Diagnostic Cytokines and Comparative Analysis Secreted from Exfoliated Deciduous Teeth, Dental Pulp, and Bone Marrow Derived Mesenchymal Stem Cells for Functional Cell-Based Therapy.

The aim of the study was to clarify the distinctive features of stem cells for effective cell-based therapy strategies in regenerative medicine. The expression levels of cytokines secreted from stem cells from exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSCs), and bone marrow derived mesenchymal stem cells (BMMSCs) were examined to identify the details of their characteristics. A total of 174 cytokines were analyzed using cytokine antibody array, and their expression levels were confirmed by an enzyme-linked immunosorbent assay. These results indicated that 11 cytokines that were related to tissue regeneration, including growth factors, chemokines, and inflammatory cytokines, were identical in SHED, DPSCs, and BMMSCs. The comparative analyses between SHED and BMMSCs revealed that hepatocyte growth factor (HGF), matrix metalloproteinase-3, and stromal cell derived factor 1 (SDF-1) were expressed 6.7-, 2.5-, and 2.1-fold higher, respectively, in SHEDs. HGF was also expressed 3.4-fold higher in DPSCs than BMMSCs. Monocyte chemoattractant protein-1, and-3 were expressed more strongly in BMMSCs. SHED contained significantly higher SDF-1 levels than DPSCs. The distinct cytokine secretion indicated that they had different character besides basic MSC features. This knowledge of diagnostic cytokines analysis secreted from SHED, DPSCs, and BMMSCs extends our understanding, and can provide a novel therapeutic paradigm shift for functional cell-based therapy.

Effect of Chemokine (C-C Motif) Ligand 7 (CCL7) and Its Receptor (CCR2) Expression on Colorectal Cancer Behaviors.

Colorectal cancer is the source of one of the most common cancer-related deaths worldwide, where the main cause of patient mortality remains metastasis. The aim of this study was to determine the role of CCL7 (chemokine (C-C motif) ligand 7) in tumor progression and finding whether it could predict survival of colorectal cancer patients. Initially, our study focused on the crosstalk between mesenchymal stem cells (MSCs) and CT26 colon carcinoma cells and resulted in identifying CCL7 as a chemokine upregulated in CT26 colon cancer cells cocultured with MSCs, compared with CT26 in monoculture in vitro. Moreover, we showed that MSCs enhance CT26 tumor cell proliferation and migration. We analyzed the effect of CCL7 overexpression on tumor progression in a murine CT26 model, where cells overexpressing CCL7 accelerated the early phase of tumor growth and caused higher lung metastasis rates compared with control mice. Microarray analysis revealed that tumors overexpressing CCL7 had lower expression of immunoglobulins produced by B lymphocytes. Additionally, using Jh mutant mice, we confirmed that in the CT26 model, CCL7 has an immunoglobulin-, and thereby, B-cell-dependent effect on metastasis formation. Finally, higher expression of CCL7 receptor CCR2 (C-C chemokine receptor type 2) was associated with shorter overall survival of colorectal cancer patients. Altogether, we showed that CCL7 is essentially involved in the progression of colorectal cancer in a CT26 mouse model and that the expression of its receptor CCR2 could be related to a different outcome pattern of patients with colorectal carcinoma.

An initial investigation into endothelial CC chemokine expression in the human rheumatoid synovium.

Rheumatoid arthritis (RA) is a destructive and chronic autoimmune inflammatory disease. Synovial inflammation is a major feature of RA and is associated with leukocyte recruitment. Leukocytes cross the endothelial cells (ECs) into the synovial tissue and fluid and this migration is mediated via a range of chemokines and adhesion molecules on the ECs. As important mediators of leukocyte extravasation, a number of chemokines from each of the chemokine families have been established as expressed in the RA joint. However, as little information is available on which chemokines are expressed/presented by the ECs themselves, the purpose of the study was to ascertain which of the CC chemokines were localised in RA ECs. Immunofluoresence was used to assess the presence of the CC-family chemokines in RA synovial ECs using von-Willebrand factor (VWF) as a pan-endothelial marker and a range of human chemokine antibodies. The percentage of VWF positive vessels which were positive for the chemokines was determined. The presence of the four most highly expressed novel chemokines were further investigated in non-RA synovial ECs and the sera and synovial fluid (SF) from patients with RA and osteoarthritis (OA). Statistical analysis of immunofluorescence data was carried out by Student's t-test. For analysis of ELISA data, Kruskal-Wallis ANOVA followed by Dunn's multiple comparison test was utilised to analyse differences in sera and SF levels for each chemokine between RA and OA. Spearman rank correlations of sera and SF chemokine levels with a range of clinical variables were also performed. Chemokine detection varied, the least abundant being CCL27 which was present in 8.3% of RA blood vessels and the most abundant being CCL19 which was present in 80%. Of the 26 chemokines studied, 19 have not been previously observed in RA ECs. Four of these novel chemokines, namely CCL7, CCL14, CCL16 and CCL22 were present on ≥60% of vessels. CCL14 and CCL22 were shown to be increased in RA ECs compared to non-RA ECs, p=0.0041 and p=0.014 respectively. EC chemokines CCL7, CCL14, CCL16 and CCL22 also occurred in RA synovial fluid and sera as established by ELISA. CCL7 was shown to be significantly increased in sera and SF from RA patients compared to that from osteoarthritis (OA) patients (p<0.01), and to have a highly significant correlation with the level of anti-CCP (R=0.93, p=0.001). Less abundant chemokines shown to be present in RA ECs were CCL1-3, CCL5, CCL10-13, CCL15, CCL17, CCL18, CCL20, CCL21 and CCL23-28. In conclusion, this initial study is the first to show the presence of a number of CC chemokines in RA ECs. It provides evidence that further validation and investigation into the presence and functionality of these novel chemokines expressed at RA synovial ECs may be warranted.

Effect of omega 3 fatty acids plus low-dose aspirin on both clinical and biochemical profiles of patients with chronic periodontitis and type 2 diabetes: a randomized double blind placebo-controlled study.

BACKGROUND AND OBJECTIVES: The aim of this study was, first, to investigate the effect of omega 3 (ω3) fatty acids plus low-dose aspirin with closed debridement in the treatment of patients with periodontitis and type 2 diabetes mellitus (DM), and second, to estimate the expression of monocyte chemoattractant protein-3 (MCP-3) in response to the supposed modulatory therapy. MATERIAL AND METHODS: Forty patients with chronic periodontitis and type 2 DM were equally divided into groups 1 (patients received ω3 plus low-dose aspirin for 6 mo) and 2 (patients received placebo during the same period). Evaluation was done clinically (pocket depth, clinical attachment loss, gingival index and plaque index) and biochemically by estimating levels of interleukin 1ß and MCP-3 in gingival crevicular fluid, plus investigating the effect of treatment on glycemic control by levels of glycated hemoglobin A1c in serum. All data were collected at baseline, 3 and 6 mo after treatment. RESULTS: Subjects of group 1 showed a highly significant reduction in pocket depth, clinical attachment loss, gingival index (p ≤ 0.01) after 3 and 6 mo compared to group 2. Glycated hemoglobin A1c levels showed a reduction in both groups at the end of the study period, with a non-significant difference (p > 0.05). Furthermore, the treatment protocol showed a significant reduction in levels of MCP-3 and interleukin 1ß at 3 and 6 mo compared to the placebo group. CONCLUSIONS: Within the limits of the present study, ω3 plus low-dose aspirin proved effective as an adjunct to closed periodontal therapy in the management of patients with periodontitis and type 2 DM. Moreover, MCP-3 was proven to be effective both in the pathogenesis of the disease and as a biomarker in evaluating the response to periodontal treatment.

Effects of interim acrylic resins on the expression of cytokines from epithelial cells and on collagen degradation.

STATEMENT OF PROBLEM: Interim acrylic resins release agents that alter cytokine expression in the surrounding tissues, which could alter extracellular matrix degradation. PURPOSE: The purpose of the study was to evaluate the responses of human epidermal keratinocytes to eluates of interim acrylic resins in regards to cytokine expression and cell-mediated collagen degradation. MATERIAL AND METHODS: Specimens of 4 different interim acrylic resins (HI-I, Jet Acrylic, SNAP acrylic, and Protemp Plus) were placed in Epilife medium for 48 hours and the eluates collected. The cells were incubated for 72 hours in nontoxic concentrations of the eluates. Cytotoxicity was evaluated with lactate dehydrogenase assays and cytokine expression with cytokine antibody arrays. Collagen degradation was determined with a collagen type I assay. The experiments were performed 3 times. Data were analyzed with 1-way and mixed-model ANOVA (α=.05). RESULTS: None of the eluates were cytotoxic. Cytokine expression from the heat-activated polymethyl methacrylate resin group was significantly less for interleukin-3, but significantly greater for interlukin-7. Expression for the chemically activated polymethyl methacrylate resin group was significantly less for growth-regulated oncogene-α, interleukin-1α, and interleukin-3. Expression for the chemically activated polyethyl methacrylate resin group was significantly less for interleukin-1α and interleukin-3, but significantly greater for interleukin-13 and monocytes chemoattractant protein-3. The cytokine expression induced by chemically activated bis-acryl composite resin was significantly greater for granulocyte-macrophage colony stimulating factor, interleukin-7, and monocytes chemoattractant protein-3, but significantly less for growth-regulated oncogene-α. Collagen degradation was not significantly different in any of the groups. CONCLUSIONS: The eluates used were not cytotoxic and did not induce cell-mediated collagen degradation. Some significant changes in cytokine expression were noted.

Peritoneal fluid concentrations of ß-chemokines in endometriosis.

OBJECTIVE: To examine whether the levels of MCP-1, RANTES and MCP-3 in the peritoneal fluid correlate with endometriosis. STUDY DESIGN: Patients with endometriosis were compared with controls. SETTING: Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA. SUBJECTS: This study involved 95 women of reproductive age who were undergoing laparoscopy for evaluation of infertility or for pelvic pain. They were divided into an endometriosis group (n=54) and a control group (n=41). INTERVENTIONS: Peritoneal fluid samples were obtained and ß-chemokines (MCP-1, RANTES and MCP-3) were measured using ELISA. STATISTICAL ANALYSIS: Mean and median values were used to present values. Due to the non-normality of chemokines, a log transformation was applied. Differences were examined using independent samples t-test. One-way ANOVA and Tukey HSD multiple comparison post hoc tests were applied. A significance level at 0.05 was set. RESULTS: The levels of MCP-1 are higher (p for log values=0.024) in the control group (mean=687.6, SD=467.7 pg/ml) than those of the endometriosis group (mean=570.4, SD=633.1 pg/ml). The same is true for the median values of MCP-1 (control median=568.5, endometriosis median=384.7 pg/ml). MCP-3 and RANTES do not differ significantly (MCP-3 p=0.787, RANTES p=0.153). The levels of MCP-1 in patients with stage II endometriosis are significantly lower in comparison with stage III (p=0.048) and stage IV (p=0.033) endometriosis. CONCLUSIONS: A decrease in the concentrations of MCP-1 in stage I endometriosis has been observed, which is even larger in stage II, in contrast to stage III and stage IV endometriosis, which exhibit concentrations similar to the controls.

Effects of resolvin D1 on cell survival and cytokine expression of human gingival fibroblasts.

BACKGROUND: Tissue breakdown in periodontitis is initiated by bacteria, such as Porphyromonas gingivalis, and is caused largely by host responses. Resolvins protect the host against acute inflammation by blocking the migration of polymorphonuclear neutrophils to initiate resolution. The effects of resolvins on human gingival fibroblasts (HGFs) are unknown. This study examines the effects of resolvin D1 on HGF survival and cytokine expression when treated with or without P. gingivalis supernatant. METHODS: Cytotoxicity of resolvin D1 on HGFs with or without a toxic level of P. gingivalis supernatant was measured with lactate dehydrogenase assays. Cytokine arrays were performed on HGF-conditioned media treated with or without resolvin D1 and with or without P. gingivalis supernatant. RESULTS: Resolvin D1 had no cytotoxic effects on HGFs at concentrations between 1 and 1,000 nM (all P > 0.05). Resolvin D1 (1,000 nM) significantly inhibited the toxic effects of 13.5% (v/v) P. gingivalis supernatant on HGFs (P = 0.002). Resolvin D1 significantly reduced the expression of interleukin (IL)-6 (P = 0.010) and monocyte chemoattractant protein (MCP)-1 (P = 0.04) in untreated fibroblasts. P. gingivalis (10%) supernatant significantly increased the expression levels of granulocyte-macrophage colony-stimulating factor (CSF), granulocyte CSF, growth-regulated oncogene (GRO), IL-5, IL-6, IL-7, IL-8, IL-10, MCP-1, MCP-2, MCP-3, and monokine induced by γ-interferon. Resolvin D1 significantly reduced the expression of GRO (P = 0.04), marginally reduced the levels of MCP-1 (P = 0.10), and marginally increased the levels of transforming growth factor (TGF)-ß1 (P = 0.07) from HGFs treated with P. gingivalis supernatant. CONCLUSIONS: Resolvin D1 altered the cytotoxicity of P. gingivalis supernatant on HGFs. Resolvin D1 significantly reduced GRO, marginally reduced MCP-1, and marginally increased TGF-ß1 from P. gingivalis-treated HGFs, which could alter the ability of P. gingivalis to induce inflammation.

Activation of innate immunity accelerates sialoadenitis in a mouse model for Sjögren's syndrome-like disease.

OBJECTIVE: Sjögren's syndrome is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the salivary and lacrimal glands. This study was undertaken to investigate the effects of innate immunity activation on sialoadenitis in a mouse strain genetically susceptible for development of SS-like disease. METHODS: Female New Zealand Black X New Zealand White F1 mice were repeatedly treated with toll-like 3 receptor agonist poly(I:C). Submandibular glands were investigated at different time points for sialoadenitis by immunohistochemistry and for gene expression of different chemokines by quantitative PCR. Submandibular gland-infiltrating cells were characterized by flow cytometry. RESULTS: Poly(I:C) treatment significantly upregulated the expression of multiple chemokines within the submandibular glands. The severity and incidence of sialoadenitis was considerably higher in poly(I:C)-treated mice. There was a preponderance of dendritic cells and NK cells in the initial inflammatory cell infiltrates, and these were followed by CD4+ T cells. CONCLUSIONS: Our data clearly demonstrate that systemic activation of innate immunity accelerates sialoadenitis in a mouse model for SS-like disease. These findings suggest that chronic activation of innate immunity can influence certain features of SS.

Monocyte chemotactic protein-3: possible involvement in apical periodontitis chemotaxis.

AIM: To study the expression of monocyte chemotactic protein-3 (MCP-3, also known as chemokine CCL-7) in tissue from apical lesions (AL) and to associate MCP-3 expression with symptomatic or asymptomatic apical periodontitis. METHODOLOGY: To determine the expression of MCP-3 in AL, biopsies obtained during tooth extraction procedures were fixed, subjected to routine processing and diagnosed as apical granuloma (AG) (n = 7) or radicular cyst (RC) (n = 5). As controls, apical periodontal ligament (PDL) specimens from healthy premolars extracted for orthodontics reasons were included (n = 7). All specimens were immunostained for MCP-3 and examined under a light microscope. In addition, homogenates from AL (n = 14) and healthy PDL samples (n = 7) were studied through immunowestern blot. Finally, periapical exudates samples were collected from root canals of teeth having diagnosis of symptomatic (n = 14) and asymptomatic apical periodontitis (n = 14) during routine endodontic treatments and analysed by immunowestern blot and densitometry. RESULTS: MCP-3 was detected in AG and RC and localized mainly to inflammatory leucocytes, whereas no expression was observed in healthy PDLs. MCP-3 was also detected in periapical exudate, and its levels were significantly higher in symptomatic than in asymptomatic apical periodontitis. CONCLUSIONS: MCP-3 was expressed in AL and its levels associated with clinical symptoms. MCP-3 might play a role in disease pathogenesis, possibly by stimulating mononuclear chemotaxis.

Mediators of inflammation in nasal lavage from aspirin intolerant patients after aspirin challenge.

The pathogenetic mechanisms underlying development of persistent inflammation in aspirin (ASA) intolerance are not fully understood. The aim of this study was to determine levels of MCP-3, RANTES, eotaxin, Il-5 and Il-3 in aspirin intolerant asthmatics (AIA) after nasal lysine-aspirin (Lys-ASA) challenge. Twenty AIA and 10 aspirin tolerant controls (ATC) were challenged with saline or 14.4mg of Lys-ASA. Lys-ASA challenge induced clinical symptoms and influx of eosinophils and basophils only in AIA group. Statistically significant higher levels of MCP-3 and RANTES were found in lavages from AIA as compared with ATC (p<0.05 in all time points). Before challenge the average level of MCP-3 was 86.95pg/ml in AIA and 47.61pg/ml in ATC, RANTES levels were 34.20pg/ml in AIA and 17.21pg/ml in ATC and did not change after the challenge in both group. The mean eotaxin's level was 11.01pg/ml in AIA and 8.03pg/ml in ATC before and increased to 20.06, 26.22pg/ml (4 and 24h in AIA) as compared to 10.51, 14.76pg/ml (4 and 24h in ATC) after the challenge (p<0.05). Interleukin-3 and Il-5 were not detectable. The highest inhibition of eosinophils' chemotaxis was induced by anti-eotaxin (47% of inhibition), followed by anti-RANTES (29%), anti-MCP-3 (19%) and anti-Il-5 (9%). In summary, we found that persistent inflammation in AIA patients is characterized by overproduction of MCP-3 and RANTES. Lack of increase in MCP-3 and RANTES levels after Lys-ASA challenge suggest that those mediators are involved in chronic rather than acute phase of ASA induced inflammation.

Different roles of odontoblasts and fibroblasts in immunity.

Odontoblasts and fibroblasts are suspected to influence the innate immune response triggered in the dental pulp by micro-organisms that progressively invade the human tooth during the caries process. To determine whether they differ in their responses to oral pathogens, we performed a systematic comparative analysis of odontoblast-like cell and pulp fibroblast responses to TLR2-, TLR3-, and TLR4-specific agonists (lipoteichoic acid [LTA], double-stranded RNA, and lipopolysaccharide [LPS], respectively). Cells responded to these agonists by differential up-regulation of chemokine gene expression. CXCL2 and CXCL10 were thus increased by LTA only in odontoblast-like cells, while LPS increased CCL7, CCL26, and CXCL11 only in fibroblasts. Supernatants of stimulated cultures increased migration of immature dendritic cells compared with controls, odontoblast-like cells being more potent attractants than fibroblasts. Analysis of these data suggests that odontoblasts and pulp fibroblasts differ in their innate immune responses to oral micro-organisms that invade the pulp tissue.