Cleavage of host cytokeratin-6 by lysine-specific gingipain induces gingival inflammation in periodontitis patients

Background/Purpose. Lysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion. Methods. K6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay. Results. We identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359–378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt. Conclusion. Kgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease

A New Serum Biomarker Set to Detect Mild Cognitive Impairment and Alzheimer’s Disease by Peptidome Technology

Background: Because dementia is an emerging problem in the world, biochemical markers of cerebrospinal fluid (CSF) and radio-isotopic analyses are helpful for diagnosing Alzheimer’s disease (AD). Although blood sample is more feasible and plausible than CSF or radiological biomarkers for screening potential AD, measurements of serum amyloid- β (Aβ), plasma tau, and serum antibodies for Aβ1 - 42 are not yet well established. Objective: We aimed to identify a new serum biomarker to detect mild cognitive impairment (MCI) and AD in comparison to cognitively healthy control by a new peptidome technology. Methods: With only 1.5μl of serum, we examined a new target plate “BLOTCHIP®” plus a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) to discriminate control (n = 100), MCI (n = 60), and AD (n = 99). In some subjects, cognitive Mini-Mental State Examination (MMSE) were compared to positron emission tomography (PET) with Pittsburgh compound B (PiB) and the serum probability of dementia (SPD). The mother proteins of candidate serum peptides were examined in autopsied AD brains. Results: Apart from Aβ or tau, the present study discovered a new diagnostic 4-peptides-set biomarker for discriminating control, MCI, and AD with 87% of sensitivity and 65% of specificity between control and AD (***p  Conclusion: The present serum biomarker set provides a new, rapid, non-invasive, highly quantitative and low-cost clinical application for dementia screening, and also suggests an alternative pathomechanism of AD for neuroinflammation and neurovascular unit damage

Potential Biomarker Peptides Associated with Acute Alcohol-Induced Reduction of Blood Pressure.

The purpose of this study was to explore the peptides that are related to acute reduction of blood pressure after alcohol drinking. Venous blood was collected from male healthy volunteers before and after drinking white wine (3 ml/kg weight) containing 13% of ethanol. Peptidome analysis for serum samples was performed using a new target plate, BLOTCHIP®. Alcohol caused significant decreases in systolic and diastolic blood pressure levels at 45 min. The peptidome analysis showed that the levels of three peptides of m/z 1467, 2380 and 2662 changed significantly after drinking. The m/z 1467 and 2662 peptides were identified to be fragments of fibrinogen alpha chain, and the m/z 2380 peptide was identified to be a fragment of complement C4. The intensities of the m/z 2380 and m/z 1467 peptides before drinking were associated with % decreases in systolic and diastolic blood pressure levels at 45 min after drinking compared with the levels before drinking, while there were no significant correlations between the intensity of the m/z 2662 peptide and % decreases in systolic and diastolic blood pressure levels after drinking. The m/z 1467 and 2380 peptides are suggested to be markers for acute reduction of blood pressure after drinking alcohol

New Biomarkers for Prediction of Disseminated Intravascular Coagulation in Patients With Sepsis

Complication of disseminated intravascular coagulation (DIC) is a determinant of the prognosis for patients with sepsis. The purpose of this study was to find DIC-related peptides in blood for prediction and early diagnosis of DIC in patients with sepsis. The participants were 20 patients with sepsis (age: 68.9 ± 11.4 years) and they were divided into 2 groups with (n = 8) and without (n = 12) a complication of DIC. Peptides in the serum of the patients were inclusively analyzed by a new method for peptidome analysis using a target plate, BLOTCHIP. By differential analysis of peptides in the blood from patients in the groups with and without DIC, we selected 13 mass spectrometry (MS) peaks as candidate marker peptides for prediction of DIC. By subsequent MS/MS structural analysis, 8 peptides were successfully identified as marker peptides for DIC in patients with sepsis. The peptides were fragments of serum amyloid A-2 protein, α2-HS-glycoprotein, fibrinogen α chain, fibrinogen β chain, serum albumin, collagen α1 (I) chain, collagen α1 (III) chain, and coagulation factor XIII A chain. In receiver–operating characteristic analysis for the relationships between the marker peptides and DIC, the area under the curve for each of these peptides was 0.594 to 0.760. We identified 8 blood marker peptides for prediction of DIC complication in patients with sepsis. Further studies by direct measurements of the serum peptide levels in larger numbers of patients with sepsis-induced DIC are needed to confirm the findings of this study

Potential Biomarker Peptides Associated with Acute Alcohol-Induced Reduction of Blood Pressure

<div><p>The purpose of this study was to explore the peptides that are related to acute reduction of blood pressure after alcohol drinking. Venous blood was collected from male healthy volunteers before and after drinking white wine (3 ml/kg weight) containing 13% of ethanol. Peptidome analysis for serum samples was performed using a new target plate, BLOTCHIP^®. Alcohol caused significant decreases in systolic and diastolic blood pressure levels at 45 min. The peptidome analysis showed that the levels of three peptides of <i>m/z</i> 1467, 2380 and 2662 changed significantly after drinking. The <i>m/z</i> 1467 and 2662 peptides were identified to be fragments of fibrinogen alpha chain, and the <i>m/z</i> 2380 peptide was identified to be a fragment of complement C4. The intensities of the <i>m/z</i> 2380 and <i>m/z</i> 1467 peptides before drinking were associated with % decreases in systolic and diastolic blood pressure levels at 45 min after drinking compared with the levels before drinking, while there were no significant correlations between the intensity of the <i>m/z</i> 2662 peptide and % decreases in systolic and diastolic blood pressure levels after drinking. The <i>m/z</i> 1467 and 2380 peptides are suggested to be markers for acute reduction of blood pressure after drinking alcohol.</p></div

Scatter plots of the relationships between ranks of serum <i>m/z</i> 2380 peptide intensity before drinking and % changes in systolic (A) and diastolic (B) blood pressure at 45 min after drinking.

<p>Spearman’s rank correlation coefficients (<i>r</i>) are given in the figures.</p

Differential profiling of serum sampled from subjects before and after drinking.

<p>Each integrated spectrum normalized with ClinPro Tools version 2.2 is the average of 10 samples (total of 44 integrations) from subjects.</p