Active matrix metalloproteinase-8 (aMMP-8) point-of-care test (POCT) in the COVID-19 pandemic.

INTRODUCTION: Active matrix metalloproteinase (aMMP)-8 utilized in point-of-care testing (POCT) is regarded as a potential biomarker for periodontal and peri-implant diseases. Various host and microbial factors eventually influence the expression, degranulation, levels and activation of aMMP-8. The type of oral fluids (saliva, mouthrinse, gingival crevicular, and peri-implant sulcular fluids [GCF/PISF], respectively) affect the analysis. AREAS COVERED: With this background, we aimed to review here the recent studies on practical, inexpensive, noninvasive and quantitative mouthrinse and GCF/PISF chair-side POCT lateral flow aMMP-8 immunoassays (PerioSafe and ImplantSafe/ORALyzer) and how they help to detect, predict, monitor the course, treatment and prevention of periodontitis and peri-implantitis. The correlations of aMMP-8 POCT to other independent and catalytic activity assays of MMP-8 are also addressed. EXPERT OPINION: The mouthrinse aMMP-8 POCT can also detect prediabetes/diabetes and tissue destructive oral side-effects due to the head and neck cancers' radiotherapy. Chlorhexidine and doxycycline can inhibit collagenolytic human neutrophil and GCF aMMP-8. Furthermore, by a set of case-series we demonstrate the potential of mouthrinse aMMP-8 POCT to real-time/online detect periodontitis as a potential risk disease for coronavirus disease 2019 (COVID-19). The clinical interdisciplinary utilization of aMMP-8 POCT requires additional oral, medical, and interdisciplinary studies.

Periodontal therapeutics: Current host-modulation agents and future directions.

With the recognition in the 1960s and 1970s of the periodontopathic importance of the microbial biofilm and its specific anaerobic microorganisms, periodontitis was treated as an infectious disease (more recently, as a dysbiosis). Subsequently, in the 1980s, host-response mechanisms were identified as the mediators of the destruction of the collagen-rich periodontal tissues (gingiva, periodontal ligament, alveolar bone), and the periodontopathogens were now regarded as the "trigger" of the inflammatory/collagenolytic response that characterizes actively destructive periodontitis. Also at this time a new pharmacologic strategy emerged, entitled "host-modulation therapy", based on 2 major findings: (1) that the ability of tetracycline antibiotics to inhibit periodontal breakdown was due (in large part) to their previously unrecognized ability to inhibit the host-derived matrix metalloproteinases (notably, the collagenases, gelatinases, macrophage metalloelastase), and by mechanisms unrelated to the antimicrobial properties of these medications; and (2) that nonsteroidal anti-inflammatory drugs, such as flurbiprofen, again by nonantimicrobial mechanisms, could reduce the severity of periodontitis (however, the adverse effects of long-term therapy precluded their development as safe and effective host-modulatory agents). Additional mechanistic studies resulted in the development of novel nonantimicrobial formulations (Periostat® [now generic] and Oracea®) and compositions of tetracyclines (notably chemically modified tetracycline-3) as host-modulator drugs for periodontitis, arthritis, cardiovascular and pulmonary diseases, cancer, and, more recently, for local and systemic bone loss in postmenopausal women. Identification of the cation-binding active site in the tetraphenolic chemically modified tetracycline molecules drove the development of a new category of matrix metalloproteinase-inhibitor compounds, with a similar active site, the biphenolic chemically modified curcumins. A lead compound, chemically modified curcumin 2.24, has demonstrated safety and efficacy in vitro, in cell culture, and in vivo in mouse, rat, rabbit, and dog models of disease. In conclusion, novel host-modulation compounds have shown significant promise as adjuncts to traditional local therapy in the clinical management of periodontal disease; appear to reduce systemic complications of this all-too-common "inflammatory/collagenolytic" disease; and Oracea® is now commonly prescribed for inflammatory dermatologic diseases.

Loss of alveolar bone density in postmenopausal, osteopenic women is associated with circulating levels of gelatinases.

OBJECTIVE: To determine whether circulating levels of two matrix metalloproteinases, MMP-2 and MMP-9, are associated with loss of alveolar bone density (ABD) or height (ABH), or with progression of periodontitis (relative clinical attachment level [RCAL]), among postmenopausal women with local and systemic bone loss. BACKGROUND: This study was planned as part of a 2-year randomized, double-blind, placebo-controlled, clinical trial examining efficacy/safety of subantimicrobial dose doxycycline (20 mg bid) in postmenopausal osteopenic women. This study examines whether serum levels of gelatinases are associated with local changes in the periodontium. METHODS: A sample of 113 women received periodontal maintenance for moderate to advanced chronic periodontitis and consented to analysis of stored serum biomarkers. Posterior vertical bitewings were taken, and serum collected, at baseline, one, and 2 years. ABD was determined by computer-assisted densitometric image analysis (CADIA), ABH by the Hausmann et al (1992, J Periodontol 63, 657) method, and RCAL by Florida Probe (every 6 months). MMPs were measured densitometrically on gelatin zymograms using denatured type I collagen as substrate and purified MMP-2 (72 kDa) and MMP-9 (92 kDa) as standards. Evidence of worsening in the periodontium at a tooth site was defined as a change from baseline of, for ABD, at least 14 densitometric units (for subcrestal locations) or 17 units (for crestal locations); of at least 0.4 mm for ABH; and of at least 1.5 mm for RCAL. Logistic regression models, while accounting for clustering, compared the odds of worsening in ABD, ABH, or RCAL, after 2 years of observation, between groups defined by baseline and concurrent levels of serum gelatinases. RESULTS: Changes in ABH and RCAL were not associated with circulating levels of MMP-2 or MMP-9. However, elevated odds of ABD loss over 24 months were associated, among smokers, with both baseline and concurrent levels of MMP-9 in the middle and highest tertile, and with concurrent levels of MMP-2 in the middle (but not the highest) tertile. Elevated odds of ABD loss were also associated, among women within 5 years of menopause, with baseline levels of MMP-2 in the highest tertile. CONCLUSION: Among postmenopausal osteopenic women, loss of ABD was associated, in smokers, with elevated circulating levels of MMP-9 and MMP-2. In those within 5 years of menopause, ABD loss was associated with elevated circulating levels of MMP-2.

A novel chemically modified curcumin reduces severity of experimental periodontal disease in rats: initial observations.

Tetracycline-based matrix metalloproteinase- (MMP-) inhibitors are currently approved for two inflammatory diseases, periodontitis and rosacea. The current study addresses the therapeutic potential of a novel pleiotropic MMP-inhibitor not based on an antibiotic. To induce experimental periodontitis, endotoxin (LPS) was repeatedly injected into the gingiva of rats on one side of the maxilla; the contralateral (control) side received saline injections. Two groups of rats were treated by daily oral intubation with a chemically modified curcumin, CMC 2.24, for two weeks; the control groups received vehicle alone. After sacrifice, gingiva, blood, and maxilla were collected, the jaws were defleshed, and periodontal (alveolar) bone loss was quantified morphometrically and by µ-CT scan. The gingivae were pooled per experimental group, extracted, and analyzed for MMPs (gelatin zymography; western blot) and for cytokines (e.g., IL-1ß; ELISA); serum and plasma samples were analyzed for cytokines and MMP-8. The LPS-induced pathologically excessive bone loss was reduced to normal levels based on either morphometric (P = 0.003) or µ-CT (P = 0.008) analysis. A similar response was seen for MMPs and cytokines in the gingiva and blood. This initial study, on a novel triketonic zinc-binding CMC, indicates potential efficacy on inflammatory mediators and alveolar bone loss in experimental periodontitis and warrants future therapeutic and pharmacokinetic investigations.

Subantimicrobial-dose doxycycline treatment increases serum cholesterol efflux capacity from macrophages.

OBJECTIVE: Subantimicrobial-dose doxycycline (SDD) treatment has been reported to reduce the severity of chronic inflammation and to increase serum high-density lipoprotein cholesterol. In a double-blind, placebo-controlled clinical trial, we determined whether SDD affects the ability of serum to facilitate cholesterol removal from macrophages. METHODS: Forty-five postmenopausal osteopenic women with periodontitis were randomly assigned to take placebo (n = 26) or doxycycline hyclate (20 mg, n = 19) tablets twice daily for 2 years. Serum samples were collected at baseline, 1-, and 2-year appointments. The cholesterol efflux capacity of serum from cultured human macrophages (THP-1) was measured. RESULTS: SDD subjects demonstrated a significant increase in serum-mediated cholesterol efflux from macrophages at both time points compared to baseline (p < 0.04 for each). Mean cholesterol efflux levels over the first year of follow-up were 3.0 percentage points (unit change) higher among SDD subjects compared to placebo subjects (p = 0.010), while there was no significant difference in 2-year changes. There were no significant differences in the changes of apolipoprotein A-I, apolipoprotein A-II, or serum amyloid A levels between the groups. CONCLUSIONS: Our results suggest that SDD treatment may reduce the risk of cardiovascular disease in this patient group by increasing the cholesterol efflux capacity of serum.

4-methoxycarbonyl curcumin: a unique inhibitor of both inflammatory mediators and periodontal inflammation.

Chronic inflammatory diseases such as periodontitis have been associated with increased risk for various medical conditions including diabetes and cardiovascular disease. Endotoxin (lipopolysaccharide, LPS), derived from gram-negative periodonto-pathogens, can induce the local accumulation of mononuclear cells in the inflammatory lesion, increasing proinflammatory cytokines and matrix metalloproteinases (MMPs). This ultimately results in the destruction of periodontal connective tissues including alveolar bone. Curcumin is the principal dyestuff in the popular Indian spice turmeric and has significant regulatory effects on inflammatory mediators but is characterized by poor solubility and low bioactivity. Recently, we developed a series of chemically modified curcumins (CMCs) with increased solubility and zinc-binding activity, while retaining, or further enhancing, their therapeutic effects. In the current study, we demonstrate that a novel CMC (CMC 2.5: 4-methoxycarbonyl curcumin) has significant inhibitory effects, better than the parent compound curcumin, on proinflammatory cytokines and MMPs in in vitro, in cell culture, and in an animal model of periodontal inflammation. The therapeutic potential of CMC 2.5 and its congeners may help to prevent tissue damage during various chronic inflammatory diseases including periodontitis and may reduce the risks of systemic diseases associated with this local disorder.

Effect of Commercial Children's Mouthrinses and Toothpastes on the Viability of Neonatal Human Melanocytes: An In Vitro Study.

In this study, we examined the cytotoxic effects of six commercial children's mouthrinses (designated as #1, #2, #3, #4, #5, and #6) and four commercial children's toothpastes (designated as #1, #2, #3, and #4) on primary human neonatal melanocytes that were used as a representative model for oral melanocytes. Mouthrinses diluted directly with culture medium (1:2, 1:5, 1:10, 1:100, and 1:1000) were added to monolayers of melanocytes for 2 min, followed by 24 h recovery, after which MTS cytotoxicity assay was conducted. The extracts of each toothpaste were prepared (50% w/v), diluted in culture medium (1:2, 1:5, 1:10, 1:50, 1:100, and 1:1000), and added to cell monolayers for 2 min (standard brushing time), followed by an analysis of cell viability after 24 h. Results showed that all mouthrinses except mouthrinse #4 showed significantly greater loss of cell viability, ascribed to cetylpyridinium chloride (CPC) that induced significant cytotoxicity to melanocytes (IC50 = 54.33 µM). In the case of toothpastes, the examination of cellular morphology showed that a 2 min exposure to all toothpaste extracts induced a concentration-dependent decline in cell viability, pronounced in toothpaste containing sodium lauryl sulfate (SLS) detergent. Further results suggested SLS to be the critical driver of cytotoxicity (IC50 = 317.73 µM). It is noteworthy that toothpaste #1 exhibited much lower levels of cytotoxicity compared to the other three toothpastes containing SLS. Taken together, these findings suggest that the melanocytotoxicity of children's mouthrinse (#4) and toothpaste (#1) is comparatively low. To the best of our knowledge, this is the first study to examine the impact of children's toothpastes and mouthrinses on neonatal primary human melanocytes. Future studies to investigate these findings in a realistic scenario replicating oral cavity conditions of the presence of microbiota, pellicle layer and saliva, and other cell types are warranted.

A novel modified-curcumin 2.24 resolves inflammation by promoting M2 macrophage polarization.

To assess resolving-like activity by a novel chemically-modified curcumin (CMC2.24) in a "two-hit" model of diabetes-associated periodontitis. Macrophages from rats were cultured in the presence/absence of either Lipopolysaccharide (LPS, 1st hit); or advanced-glycation-end products (AGE, 2nd hit); or both combined. CMC2.24 was added as treatment. The conditioned media were analyzed for MMP-9, cytokines (IL-1ß, IL-6, TNF-α), resolvins (RvD1, RvE1, lipoxin A4), and soluble receptor for AGE (sRAGE). The phenotypes of M1/M2 macrophage were analyzed by flow cytometry. Both LPS/AGE-alone, and two-combined, dramatically increased the secretion of MMP-9 by macrophages. CMC2.24 "normalized" the elevated levels of MMP-9 under all conditions. Moreover, CMC2.24 significantly reduced the secretion of IL-1ß and IL-6 with a fewer effects on TNF-α. Importantly, CMC2.24 increased RvD1 and sRAGE secretion by macrophages exposed to LPS/AGE; and both treatment groups exhibited increased M2 relative to M1 populations. Furthermore, scatter-diagram showed the macrophages gradually shifted from M1 towards M2 with CMC2.24-treated, whereas LPS/AGE-alone groups remained unchanged. CMC2.24 "normalized" cytokines and MMP-9, but also enhanced RvD1 and sRAGE in macrophages. Crucially, CMC2.24 appears to be a potent inhibitor of the pro-inflammatory M1 phenotype; and a promotor of the pro-resolving M2 phenotype, thus acting like a crucial "switch" to reduce inflammation.

Efficacy of a Novel Pleiotropic MMP-Inhibitor, CMC2.24, in a Long-Term Diabetes Rat Model with Severe Hyperglycemia-Induced Oral Bone Loss.

Purpose: CMC2.24, a novel 4-(phenylaminocarbonyl)-chemically-modified-curcumin, is a pleiotropic MMP-Inhibitor of various inflammatory/collagenolytic diseases including periodontitis. This compound has demonstrated efficacy in host modulation therapy along with improved resolution of inflammation in various study models. The objective of current study is to determine the efficacy of CMC2.24 in reducing the severity of diabetes, and its long-term role as an MMP-inhibitor, in a rat model. Methods: Twenty-one adult male Sprague-Dawley rats were randomly distributed into three groups: Normal (N), Diabetic (D) and Diabetic+CMC2.24 (D+2.24). All three groups were orally administered vehicle: carboxymethylcellulose alone (N, D), or CMC2.24 (D+2.24; 30mg/kg/day). Blood was collected at 2-months and 4-months' time-point. At completion, gingival tissue and peritoneal washes were collected/analyzed, and jaws examined for alveolar bone loss by micro-CT. Additionally, sodium hypochlorite(NaClO)-activation of human-recombinant (rh) MMP-9 and its inhibition by treatment with 10µM CMC2.24, Doxycycline, and Curcumin were evaluated. Results: CMC2.24 significantly reduced the levels of lower-molecular-weight active-MMP-9 in plasma. Similar trend of reduced active-MMP-9 was also observed in cell-free peritoneal and pooled gingival extracts. Thus, treatment substantially decreased conversion of pro- to actively destructive proteinase. Normalization of the pro-inflammatory cytokine (IL-1ß, resolvin-RvD1), and diabetes-induced osteoporosis was observed in presence of CMCM2.24. CMC2.24 also exhibited significant anti-oxidant activity by inhibiting the activation of MMP-9 to a lower-molecular-weight (82kDa) pathologically active form. All these systemic and local effects were observed in the absence of reduction in severity of hyperglycemia. Conclusion: CMC2.24 reduced activation of pathologic active-MMP-9, normalized diabetic osteoporosis, and promoted resolution of inflammation but had no effect on the hyperglycemia in diabetic rats. This study also highlights the role of MMP-9 as an early/sensitive biomarker in the absence of change in any other biochemical parameter. CMC2.24 also inhibited significant activation of pro-MMP-9 by NaOCl (oxidant) adding to known mechanisms by which this compound treats collagenolytic/inflammatory diseases including periodontitis.

SAGE: Novel Therapy to Reduce Inflammation in a Naturally Occurring-Dog Model of Periodontal Disease.

Objective: To determine the effect of a semi-synthetic-glycosaminoglycan Ether (SAGE) as a universal therapeutic benefit to reduce periodontal inflammation and alveolar bone loss in naturally occurring-beagle-dog model of periodontal disease as a surrogate for human non-risk associated natural periodontitis. Methods: Six adult female dogs with generalized periodontitis were distributed into two groups: control and SAGE treatment (n=3/group). After a 1-hour full-mouth scaling and root planning (SRP) at baseline, control or SAGE treatment (50mg/mL) bioadhesive gel formulation was locally applied for 12 weeks. Various clinical periodontal measurements (probing depth, CAL) were measured at different time periods (baseline, 4, 8 and 12 weeks), and gingival crevicular fluid (GCF), blood samples and gingival tissue biopsies (12 week) were analyzed for inflammatory mediators, collagenases and cell-signaling molecules. Standardized radiographs were taken at baseline and 12week period. Results: SAGE treatment significantly reduced gingival inflammation (GCF flow), pocket depth (PD), and clinical attachment loss (CAL) compared to control. SAGE also considerably reduced alveolar bone loss and reduced MMP-9, IL-6, CRP levels in gingival tissue, GCF and plasma. Cell-signaling molecules in the inflammatory cascade system TLR-2, TLR-4, p38 MAPK, ERK1/2 and NF-kB responded to SAGE in a pattern consistent with reductions at the active phase of the inflammatory process and collagenolysis. Conclusion: In the beagle dog model of periodontitis, local SAGE administration significantly attenuated clinical measures of periodontitis, pro-inflammatory cytokines, MMPs, and signal transduction molecules. All our studies, using in vitro and in vivo models, support the therapeutic potential of SAGE as an innovative adjunct to SRP in the treatment of chronic periodontal disease.

The Maximum-Tolerated Dose and Pharmacokinetics of a Novel Chemically Modified Curcumin in Rats.

PURPOSE: CMC 2.24, a chemically modified curcumin, was developed as a novel, pleiotropic MMP-inhibitor to treat various inflammatory/collagenolytic diseases including periodontitis. To date, this compound has shown efficacy in vitro, in cell culture, and in vivo (oral administration) in mice, rats and dogs. In preparation for possible Phase I human clinical trials, the current study describes the maximum-tolerated-dose (MTD), pharmacokinetics (PK), and toxicology of CMC 2.24 in the rat model. METHODS: For the MTD study, 30 Sprague-Dawley rats were randomly distributed into 5 groups (3M/3F per group): Placebo (vehicle; carboxymethylcellulose) and CMC 2.24 at various doses (50, 100, 500, 1000 mg/kg/day), were administered once daily by oral gavage for 5 days. For the PK study, 24 rats were administered either Placebo or CMC 2.24 (100mg/kg/day) once daily for 28 days or only once (500 or 1000 mg/kg). Analysis of this test compound was done using LC/MS/MS for PK evaluation on blood samples drawn from rats at multiple time points. The animals were sacrificed after 5 or 28 days of treatment, and blood chemistry and serology were analyzed. Major organs (heart, lung, liver, kidney, spleen, intestine, brain) were histologically examined at necropsy. RESULTS: Orally administered, CMC 2.24 did not produce significant changes in body weight, food consumption or adverse events in the MTD and toxicology studies. Moreover, no obvious pathologic changes were observed based on histology, hematology, serum biochemistry, or necropsy compared to placebo-treated controls. The PK study demonstrated a peak-blood concentration (Cmax) at 45 mins after oral administration of 2.24 and a serum half-life of 10 hours. CONCLUSION: In conclusion, CMC 2.24, orally administered to rats once a day, appears to be safe and effective at a wide range of doses, consistent with efficacy previously demonstrated in studies on animal models of various collagenolytic diseases, such as periodontitis, diabetes and cancer.

Chemically modified tetracycline-3 (CMT-3): a novel inhibitor of the serine proteinase, elastase.

Two classes of enzymes play an important role in connective tissue breakdown during various inflammatory diseases: serine proteinases and matrix metalloproteinases (MMPs). Tetracyclines (TCs) exhibit important anti-inflammatory and MMP-inhibitory properties that are unrelated to their antibacterial activities. Of the various TCs and their chemically modified NON-antibiotic analogs (CMTs) tested in vitro and in vivo, CMT-3 (6-demethyl-6-deoxy 4 de-dimethylamino tetracycline) has repeatedly been shown to be the most potent inhibitor of MMP activity and cytokine production. In addition to its anti-MMP function, we have shown that among all CMTs, CMT-3 is the only CMT that can also directly inhibit both the amidolytic activity of human leukocyte elastase (HLE, a serine proteinase) and the extracellular matrix degradation mediated by HLE. In addition, CMT-3 has been found to reduce leukocyte elastase activity in vivo in gingival extracts of rats with experimental periodontal disease. Thus, CMT-3 can inhibit pathologic connective tissue breakdown by (at least) two mechanisms: direct inhibition of neutral proteinases (elastase and MMPs); and protecting their endogenous inhibitors, α(1)-PI and TIMPs, from being digested and inactivated by MMPs and HLE, respectively. The pleiotropic properties of CMT-3 including (but not limited to) inhibition of serine proteinases, MMPs, and cytokines provide impressive therapeutic potential to reduce excessive connective tissue breakdown during various pathologic processes including inflammatory diseases, cancer metastasis and metabolic bone diseases.

Non-antibacterial tetracyclines modulate mediators of periodontitis and atherosclerotic cardiovascular disease: a mechanistic link between local and systemic inflammation.

Periodontitis, one of the most common chronic inflammatory diseases afflicting man, is increasingly being recognized as a risk factor for atherosclerotic cardiovascular disease (ASCVD). Non-antimicrobial tetracyclines are known to have inhibitory effects on inflammatory mediators and effector molecules, including cytokines and matrix metalloproteinases (MMPs), associated with both diseases. In this paper, we discuss the evidence that doxycycline and related non-antibiotic chemically modified tetracyclines (e.g., CMT-3) can effectively reduce cytokine (TNF-α, IL-6, and MCP-1) production by human mononuclear inflammatory cells when stimulated either by endotoxin (LPS) or by a complex of C-reactive protein/oxidized LDL cholesterol relevant to the pathogenesis of periodontal disease and ASCVD, respectively. This inhibition by tetracycline compounds appears to be mediated at least in part by a suppression of the phosphorylation/activation of the NFκB cell signaling pathway. We are currently conducting clinical trials on patients who exhibit both diseases, and our preliminary data suggest that virtually all acute coronary syndrome (ACS) patients exhibit moderate-to-severe periodontitis, a higher incidence of this oral inflammatory disease than that seen in the population at large. In other studies, a non-antimicrobial formulation of doxycycline (SDD) has been found to dramatically reduce hsCRP, IL-6 and MMP-9 levels in plasma of ACS patients, and SDD has also been found to significantly increase serum levels of both cardio-protective HDL cholesterol and its core molecule apolipoprotein A-I in ASCVD-vulnerable patients with periodontitis. Our current research suggests that one mechanism involved may be the ability of SDD to inhibit MMP-mediated HDL loss by protecting apolipoprotein A-I from proteinase attack. These pleiotropic mechanisms of non-antimicrobial tetracyclines provide significant therapeutic potential to treat chronic inflammatory diseases including both periodontitis and ASCVD.

Therapeutic potential of a novel semi-synthetic-sulfated-polysaccharide to suppress inflammatory mediators in P. gingivalis LPS stimulated human monocytes/macrophages.

BACKGROUND: Chronic periodontitis is associated with an increased risk for systemic conditions such as cardiovascular disease, diabetes, and osteoporosis. During chronic periodontitis, endotoxin (lipopolysaccharide, LPS) produced by P. gingivalis provokes monocyte accumulation and differentiation into macrophages and increased secretion of pro-inflammatory cytokines and matrix metalloproteases (MMPs). While normal levels of MMPs are important in cellular function, increased levels of cytokines and MMPs can cause connective tissue destruction. RESULTS: In the current study, we investigated the therapeutic capability of a novel semi-synthetic sulfated polysaccharide (SAGE) on the production of cytokines and MMPs by cultured human mononuclear cells and macrophages stimulated with endotoxin LPS produced by P. gingivalis, a periodontally-relevant cell culture model. Our research demonstrated SAGE inhibited the LPS induced synthesis of inflammatory mediators including TNF-α, IL-1ß, PGE2, and MMP-9 in this periodontal-relevant cell culture model. In addition, TLR-2 and TLR-4 levels were also reduced with the SAGE treatment. CONCLUSIONS: The therapeutic potential of this novel semi-synthetic sulfated polysaccharide compound may help to prevent tissue damage and bone loss in patients with periodontal disease or other inflammatory diseases.

A Novel Modified-Curcumin Promotes Resolvin-Like Activity and Reduces Bone Loss in Diabetes-Induced Experimental Periodontitis.

PURPOSE: Clinically, it is challenging to manage diabetic patients with periodontitis. Biochemically, both involve a wide range of inflammatory/collagenolytic conditions which exacerbate each other in a "bi-directional manner." However, standard treatments for this type of periodontitis rely on reducing the bacterial burden and less on controlling hyper-inflammation/excessive-collagenolysis. Thus, there is a crucial need for new therapeutic strategies to modulate this excessive host response and to promote enhanced resolution of inflammation. The aim of the current study is to evaluate the impact of a novel chemically-modified curcumin 2.24 (CMC2.24) on host inflammatory response in diabetic rats. METHODS: Type I diabetes was induced by streptozotocin injection; periodontal breakdown then results as a complication of uncontrolled hyperglycemia. Non-diabetic rats served as controls. CMC2.24, or the vehicle-alone, was administered by oral gavage daily for 3 weeks to the diabetics. Micro-CT was used to analyze morphometric changes and quantify bone loss. MMPs were analyzed by gelatin zymography. Cell function was examined by cell migration assay, and cytokines and resolvins were measured by ELISA. RESULTS: In this severe inflammatory disease model, administration of the pleiotropic CMC2.24 was found to normalize the excessive accumulation and impaired chemotactic activity of macrophages in peritoneal exudates, significantly decrease MMP-9 and pro-inflammatory cytokines to near normal levels, and markedly increase resolvin D1 (RvD1) levels in the thioglycolate-elicited peritoneal exudates (tPE). Similar effects on MMPs and RvD1 were observed in the non-elicited resident peritoneal washes (rPW). Regarding clinical relevance, CMC2.24 significantly inhibited the loss of alveolar bone height, volume and mineral density (ie, diabetes-induced periodontitis and osteoporosis). CONCLUSION: In conclusion, treating hyperglycemic diabetic rats with CMC2.24 (a tri-ketonic phenylaminocarbonyl curcumin) promotes the resolution of local and systemic inflammation, reduces bone loss, in addition to suppressing collagenolytic MMPs and pro-inflammatory cytokines, suggesting a novel therapeutic strategy for treating periodontitis complicated by other chronic diseases.

Suppressive effects of formoterol and salmeterol on eotaxin-1 in bronchial epithelial cells.

Eotaxin-1 (CCL11), an eosinophil-specific C-C chemokine, is a potent chemoattractant for mobilization of eosinophils into airways after allergic stimulation. Eotaxin-1 recruits eosinophils into inflammatory sites, and may play a role in the pathogenesis of asthma. Formoterol and salmeterol are two inhaled long acting beta(2) adrenoceptor agonists (LABAs), widely used for the local treatment of asthma. However, little is known about their effects on the eotaxin-1 expression of bronchial epithelial cells. BEAS-2B cells were stimulated by adding IL-4 with or without 2 h pre-treatment of formoterol or salmeterol. The protein and mRNA expression of eotaxin-1 were measured by ELISA assay and real-time PCR, respectively. Effects of formoterol and salmeterol on nuclear and cytosolic pSTAT-6 expression were evaluated by Western blot and immunofluorescence study. Formoterol and salmeterol (10(-7)-10(-10) m) significantly down-regulated IL-4- induced eotaxin-1 expression in BEAS-2B cells. A specific beta(2) adrenoceptor antagonist (ICI 118,551) reversed their suppression of eotaxin-1 production. Forskolin, an cAMP activator, could also suppress the expression of eotaxin-1 by IL-4 in a dose dependent manner (10(-7)-10(-10 )m). The western blot and immunofluorescence studies demonstrated that formoterol 10(-7 )m suppressed the nuclear expression of pSTAT-6. Formoterol and salmeterol, two inhaled long-acting beta(2) agonists, down-regulated IL-4- induced eotaxin-1 expression in BEAS-2B cells. The effect was mediated via the beta(2) adrenoceptor, and cAMP. Formoterol significantly down-regulated pSTAT6 at higher concentration, and further turned off the IL-4 signaling pathway.

Chemically-Modified Curcumin 2.24: A Novel Systemic Therapy for Natural Periodontitis in Dogs.

PURPOSE: To determine the effect of a pleiotropic MMP-inhibitor, a novel chemically-modified curcumin 2.24 (CMC2.24), on the clinical and biological measures of naturally-occurring periodontitis in the beagle dog. METHODS: Eight adult female dogs with generalized periodontitis were distributed into two groups: Placebo and Treatment (n=4/group). After a 1-hr full-mouth scaling and root planing (SRP) at time 0, placebo or CMC2.24 (10mg/kg) capsules were orally administered once/day for 3 months. Various clinical periodontal parameters (e.g., pocket depth, gingival index) were measured at different time periods (0, 1, 2 and 3 months), and gingival crevicular fluid (GCF) samples and gingival tissue biopsies (3-month) were analyzed for cytokines, MMPs and cell-signaling molecules. Standardized radiographs were taken at 0 and 3-month; in addition, peripheral blood monocytes/macrophages from these dogs at 3-month were cultured and analyzed for the pro-, activated-, and total-forms of both MMP-2 and MMP-9. RESULTS: CMC2.24 treatment significantly reduced gingival inflammation (gingival index, GCF flow), pocket depth (PD), and the numbers of pockets (PD≥4mm), compared to placebo. CMC2.24 also significantly reduced MMP-9 and MMP-2 (primarily in the activated-form) in gingival tissue, alveolar bone loss, and reduced GCF IL-1ß. Cell-signaling molecules, TLR-2 (but not TLR-4) and p38 MAPK, responded to CMC2.24 in a pattern consistent with reductions in inflammation and collagenolysis. In culture, CMC2.24 had no effect on pro-MMP-9 but essentially completely blocked the conversion of pro- to activated-MMP-9 in systemic blood-derived monocytes/macrophages from these dogs. CONCLUSION: In the beagle dog model of natural periodontitis, orally administered CMC2.24 (a novel triketonic phenylaminocarbonyl-curcumin) significantly decreased clinical measures of periodontitis as well as pro-inflammatory cytokines, MMPs, and cell-signaling molecules. These and previous studies, using other in vitro and in vivo models, support the clinical potential of CMC2.24 as a novel adjunct to SRP in the treatment of chronic periodontitis.

Osteoimmunology of Oral and Maxillofacial Diseases: Translational Applications Based on Biological Mechanisms.

The maxillofacial skeleton is highly dynamic and requires a constant equilibrium between the bone resorption and bone formation. The field of osteoimmunology explores the interactions between bone metabolism and the immune response, providing a context to study the complex cellular and molecular networks involved in oro-maxillofacial osteolytic diseases. In this review, we present a framework for understanding the potential mechanisms underlying the immuno-pathobiology in etiologically-diverse diseases that affect the oral and maxillofacial region and share bone destruction as their common clinical outcome. These otherwise different pathologies share similar inflammatory pathways mediated by central cellular players, such as macrophages, T and B cells, that promote the differentiation and activation of osteoclasts, ineffective or insufficient bone apposition by osteoblasts, and the continuous production of osteoclastogenic signals by immune and local stromal cells. We also present the potential translational applications of this knowledge based on the biological mechanisms involved in the inflammation-induced bone destruction. Such applications can be the development of immune-based therapies that promote bone healing/regeneration, the identification of host-derived inflammatory/collagenolytic biomarkers as diagnostics tools, the assessment of links between oral and systemic diseases; and the characterization of genetic polymorphisms in immune or bone-related genes that will help diagnosis of susceptible individuals.

Enhanced efficacy of chemically modified curcumin in experimental periodontitis: systemic implications.

INTRODUCTION: Dental microbial biofilm initiates gingival inflammation, and its suppression is the current dominant strategy for treating periodontitis. However, the host response to the biofilm is largely responsible for the connective tissue breakdown including alveolar bone loss, which is mediated by proinflammatory cytokines and matrix metalloproteinases (MMPs). METHODS: The current study compared the efficacy of a novel host-modulation compound, a chemically modified curcumin (CMC 2.24), to that of its parent compound (natural curcumin), in both lipopolysaccharide (LPS) (a bacterial endotoxin)-induced cell culture and in vivo models of periodontitis. RESULTS: In cell culture, both CMC 2.24 and curcumin appeared similarly effective in suppressing LPS-induced cytokine (IL-1ß and TNF-α) secretion by mononuclear inflammatory cells; however, CMC 2.24 significantly reduced MMP-9 secretion by 78% (P<0.05) whereas curcumin was ineffective. In vivo, CMC 2.24 administration was more effective than curcumin in suppressing (a) IL-1ß in gingival tissue and (b) MMP-9 in both gingiva and plasma, the latter indicating a reduced severity of systemic inflammation. The difference in primary clinical outcome between the two treatments was that CMC 2.24 reduced the pathologically excessive alveolar bone loss, assessed morphometrically at multiple sites, by 80%-90% (P<0.01), whereas curcumin, surprisingly, either increased (P<0.05) or had no effect on alveolar bone loss at these sites. CONCLUSION: These data, plus that from previous studies, support the therapeutic potential of CMC 2.24 in the management of inflammatory periodontal disease and its ability to reduce the risk of associated systemic diseases. The current study also indicates that the MMP-9 inhibitor efficacy is associated with the ability of CMC 2.24 (but not curcumin) to inhibit alveolar bone loss in this rat model of periodontitis.

Subantimicrobial-dose doxycycline modulates gingival crevicular fluid biomarkers of periodontitis in postmenopausal osteopenic women.

BACKGROUND: We recently demonstrated that a 2-year subantimicrobial-dose doxycycline (SDD) regimen (double-masked, placebo-controlled clinical trial) in postmenopausal (PM) women exhibiting mild systemic bone loss (osteopenia) and local bone loss (periodontitis) reduced the progression of periodontal attachment loss (intent-to-treat analysis) and the severity of gingival inflammation and alveolar bone loss (subgroups) without producing antibiotic side effects. We now describe SDD effects on biomarkers of collagen degradation and bone resorption in the gingival crevicular fluid (GCF) of the same vulnerable subjects. METHODS: GCF was collected from SDD- and placebo-treated PM subjects (n=64 each) at the baseline and 1- and 2-year appointments; the volume was determined; and the samples were analyzed for collagenase activity (using a synthetic peptide as substrate), relative levels of three genetically distinct collagenases (Western blot), a type-1 collagen breakdown product/bone resorption marker (a carboxyterminal telopeptide cross-link fragment of type I collagen [ICTP]; radioimmunoassay), and interleukin-1beta (enzyme-linked immunosorbent assay). Statistical analyses were performed using generalized estimating equations; primary analyses were intent-to-treat. RESULTS: Collagenase activity was significantly reduced by SDD treatment relative to placebo based on intent-to-treat (P=0.01). ICTP showed a similar pattern of change during SDD treatment, and GCF collagenase activity and ICTP were positively correlated at all time periods (P<0.001). Matrix metalloproteinase (MMP)-8 accounted for approximately 80% of total collagenase in GCF, with much less MMP-1 and -13, and SDD reduced the odds of elevated MMP-8 by 60% compared to placebo (P=0.006). CONCLUSION: These observations support the therapeutic potential of long-term SDD therapy to reduce periodontal collagen breakdown and alveolar bone resorption in PM women; effects on serum biomarkers of systemic bone loss in these subjects are being analyzed.