bioinformatic analysis of differentially abundant proteins and initial western blotting revealed increased abundance of myosin and actin in patients with malignant lesions. We validated those results by additional western blotting of individual whole saliva samples from twelve other subjects with pre-malignant oral lesions and twelve with malignant oral lesions. Sensitivity/specificity values for distinguishing between different lesion types were 100%/75% (p = 0.002) for actin, and 67%/83% (p<0.00001) for myosin in soluble saliva. Exfoliated epithelial cells from subjects' saliva also showed increased myosin and actin abundance in those with malignant lesions, linking our observations in soluble saliva to abundance differences between pre-malignant and malignant cells.
Salivary actin and myosin abundances distinguish oral lesion types with sensitivity and specificity rivaling other non-invasive oral cancer tests. Our findings provide a promising starting point for the development of non-invasive and inexpensive salivary tests to reliably detect oral cancer early.
PMCID: PMC2887353  PMID: 20567502
13.  Systematic comparison of the human saliva and plasma proteomes 
The proteome of human salivary fluid has the potential to open new doors for disease biomarker discovery. A recent study to comprehensively identify and catalog the human ductal salivary proteome led to the compilation of 1166 proteins. The protein complexity of both saliva and plasma is large, suggesting that a comparison of these two proteomes will provide valuable insight into their physiological significance and an understanding of the unique and overlapping disease diagnostic potential that each fluid provides. To create a more comprehensive catalog of human salivary proteins, we have first compiled an extensive list of proteins from whole saliva (WS) identified through MS experiments. The WS list is thereafter combined with the proteins identified from the ductal parotid, and submandibular and sublingual (parotid/SMSL) salivas. In parallel, a core dataset of the human plasma proteome with 3020 protein identifications was recently released. A total of 1939 nonredundant salivary proteins were compiled from a total of 19 474 unique peptide sequences identified from whole and ductal salivas; 740 out of the total 1939 salivary proteins were identified in both whole and ductal saliva. A total of 597 of the salivary proteins have been observed in plasma. Gene ontology (GO) analysis showed similarities in the distributions of the saliva and plasma proteomes with regard to cellular localization, biological processes, and molecular function, but revealed differences which may be related to the different physiological functions of saliva and plasma. The comprehensive catalog of the salivary proteome and its comparison to the plasma proteome provides insights useful for future study, such as exploration of potential biomarkers for disease diagnostics.
PMCID: PMC2773554  PMID: 19898684
Biomarkers; Body fluid; MS; Plasma; Saliva

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