Elucidating the molecular healing of intrabony defects following non-surgical periodontal therapy: A pilot study.

OBJECTIVE: To elucidate the molecular healing of intrabony defects following non-surgical periodontal therapy (NSPT) using gingival crevicular fluid (GCF). BACKGROUND DATA: Currently limited information is available regarding the GCF of intrabony defects and the change in biomarker levels in the GCF at early time points following treatment interventions. METHODS: Twenty-one patients (Periodontitis Stage III or IV) who have received NSPT, contributing one intrabony defect and one healthy site were included in this study. GCF sampling was performed at baseline, 1 day, 5 days and 3 months after NSPT. Multiplex bead immunoassays allowed the profiling of GCF for 27 markers, associated with inflammation and repair/regeneration. A mixed effects model with Bonferroni correction for multiple comparisons was employed to compare the changes in the levels of GCF markers over time. RESULTS: Following NSPT, changes were observed for several GCF markers, marked by significant increases 1 day post-intervention, before returning to baseline levels by 3 months. Specifically, GCF concentrations of IL-2, IL-4, IL-6, IL-8, MMP-1, MMP-3, TIMP-1 and FGFb significantly increased 1 day after NSPT. Signs of activation of cellular senescence were observed 1 day following treatment of intrabony defects, rapidly regressing by 5 days. CONCLUSION: Significant molecular changes are observed as early as 1 day following NSPT in intrabony defects, along with activation of cellular senescence.

A review on clinical use of CAD/CAM and 3D printed dentures.

Aim The aim of this article was to review the current clinical application of computer-aided design/computer-aided manufacturing (CAD/CAM) and three-dimensional (3D) printed dentures in dental clinics.Methods A systematic approach for searching PubMed, Embase, Scopus, and Web of Science databases. The search was performed using a variety of keywords including clinical use AND 3D printed removable dentures OR clinical use AND CAD/CAM removable dentures OR clinical use AND digital removable dentures. Selection criteria included articles written in English and reporting information on clinical applications of digital dentures between 2010 to January 2022.Results The findings outlined the main clinical advantages of digital dentures such as saving working time, satisfying clinical results and securing patients' records, and also requirement of additional visits to secure aesthetic patient satisfaction, good retention and ideal vertical dimension. Many studies recommended performing clinical try-in with regards to providing better results. It was also established that 3D printers are less expensive than milling centres and therefore can be afforded by individual dental professionals.Conclusion Digital dentures are a promising option in treating edentulous patients, especially in remote areas where skilful technicians are rare. However, there are some limitations in their applications.

Extracellular Prostaglandins E1 and E2 and Inflammatory Cytokines Are Regulated by the Senescence Program in Potentially Premalignant Oral Keratinocytes.

Potentially pre-malignant oral lesions (PPOLs) are composed of keratinocytes that are either mortal (MPPOL) or immortal (IPPOL) in vitro. We report here that MPPOL, but not generally IPPOL, keratinocytes upregulate various extracellular tumor-promoting cytokines (interleukins 6 and 8) and prostaglandins E1 (ePGE1) and E2 (ePGE2) relative to normal oral keratinocytes (NOKs). ePGE upregulation in MPPOL was independent of PGE receptor status and was associated with some but not all markers of cellular senescence. Nevertheless, ePGE upregulation was dependent on the senescence program, cyclo-oxygenase 2 (COX2) and p38 mitogen-activated protein kinase and was partially regulated by hydrocortisone. Following senescence in the absence of p16INK4A, ePGEs accumulated in parallel with a subset of tumor promoting cytokine and metalloproteinase (MMP) transcripts, all of which were ablated by ectopic telomerase. Surprisingly, ataxia telangiectasia mutated (ATM) function was not required for ePGE upregulation and was increased in expression in IPPOL keratinocytes in line with its recently reported role in telomerase function. Only ePGE1 was dependent on p53 function, suggesting that ePGEs 1 and 2 are regulated differently in oral keratinocytes. We show here that ePGE2 stimulates IPPOL keratinocyte proliferation in vitro. Therefore, we propose that MPPOL keratinocytes promote the progression of IPPOL to oral SCC in a pre-cancerous field by supplying PGEs, interleukins and MMPs in a paracrine manner. Our results suggest that the therapeutic targeting of COX-2 might be enhanced by strategies that target keratinocyte senescence.

Analysis of gingival crevicular fluid biomarkers in patients with metabolic syndrome.

OBJECTIVES: To assess associations between gingival crevicular fluid (GCF) markers in patients with metabolic syndrome, with or without concomitant periodontitis. METHODS: A total of 95 patients with Metabolic Syndrome (MetS) had a periodontal examination and gingival crevicular fluid samples taken. Proteomic analysis of gingival crevicular fluid (GCF) was carried out by Human XL Cytokine protein arrays in 12 selected patients, followed by multiplex ELISA of 11 analytes in 95 participants. RESULTS: Increased levels of Aggrecan, IL-6 and IL-8 were found in patients with periodontal health compared with moderate and severe periodontitis. The inverse stepwise association between severity of periodontitis and reduced Aggrecan levels was also observed at adjusted linear regression analysis. Diagnosis of diabetes was associated with higher GCF levels of IL-8 and MMP-8. CONCLUSION: Diabetes may affect GCF levels of cytokines, irrespective of periodontal status. Periodontal status may be associated with Aggrecan levels in the GCF of patients affected by metabolic syndrome. CLINICAL SIGNIFICANCE: Investigation of GCF biomarkers may potentially help have diagnostic potential in patients with MetS.

Molecular profiling of intrabony defects' gingival crevicular fluid.

AIM: To profile, for the first time, the gingival crevicular fluid (GCF) of intrabony defects against a wide array of inflammatory and regenerative markers. MATERIALS AND METHODS: Twenty-one patients contributed one intrabony defect and one periodontally healthy site. Clinical and radiographic measures were obtained. GCF samples were analyzed with multiplex bead immunoassays over 27 markers previously identified by our group. Comparisons were performed using Wilcoxon matched-pairs signed-ranks tests, using a Bonferroni corrected α = 0.05/27 = 0.0019. RESULTS: Intrabony defect sites presented significantly increased GCF volume and disease-associated clinical and radiographic characteristics (p < .05). Intrabony defect sites presented significantly increased IL-1α, IL-1ß, IL-6, IFN-γ, and MMP-8 levels compared with periodontally healthy sites (p < .0019). For regeneration markers, significantly higher FGF basic and VEGF levels were observed (p < .0019). Notably, traits of cell senescence were identified for the first time in the GCF. CONCLUSIONS: The differentiation of intrabony defects from periodontally healthy control sites can be based on clinical and radiographic measures and on a differentiated GCF profile that is site-specific. Alongside catabolic processes, through significant up-regulation of inflammation and connective tissue remodeling, unique molecular characteristics of intrabony defects may render them a microenvironment amenable to regeneration. Traits of the senescence-associated secretory phenotype may suggest the existence of senescent cells during periodontal inflammation in intrabony defects.

Saliva and Serum Cytokine Profiles During Oral Ulceration in Behçet's Disease.

Behçet's disease (BD) is a chronic, multi-systemic disorder of unknown aetiology typified by recurrent oral and genital mucocutaneous lesions, uveitis and vasculitis. Innate and adaptive immune system dysregulation has been implicated in pathogenesis with alterations in serum cytokine profiles. Few studies have investigated salivary cytokines in BD, despite more than 90% of BD patients first presenting with oral ulceration. The aim of this pilot study was twofold; firstly to investigate whether cytokine levels in matched serum and saliva samples show a differential profile in BD (with and without oral ulcers), recurrent aphthous stomatitis (RAS) and healthy controls (HCs), and secondly, to explore if any differential profiles in serum and/or saliva could provide a panel of cytokines with diagnostic and therapeutic potential for BD. Concentrations of 12 cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IFN-γ, TNF-α, TNF-ß) were measured using the Human Th1/Th2 11-Plex FlowCytomix™ kit with IL-17A, in BD (N=20), RAS (N=6) and HCs (N=10). A differential range of cytokines was detected in serum and saliva with the majority of cytokine levels higher in saliva. The most prevalent salivary cytokines were IL-1ß, IL-2, IL-8, IL-10 and TNF-α present in all samples in contrast to serum where the most prevalent cytokine detected was IL-8 (91.9%). The least abundant cytokine was IFN-γ in both saliva (43.2%) and serum (2.7%). After normalizing saliva for protein content, BD patients with oral ulcers (BD-MA) had significantly higher levels of salivary IL-1ß (p=0.01), IL-8 (p=0.02), TNF-α (p=0.004) and IL-6 (p=0.01) than HCs. Notably, BD patients without oral ulcers (BD-MQ) also had significantly higher salivary IL-1ß, IL-8 and TNF-α (p ≤ 0.05) than HCs. During relapsed (BD-RE) and quiet (BD-Q) systemic episodes, salivary IL-ß and TNF-α were also significantly increased with IL-8 significantly higher only in BD-Q (p=0.02). BD oral ulcers signify a potential reactivation of systemic inflammation. Identifying cytokines released during asymptomatic episodes and oral ulceration might lead to targeted drug therapy to prevent recurrent oral ulcers and possible disease relapse. This is the first study to report salivary cytokine levels in BD. The detectable levels suggests cytokine profiling of BD saliva may provide an alternative, less invasive, sensitive procedure for frequent monitoring of disease activity and progression.

Expression of inflammatory biomarkers and growth factors in gingival crevicular fluid at different healing intervals following non-surgical periodontal treatment: A systematic review.

BACKGROUND: Non-surgical periodontal treatment (NSPT) is widely employed for the treatment of periodontal disease and yields significant clinical improvements. Gingival crevicular fluid (GCF) can be used to profile health and disease, and recent technological advances, such as multiplex bead immunoassays, are promising in identifying a wider array of GCF factors with the ultimate aim to predict the treatment response. OBJECTIVE: The aim of this systematic review was to compare the expression of GCF markers using multiplex bead immunoassays before treatment and during early, average, or late healing period, following non-surgical periodontal treatment (NSPT). METHODS: An electronic literature search was conducted by two independent examiners (VK and NC) in MEDLINE, EMBASE, OpenGrey, LILACS, and Cochrane Library up to January 2020. The PICO question formulated was as follows: "In patients with periodontal disease, does the expression of gingival crevicular fluid (GCF) markers detected using multiplex bead immunoassay differ at baseline compared with early (≤30 days), average (6-8 weeks), or late (≥3 months) healing after intervention?" RESULTS: A total of 366 publications were obtained and reviewed for eligibility for inclusion. Of these, 12 publications fulfilled the inclusion criteria and were included in the present review. Data for a total of 31 different GCF markers were extracted and summarized for early, average, or late healing after NSPT. Early healing following NSPT (≤ 30 days) indicated an increase in IL-1ß, TNFα, and IL-10. At the average healing period (6-8 weeks), IL-1ß, IL-1α, IL-6, TNF-α, IFN-γ, GM-CSF, MCP-1, and MIP-1α were all reduced, compared to their respective baseline values. Three months after NSPT, IL-1ß, IL-4, IL-6, IL-10, TNF-α, and IFN-γ were detected at reduced levels, compared to pre-treatment levels. Overall, the changes following treatment indicated a reduction of inflammation present at baseline. CONCLUSION: Following non-surgical periodontal treatment, an upregulation of inflammation markers is noted early post-operatively and a subsequent reduction of their levels three months following treatment. The investigation of levels of GCF markers associated with inflammation and regeneration, especially using multiplex bead immunoassay technologies, is a valuable tool to better understand the processes associated with healing following periodontal treatment.

The seroprevalence and salivary shedding of herpesviruses in Behçet's syndrome and recurrent aphthous stomatitis.

BACKGROUND: Behçet's syndrome (BS) is one of the multisystemic diseases that presents with oral ulceration and several other systemic manifestations including genital ulceration, folliculitis, erythema nodosum-like lesions, uveitis, and arthropathy. Ocular manifestation, central nervous system involvement, and gastrointestinal manifestation account for most of the complications of this disease, whereas orogenital ulceration and dermatological involvement affects the quality of life. The cause of the disease is not fully elucidated; however, herpesviruses have long been thought to play a pivotal role in the disease pathogenesis. OBJECTIVE: To investigate the seroprevalence and salivary shedding of herpesviruses in BS. METHOD: The levels of specific immunoglobulin G in six different herpesviruses in serum samples collected from 54 BS, 28 healthy controls (HC), and 7 recurrent aphthous stomatitis (RAS) patients were investigated. Salivary viral load was also quantified for these viruses in matched saliva samples using quantitative real-time polymerase chain reaction. RESULTS: The BS had lower cytomegalovirus (CMV) IgG level in comparison to HC (p=0.0226) and RAS (p=0.0450). There was statistically significant higher salivary shedding of Epstein-Barr virus (EBV) in BS in comparison to HC (p=0.0052), but not RAS (p=0.3318). CONCLUSIONS: A high EBV shedding was observed in both BS and RAS and a lower level of CMV IgG was observed in BS only. The reason for the observed lower level of CMV IgG in BS is not clear. However, one explanation might be a defect in the cross-talk between innate and adaptive immune responses which was suggested by a previously described defect in the toll-like receptor 1 and 2 heterodimer formation and function, this being the initial receptor sensing of CMV.

Differential potentiation of the virulence of the Pseudomonas aeruginosa cystic fibrosis liverpool epidemic strain by oral commensal Streptococci.

BACKGROUND: The Pseudomonas aeruginosa Liverpool epidemic strain (LES) is an important cystic fibrosis (CF) pathogen and is associated with increased morbidity and a worsened prognosis, compared with other CF-associated strains. However, interactions of common LES phenotypic variants with other members of the polymicrobial biofilms associated with chronic CF respiratory disease, such as oral commensal streptococci, have not been investigated. METHODS: Biofilm population dynamics, virulence factor production, and pathogenicity in Galleria mellonella larvae of common LES phenotypes (ie, low production, intermediate production, and overproduction of pyocyanin) in the presence or absence of anginosus group streptococci (AGS) were compared. RESULTS: AGS populations isolated from biofilm cocultures were P. aeruginosa phenotypic variant dependent, with higher AGS cell densities than those in monoculture frequently observed. Coexistence of AGS with a producer of low or intermediate levels of pyocyanin was found to result in enhancement of virulence factor production. In addition, the LES formed pathogenic partnerships with AGS in the G. mellonella infection model, with killing dependent on LES phenotype and AGS species. CONCLUSIONS: The pathogenic potential of LES phenotypic variants can be enhanced by the presence of oral commensal streptococci. As adaptive mutations leading to reduced virulence factor production are commonplace, the observations made are relevant in the general context of the biology of P. aeruginosa infection during CF.

The role of TLR2 and 4 in Behçet's disease pathogenesis.

TLRs are PRRs that play a pivotal role in sensing exogenous pathogens and endogenous danger signals. Their role in the pathogenesis of inflammatory and immune-related diseases is gradually being unravelled. TLR2 and TLR4 are capable of sensing the oral microbial community, which is considered a potential trigger for Behçet's disease (BD). This study aimed to investigate the expression and function of TLR2 and TLR4 in the oral mucosa of BD. A total of 87 patients was included: 55 BD, 24 healthy controls and eight recurrent aphthous stomatitis. Total RNA was purified from non-lesional oral mucosal brush biopsies and analysed for the presence of TLR2 and TLR4 mRNA, along with their splice variants. The response of peripheral blood mononuclear cells to classical TLR2 and TLR4 agonists was also investigated. TLR2b, TLR2d, TLR2e, TLR4.3 and TLR4.4 were significantly elevated in relapsed BD. A significant defect in the response to cognate agonists of TLR1/2 heterodimer and TLR4 was also observed in BD. The expression of unusual splice variants of TLR2 and TLR4 might explain the observed defect in these receptors' function in BD.

Quantitative analysis of optical coherence tomography and histopathology images of normal and dysplastic oral mucosal tissues.

Selecting the most representative site for biopsy is crucial in establishing a definitive diagnosis of oral epithelial dysplasia. The current process involves clinical examination that can be subjective and prone to sampling errors. The aim of this study was therefore to investigate the use of optical coherence tomography (OCT) for differentiation of normal and dysplastic oral epithelial samples, with a view to developing an objective and reproducible approach for biopsy site selection. Biopsy samples from patients with fibro-epithelial polyps (n = 13), mild dysplasia (n = 2), and moderate/severe dysplasia (n = 4) were scanned at 5-µm intervals using an OCT microscope and subsequently processed and stained with hematoxylin and eosin (H&E). Epithelial differentiation was measured from the rate of change (gradient) of the backscattered light intensity in the OCT signal as a function of depth. This parameter is directly related to the density of optical scattering from the cell nuclei. OCT images of normal oral epithelium showed a clear delineation of the mucosal layers observed in the matching histology. However, OCT images of oral dysplasia did not clearly identify the individual mucosal layers because of the increased density of abnormal cell nuclei, which impeded light penetration. Quantitative analysis on 2D-OCT and histology images differentiated dysplasia from normal control samples. Similar analysis on 3D-OCT datasets resulted in the reclassification of biopsy samples into the normal/mild and moderate/severe groups. Quantitative differentiation of normal and dysplastic lesions using OCT offers a non-invasive objective approach for localizing the most representative site to biopsy, particularly in oral lesions with similar clinical features.

Candida albicans strain-dependent modulation of pro-inflammatory cytokine release by in vitro oral and vaginal mucosal models.

Candida albicans is a commensal organism at several sites and is a versatile, opportunistic pathogen. The underlying factors of pathogen and host associated with commensalism and pathogenicity in C. albicans are complex and their importance is largely unknown. We aimed to study the responses of oral epithelial (OEM) and vaginal epithelial models (VEM) to infection by oral and vaginal C. albicans strains to obtain evidence of inter-strain differences in pathogenicity and of site-specificity. Following inoculation of models, proinflammatory cytokines IL-1α, IL-1ß, IL-6, IL-8 and prostaglandin E2 (PGE2) release were monitored and histological staining undertaken. Striking differences in strain behaviour and epithelial responses were observed. IL-1α, IL-1ß and IL-8 release were significantly increased from the OEM in response to denture stomatitis strain NCYC 1467. Increased IL-8 release also followed infection of the OEM with both vaginal strains. Overall the VEM was relatively unresponsive to infection with either oral or vaginal strains under these conditions. Adherence and hyphal development were observed for all strains on both models although extensive, uniform tissue penetration was seen only with stomatitis strain NCYC 1467 on the OEM. Candidal strains were assayed for phospholipase (PL) and secreted aspartyl proteinase (SAP) activities where phospholipase (PL) activity was highest for strain NCYC 1467 although highest SAP activity was observed for vaginal strain NCPF 8112 in this assay. This is the first study to concurrently investigate cytokine production from oral and epithelial models using candidal strains originating from these respective mucosal sites from healthy and disease states. These data demonstrate significant differences in inflammatory responses of host epithelia to individual C. albicans strains.

Age, gender and disease-related platelet and neutrophil activation ex vivo in whole blood samples from patients with Behçet's disease.

OBJECTIVES: Behçet's disease (BD) is more severe among young males and disease severity decreases with age. Therefore, the effect of disease activity, gender and age on platelet and neutrophil activation in whole blood taken from patients with BD was investigated. METHODS: Using an anti-coagulant Tripotassium ethylenediaminetetra acetic acid (K3EDTA) plus citrate-theophylline-adenosine-dipyridamole (CTAD) (K3EDTA/CTAD) that preserves the degree of platelet activation that exists in vivo, we assessed neutrophil and platelet activation, microparticles, and monocyte and neutrophil-platelet aggregate formation in 43 BD patients using flow cytometry. This is the first description of platelet activation and microparticles in BD patients using this methodology. RESULTS: Inactive [2.78 (0.56)%, P = 0.0009; 3.11 (0.78)%, P < 0.0001] and active [2.28 (0.84)%, P < 0.0001; 3.071 (0.67)%, P = 0.0031] BD patients had significantly higher percentages of CD62P-expressing platelets and CD62P+ platelet microparticles as compared with healthy controls (HCs) [0.84 (0.1)% and 1.23 (0.14)%], respectively. The percentages of CD62P+ platelets and CD62P+ platelet microparticles in female and male BD patients were also significantly higher than those expressed by female and male HCs. The percentages of CD62P+ microparticles were significantly increased in the 20-30-(P = 0.0301) and 31-50-(P < 0.0162) year age ranges, but not in the >50-year age group of BD patients. CONCLUSION: BD is a rare, chronic multi-systemic vasculitis and interaction of activated platelets with leucocytes has been linked to pathological disorders associated with vascular inflammation. Importantly, this study demonstrates that platelet microparticle activation is increased in BD. Also, this is the first report in which changes in platelet activation in BD are concordant with the observations that BD disease activity diminishes with age.

Scattering attenuation microscopy of oral epithelial dysplasia.

We present a new method for quantitative visualization of premalignant oral epithelium called scattering attenuation microscopy (SAM). Using low-coherence interferometry, SAM projects measurements of epithelial optical attenuation onto an image of the tissue surface as a color map. The measured attenuation is dominated by optical scattering that provides a metric of the severity of oral epithelial dysplasia (OED). Scattering is sensitive to the changes in size and distribution of nuclear material that are characteristic of OED, a condition recognized by the occurrence of basal-cell-like features throughout the epithelial depth. SAM measures the axial intensity change of light backscattered from epithelial tissue. Scattering measurements are obtained from sequential axial scans of a 3-D tissue volume and displayed as a 2-D SAM image. A novel segmentation method is used to confine scattering measurement to epithelial tissue. This is applied to oral biopsy samples obtained from 19 patients. Our results show that imaging of tissue scattering can be used to discriminate between different dysplastic severities and furthermore presents a powerful tool for identifying the most representative tissue site for biopsy.

An altered keratinocyte phenotype in oral submucous fibrosis: correlation of keratin K17 expression with disease severity.

Oral submucous fibrosis (OSF) is characterized by abnormal collagen metabolism in the submucosal connective tissue. Its influence on the overlying epithelium is not known but about 14% of OSF cases undergo malignant transformation to squamous cell carcinoma indicating association with abnormality of the epithelium. Here, we have defined the keratin expression profile, by immunohistochemistry and quantitative image analysis, using a panel of 22 anti-keratin monoclonal antibodies on 28 OSF samples. We observed an increase of K1 and K10 in the suprabasal layers, induction of K6 in the basal layer and complete loss of K19 in the epithelium. Furthermore, there was increased K17 expression in the suprabasal layers, which correlated with disease severity. In a subset of the most severe OSF cases (14%), K17 expression was completely lost in the basal layer which might define them to be at most risk to undergo malignant transformation. There was no detectable expression of K8, K18, K7 and K9 and the expression of K4, K13, K14, K15 and K16 did not change in OSF. We propose that the altered keratin profiles could be useful as histological diagnostic markers and provide important insights into the pathogenesis of the disease and its predisposition to malignancy.

Presentation of ICAM-1 protein at the cell surface of oral keratinocytes in the presence of adrenomedullin and corticotrophin.

Increasing evidence suggests that adrenomedullin (AM) and corticotrophin (ACTH) are immunomodulatory. Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the recruitment of leukocytes not only from peripheral blood into inflamed tissues but also into epithelia. We have investigated the effects of AM and ACTH on the expression of ICAM-1 by human oral keratinocytes. The human oral keratinocyte cell line H357 was incubated with either AM or ACTH for up to 8 hrs and ICAM-1 expression was measured by cell surface ELISA. ICAM-1 was up regulated by both peptides and this was attenuated by the adenylyl cyclase inhibitor SQ22,536 and the NF-kappaB inhibitor SN-50. H357 cells constitutively express ICAM-1 mRNA and expression of this gene was significantly modulated by AM and ACTH. Furthermore AM caused translocation of NF-kappaB from the cytoplasm to the nucleus. This is the first report describing up regulation of ICAM-1 in oral keratinocytes by AM and ACTH and the results suggest both cAMP and NF-kappaB may play a role. These results further suggest both peptides may have an immunostimulatory role in oral muocsa and skin.

Adrenomedullin signals through NF-kappaB in epithelial cells.

Adrenomedullin is a peptide found in a variety of cells and tissues and involved in a multitude of biological processes. Recently, adrenomedullin has been identified as a host defense peptide and as such it plays a role in the inflammatory response. The transcription factor NF-kappaB is a major regulator of genes involved in the inflammatory response and the aim of this study was to determine whether NF-kappaB played a role in the inflammatory process triggered by adrenomedullin. Skin epithelial cells (HaCaTs) were used as our model in vitro. Western blot analysis from adrenomedullin-stimulated HaCaT cells revealed a rapid degradation of NF-kappaB inhibitor alpha and beta followed by the translocation of free NF-kappaB to the nucleus, where it was detected by Texas Red immunostaining after incubation with adrenomedullin for 15 min. Electromobility shift assay showed that NF-kappaB present in the nucleus was active, since it bound to a probe containing an NF-kappaB binding site. Supershift assays indicated that p50 and p65, members of the NF-kappaB family, were both part of the NF-kappaB dimmers involved in adrenomedullin cell signaling. HaCaTs secreted interleukin-6 in response to AM, which was significantly attenuated by the NF-kappaB inhibitor SN-50. Taken together, the data lend support for an immunoregulatory role for AM.

Stimulation of adhesion molecule expression in human endothelial cells (HUVEC) by adrenomedullin and corticotrophin.

Adrenomedullin (AM) and corticotrophin (ACTH) are both vasoactive peptides produced by a variety of cell types, including endothelial cells. Although AM and ACTH are considered to be important in the control of blood pressure and the response to stress, respectively, their role in inflammation and the immune response has not been clarified. This study shows, with the use of a cell-based ELISA, that AM and ACTH induce cell surface expression of the adhesion molecules E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVEC). Furthermore, this effect appears to be mediated in part via elevation of cAMP, given that both peptides elevate cAMP, the cell-permeable cAMP analog dibutyryl cAMP is able to mimic induction of all three cell adhesion molecules and the effect of AM and ACTH is inhibited by the adenylyl cyclase inhibitor SQ-22536. These findings demonstrate a role for AM and ACTH in the regulation of the immune and inflammatory response.