Proteomic Comparison between Periodontal Pocket Tissue and Other Oral Samples in Severe Periodontitis: The Meeting of Prospective Biomarkers

Periodontitis is characterized by gingival regression, alveolar bone resorption and the development of deep periodontal pockets that, if left untreated, can lead to tooth loss. Currently, specific biomarkers are needed for the early, objective diagnosis, monitoring, and management of periodontal patients. In this proteomic study, periodontal pocket tissues from patients with severe periodontitis were analyzed in comparison to periodontally healthy sites with the aim of discovering distinctive protein targets. Gingival tissues were fragmented using a motorized mechanical method and mixture protein was separated via mono-dimensional gel electrophoresis. The examination of protein bands using definite 1D image analysis software allowed for the detection of 22 differentially expressed proteins between pathological and healthy samples that were identified through mass spectrometry. A comparative assessment of these proteins with those previously reported in other studies conducted on periodontal diseases in various types of oral specimens, such as gingival crevicular fluid, dentin, tooth pulp, root canal content, salivary gland secretions, saliva, periodontal ligament cells, and dental stem cells, highlighted a great number of significant common matches. The discovery of a selective cluster of periodontitis-related biomarkers could become particularly important before the clinical manifestation of the disease to promptly stop its progression for a timely preventive diagnosis

Proteomics Disclose the Potential of Gingival Crevicular Fluid (GCF) as a Source of Biomarkers for Severe Periodontitis

: Periodontal disease is a widespread disorder comprising gingivitis, a mild early gum inflammation, and periodontitis, a more severe multifactorial inflammatory disease that, if left untreated, can lead to the gradual destruction of the tooth-supporting apparatus. To date, effective etiopathogenetic models fully explaining the clinical features of periodontal disease are not available. Obviously, a better understanding of periodontal disease could facilitate its diagnosis and improve its treatment. The purpose of this study was to employ a proteomic approach to analyze the gingival crevicular fluid (GCF) of patients with severe periodontitis, in search of potential biomarkers. GCF samples, collected from both periodontally healthy sites (H-GCF) and the periodontal pocket (D-GCF), were subjected to a comparison analysis using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A total of 26 significantly different proteins, 14 up-regulated and 12 down-regulated in D-GCF vs. H-GCF, were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The main expressed proteins were inflammatory molecules, immune responders, and host enzymes. Most of these proteins were functionally connected using the STRING analysis database. Once validated in a large scale-study, these proteins could represent a cluster of promising biomarkers capable of making a valuable contribution for a better assessment of periodontitis

Diagnostic proteomic biomarkers to detect kidney diseases

Urinary proteomics is primarily applied to the study of renal and urogenital tract disorders. Here are reported two distinct successful examples of this approach for the discovery of early urinary biomarkers of kidney­ related dysfunctions: diabetic nephropathy (DN), a well ­known complication of diabetes frequently leading to dialysis, and drug­induced nephrotoxicity, a possible condition caused by medication ­overuse headache (MOH). Early detection of kidney disorders based on selective biomarkers could permit to diagnose patients at the initial stage of the disease, where the therapy is still possible to stop or prevent occurrence of advance disease. Urine samples were first concentrated and desalted. Subsequently, they were subjected to two­-dimensional gel electrophoresis (2­DE) coupled to mass spectrometry (MS) for protein identification. Furthermore, some proteins were verified by Western blot and ELISA test. In diabetes-­related study, 11 differentially expressed proteins were detected (8 up­regulated and 3 down­regulated) in type 2 diabetic (T2D) and T2DN patients compared to the healthy control subjects. In MOH study, a total of 21 over­excreted proteins was revealed in urine of non­-steroidal anti­inflammatory drugs (NSAIDs) and mixtures abusers vs controls. Particularly, 4 proteins were positively validated by immunoblotting and ELISA. Urinary proteomics allows non­invasive assessment of renal diseases at an early stage by the identification of characteristic protein pattern

Identification of the most abundant proteins in equine amniotic fluid by a proteomic approach

Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4\u201312% gels. A pool of the 24 samples, after SDS-PAGE, was cut in 25 slices, trypsin-digested and analysed by mass spectrometry (MS) for protein identification. Mean AF protein concentration was 1.96\ua0\ub1\ua01.12\ua0g/L. Thirty-four proteins were successfully identified by MS and subsequently categorised according to Gene Ontology (GO). Twelve proteins (e.g. fibronectin, lumican, thrombospondin and fibulin) belonged to or interacted with the extracellular matrix (ECM) playing an important role in the development of foetal tissues. Most of the remaining proteins were classified as transport (e.g. albumin, major allergen Equ c1 and alpha-fetoprotein) delivering nutrients, ions and lipids essential for foetal growth and development. Among these proteins, major allergen Equ c1 is widely studied in human medicine because it induces Ig-E mediated type I allergic reaction. The absence of immunoglobulins in equine AF was also confirmed

Exploration of candidate serum biomarkers potentially related to the chronic pain condition in Medication-overuse headache

Background Medication Overuse Headache (MOH) is a prevalent and disabling disorder resulting from the overuse of analgesic drugs, triptans or other acute headache medications. In previous proteomic studies, several proteins have been found at high concentrations in the urine of MOH patients and in the serum of rats with neuropathic pain. The aim of this study was to compare the serum levels of lipocalin-type Prostaglandin D2 synthase (L-PGDS), Vitamin D-binding protein (VDBP), apolipoprotein E (APOE) and apolipoprotein A1 (APOA1) in MOH patients and healthy individuals, further exploring their relationship with cutaneous pain thresholds (CPTs) in the territories innervated by the trigeminal nerve. Methods 69 MOH patients and 42 age- and sex-matched healthy volunteers were enrolled in the study. Von Frey-like filaments were applied to the skin territories innervated by the trigeminal nerve, to determine the CPTs. L-PGDS, VDBP, APOE and APOA1 were quantified in the serum by Enzyme-linked Immunosorbent Assay (ELISA). Clinical and laboratory data were collected. Comparisons between MOH patients and healthy individuals were performed using independent t test or χ2 test. To correlate serum proteins with CPTs, Pearson correlation coefficient or Spearman's rank correlation coefficient were used. Results CPTs were lower among MOH patients. L-PGDS, VDBP and APOE had significantly different serum concentrations between groups (p < 0.01), but no correlation was found with CPTs. APOA1 serum concentrations did not differ between patients and healthy individuals. Conclusions L-PGDS, VDBP and APOE had abnormal serum levels in MOH patients, confirming their alteration in some conditions of chronic headache and neuropathic pain. The in-depth study of target proteins represents a promising approach for a better understanding of MOH, as well as the detection of candidate biomarkers for chronic headache or the risks associated with overuse medications

Validation of potential candidate biomarkers of drug-induced nephrotoxicity and allodynia in medication-overuse headache

Medication-overuse headache (MOH) is a chronic disorder that results from the overuse of analgesics drugs, triptans or other acute headache compounds. Although the exact mechanisms underlying MOH remain still unknown, several studies suggest that it may be associated with development of "central sensitization", which may cause cutaneous allodynia (CA). Furthermore, the epidemiology of drug-induced disorders suggests that medication overuse could lead to nephrotoxicity. The aim of this work was to confirm and validate the results obtained from previous proteomics studies, in which we analyzed the urinary proteome of MOH patients in comparison with healthy non-abusers individuals

Serum levels of allopregnanolone, progesterone and testosterone in menstrually-related and postmenopausal migraine: A cross-sectional study

Background: Reduced blood or cerebrospinal fluid levels of allopregnanolone are involved in menstrual cycle-linked CNS disorders, such as catamenial epilepsy. This condition, like menstrually-related migraine, is characterized by severe, treatment-resistant attacks. We explored whether there were differences in allopregnanolone, progesterone and testosterone serum levels between women with menstrually-related migraine (MM, n¼30) or postmenopausal migraine without aura who had suffered from menstrually-related migraine during their fertile age (PM, n¼30) and non-headache control women in fertile age (FAC, n¼30) or post-menopause (PC, n¼30). Methods: Participants were women with migraine afferent to a headache centre; controls were female patients’ acquaintances. Serum samples obtained were analyzed by HPLC-ESI-MS/MS. Results: In menstrually-related migraine and postmenopausal migraine groups, allopregnanolone levels were lower than in the respective control groups (fertile age and post-menopause) (p<0.001, one-way analysis of variance followed by Tukey-Kramer post-hoc comparison test) while progesterone and testosterone levels were similar. By grouping together patients with migraine, allopregnanolone levels were inversely correlated with the number of years and days of migraine/ 3 months (p 0.005, linear regression analysis). Conclusion: Decreased GABAergic inhibition, due to low allopregnanolone serum levels, could contribute to menstrually-related migraine and persistence of migraine after menopause. For the management of these disorders, a rise in the GABAergic transmission by increasing inhibitory neurosteroids might represent a novel strategy

Analysis of protein expression in periodontal pocket tissue: a preliminary study

The periodontal disease is caused by a set of inflammatory disorders characterized by periodontal pocket formation that lead to tooth loss if untreated. The proteomic profile and related molecular conditions of pocket tissue in periodontally-affected patients are not reported in literature. To characterize the proteomic profile of periodontally-affected patients, their interproximal periodontal pocket tissue was compared with that of periodontally-healthy patients. Pocket-associated and healthy tissue samples, harvested during surgical therapy, were treated to extract the protein content. Tissues were always collected at sites where no periodontal-pathogenic bacteria were detectable. Proteins were separated using two-dimensional gel electrophoresis and identified by liquid chromatography/mass spectrometry. After identification, four proteins were selected for subsequent Western Blot quantitation both in pathological and healty tissues

Discovery by a proteomic approach of possible early biomarkers of drug-induced nephrotoxicity in medication-overuse headache

BACKGROUND: Medication-overuse headache (MOH) is a chronic headache condition that results from the overuse of analgesics drugs, triptans, or other antimigraine compounds. The epidemiology of drug-induced disorders suggests that medication overuse could lead to nephrotoxicity, particularly in chronic patients. The aim of this work was to confirm and extend the results obtained from a previous study, in which we analyzed the urinary proteome of 3 MOH patients groups: non-steroidal anti-inflammatory drugs (NSAIDs), triptans and mixtures abusers, in comparison with non-abusers individuals (controls). METHODS: In the present work we employed specialized proteomic techniques, namely two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS), and the innovative Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry (SELDI-TOF-MS), to discover characteristic proteomic profiles associated with MOH condition. RESULTS: By 2-DE and MS analysis we identified 21 over-excreted proteins in MOH patients, particularly in NSAIDs abusers, and the majority of these proteins were involved in a variety of renal impairments, as resulted from a literature search. Urine protein profiles generated by SELDI-TOF-MS analysis showed different spectra among groups. Moreover, significantly higher number of total protein spots and protein peaks were detected in NSAIDs and mixtures abusers. CONCLUSIONS: These findings confirm the presence of alterations in proteins excretion in MOH patients. Analysis of urinary proteins by powerful proteomic technologies could lead to the discovery of early candidate biomarkers, that might allow to identify MOH patients prone to develop potential drug overuse-induced nephrotoxicity

Proteomic research of proteins involved in pain expression in an animal model of chronic pain

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