Anti-Metastatic Properties of a Marine Bacterial Exopolysaccharide-Based Derivative Designed to Mimic Glycosaminoglycans

Osteosarcoma is the most frequent malignant primary bone tumor characterized by a high potency to form lung metastases. In this study, the effect of three oversulfated low molecular weight marine bacterial exopolysaccharides (OS-EPS) with different molecular weights (4, 8 and 15 kDa) were first evaluated in vitro on human and murine osteosarcoma cell lines. Different biological activities were studied: cell proliferation, cell adhesion and migration, matrix metalloproteinase expression. This in vitro study showed that only the OS-EPS 15 kDa derivative could inhibit the invasiveness of osteosarcoma cells with an inhibition rate close to 90%. Moreover, this derivative was potent to inhibit both migration and invasiveness of osteosarcoma cell lines; had no significant effect on their cell cycle; and increased slightly the expression of MMP-9, and more highly the expression of its physiological specific tissue inhibitor TIMP-1. Then, the in vivo experiments showed that the OS-EPS 15 kDa derivative had no effect on the primary osteosarcoma tumor induced by osteosarcoma cell lines but was very efficient to inhibit the establishment of lung metastases in vivo. These results can help to better understand the mechanisms of GAGs and GAG-like derivatives in the biology of the tumor cells and their interactions with the bone environment to develop new therapeutic strategies.

Effect Of Calendula Officinalis Cream On Achilles Tendon Healing.

In recent years, the scientific community has undertaken research on plant extracts, searching for compounds with pharmacological activities that can be used in diverse fields of medicine. Calendula officinalis L. is known to have antioxidant, anti-inflammatory, antibacterial, and wound healing properties when used to treat skin burns. Therefore, the purpose of this study was to analyze the effects of C. officinalis on the initial phase of Achilles tendon healing. Wistar rats were separated in three groups: Calendula (Cal)-rats with a transected tendon were treated with topical applications of C. officinalis cream and then euthanized 7 days after injury; Control (C)-rats were treated with only vehicle after transection; and Normal (N)-rats without tenotomy. Higher concentrations of hydroxyproline (an indicator of total collagen) and non-collagenous proteins were observed in the Cal group in relation to the C group. Zymography showed no difference in the amount of the isoforms of metalloproteinase-2 and of metalloproteinase-9, between C and Cal groups. Polarization microscopy images analysis showed that the Cal group presented a slightly higher birefringence compared with the C group. In sections of tendons stained with toluidine blue, the transected groups presented higher metachromasy as compared with the N group. Immunocytochemistry analysis for chondroitin-6-sulfate showed no difference between the C and Cal groups. In conclusion, the topical application of C. officinalis after tendon transection increases the concentrations of collagen and non-collagenous proteins, as well as the collagen organization in the initial phase of healing.

Antitumor Activity Of Irradiated Riboflavin On Human Renal Carcinoma Cell Line 786-o.

Riboflavin (vitamin B2) is a precursor for coenzymes involved in energy production, biosynthesis, detoxification, and electron scavenging. Previously, we demonstrated that irradiated riboflavin (IR) has potential antitumoral effects against human leukemia cells (HL60), human prostate cancer cells (PC3), and mouse melanoma cells (B16F10) through a common mechanism that leads to apoptosis. Hence, we here investigated the effect of IR on 786-O cells, a known model cell line for clear cell renal cell carcinoma (CCRCC), which is characterized by high-risk metastasis and chemotherapy resistance. IR also induced cell death in 786-O cells by apoptosis, which was not prevented by antioxidant agents. IR treatment was characterized by downregulation of Fas ligand (TNF superfamily, member 6)/Fas (TNF receptor superfamily member 6) (FasL/Fas) and tumor necrosis factor receptor superfamily, member 1a (TNFR1)/TNFRSF1A-associated via death domain (TRADD)/TNF receptor-associated factor 2 (TRAF) signaling pathways (the extrinsic apoptosis pathway), while the intrinsic apoptotic pathway was upregulated, as observed by an elevated Bcl-2 associated x protein/B-cell CLL/lymphoma 2 (Bax/Bcl-2) ratio, reduced cellular inhibitor of apoptosis 1 (c-IAP1) expression, and increased expression of apoptosis-inducing factor (AIF). The observed cell death was caspase-dependent as proven by caspase 3 activation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. IR-induced cell death was also associated with downregulation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian)/protein serine/threonine kinase B/extracellular signal-regulated protein kinase 1/2 (Src/AKT/ERK1/2) pathway and activation of p38 MAP kinase (p38) and Jun-amino-terminal kinase (JNK). Interestingly, IR treatment leads to inhibition of matrix metalloproteinase-2 (MMP-2) activity and reduced expression of renal cancer aggressiveness markers caveolin-1, low molecular weight phosphotyrosine protein phosphatase (LMWPTP), and kinase insert domain receptor (a type III receptor tyrosine kinase) (VEGFR-2). Together, these results show the potential of IR for treating cancer.

Insights into preventive measures for dental erosion

Dental erosion is defined as the loss of tooth substance by acid exposure not involving bacteria. The etiology of erosion is related to different behavioral, biological and chemical factors. Based on an overview of the current literature, this paper presents a summary of the preventive strategies relevant for patients suffering from dental erosion. Behavioral factors, such as special drinking habits, unhealthy lifestyle factors or occupational acid exposure, might modify the extent of dental erosion. Thus, preventive strategies have to include measures to reduce the frequency and duration of acid exposure as well as adequate oral hygiene measures, as it is known that eroded surfaces are more susceptible to abrasion. Biological factors, such as saliva or acquired pellicle, act protectively against erosive demineralization. Therefore, the production of saliva should be enhanced, especially in patients with hyposalivation or xerostomia. With regard to chemical factors, the modification of acidic solutions with ions, especially calcium, was shown to reduce the demineralization, but the efficacy depends on the other chemical factors, such as the type of acid. To enhance the remineralization of eroded surfaces and to prevent further progression of dental wear, high-concentrated fluoride applications are recommended. Currently, little information is available about the efficacy of other preventive strategies, such as calcium and laser application, as well as the use of matrix metalloproteinase inhibitors. Further studies considering these factors are required. In conclusion, preventive strategies for patients suffering from erosion are mainly obtained from in vitro and in situ studies and include dietary counseling, stimulation of salivary flow, optimization of fluoride regimens, modification of erosive beverages and adequate oral hygiene measures.

Effect of Laser Phototherapy on the Release of TNF-alpha and MMP-1 by Endodontic Sealer-Stimulated Macrophages

Objective: Our aim was to analyze the effect of laser phototherapy on the secretory activity of macrophages activated by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), and stimulated by substances leached from an epoxy resin-based sealer (AH-Plus) and a calcium hydroxide-based sealer (Sealapex). Background Data: Laser phototherapy can modulate the inflammatory process, improving wound healing. This type of therapy could be useful for modulating postoperative symptoms seen after endodontic treatment. Materials and Methods: Cytotoxicity was indirectly assessed by measuring mitochondrial activity. Macrophages were stimulated by the leached substances or not (controls), and the groups were then irradiated or not. The secretion of pro-inflammatory cytokines (TNF-alpha and MMP-1) was analyzed using ELISA. Two irradiations at 6-h intervals were done with an As-Ga-Al diode laser (780 nm, 70 mW, spot size 4.0 mm(2), 3 J/cm(2), for 1.5 sec) in contact mode. Results: The sealers were non-cytotoxic to macrophages. The production of TNF-alpha was significantly decreased by laser phototherapy, regardless of experimental group. The level of secretion of MMP-1 was similar in all groups. Conclusion: Based on the conditions of this study we concluded that in activated macrophages, laser phototherapy impairs the secretion of the pro-inflammatory cytokine TNF-alpha, but has no influence on MMP-1 secretion.

Evaluation of Protective Effect of a Water-In-Oil Microemulsion Incorporating Quercetin Against UVB-Induced Damage in Hairless Mice Skin

Purpose. Histological aspects were considered in order to evaluate the in vivo photoprotective effect of a w/o microemulsion containing quercetin against UVB irradiation-induced dermal damages. The toxicity in cell culture and the potential skin irritation resulting from topical application of this formulation were investigated. Methods. Mouse dorsal surfaces were treated topically with 300 mg of the unloaded and quercetin-loaded (0.3%, w/w) microemulsions before and after exposure to UVB (2.87 J/cm(2)) irradiation. The untreated control groups irradiated and non-irradiated were also evaluated. UVB-induced histopathological changes as well as the photoprotective effect of this formulation were evaluated considering the parameters of infiltration of inflammatory cells, epidermis thickening (basale and spinosum layers) and collagen and elastic fiber contents. The cytotoxicity of the reported formulation was evaluated in L929 mice fibroblasts by MTT assay and the skin irritation was investigated after topical application of both unloaded and quercetin-loaded microemulsions once a day for 15 days. Results. The results demonstrated that the w/o microemulsion containing quercetin reduced the incidence of histological skin alterations, mainly the connective-tissue damage, induced by exposure to UVB irradiation. This suggests that protective effects of this formulation against UV-induced responses are not secondary to the interference of UV transmission (i.e., blocking the UVB radiation from being absorbed by the skin), as is usually implied with UVB absorbers and sunscreens, but is instead due to different biological effects of this flavonoid. Furthermore, by evaluating the cytotoxic effect on L929 cells and histological aspects such as infiltration of inflammatory cells and epidermis thickness of hairless mice, the present study also demonstrated the lack of toxicity of the proposed system. Conclusion. Based on these mice models, a detailed characterization of the w/o microemulsion incorporating quercetin effects as a photochemoprotective agent on human skin is presented.

Stromal cells play a role in cervical cancer progression mediated by MMP-2 protein

Metalloproteinases, especially metal loprotemase-2 (MMP-2), are known for their role in the degradation of the extracellular matrix. Nevertheless, a thorough understanding of MMP-2 expression in neoplastic lesions of the uterine cervix has yet to be accomplished. This study aimed to analyze the MMP-2 expression in cervical intraepithelial neoplasia III (CIN3) and in cervical squamous cell carcinoma, in tumor cells and adjacent stromal cells. MMP-2 expression was assessed by an immunohistochernical technique. MMP-2 expression was greater in the stromal cells of invasive carcinomas than in CIN3 (p < 0.0001). MMP-2 expression in stromal cells correlates with the clinical stage, gradually increasing as the tumor progresses (p = 0.04). This study corroborates that stromal cells play an important role in tumor invasion and progression, mediated by the progressive enhancement of MMP-2 expression from CIN3 to advanced invasive tumor. The intense MMP-2 expression most probably is associated with poor tumor prognosis.

Quercetin inhibits UV irradiation-induced inflammatory cytokine production in primary human keratinocytes by suppressing NF-kappa B pathway

Background: Topical flavonoids, such as quercetin, have been shown to reduce ultraviolet (UV) irradiation-mediated skin damage. However, the mechanisms and signaling pathways involved in this protective effect are not clear. UV irradiation leads to activation of two major signaling pathways, namely nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) pathways. Activation of NF-kappa B pathway by UV irradiation stimulates inflammatory cytokine expression, whereas activation of AP-1 pathway by UV irradiation promotes matrix metalloproteinase (MMP) production. Both pathways contribute to UV irradiation-induced skin damage, such as photoaging and skin tumor formation. Objective: To elucidate the underlying mechanism, we examined the effect of quercetin on UV irradiation induced activation of NF-kappa B and AP-1 pathways. Methods: Primary human keratinocytes, the major skin cell type subjected to physiological solar UV irradiation, were used to study the effects of quercetin on UV irradiation-induced signal transduction pathways. Results: Quercetin decreased UV irradiation-induced NF-kappa B DNA-binding by 80%. Consequently, quercetin suppressed UV irradiation-induced expression of inflammatory cytokines IL-1 beta (similar to 60%), IL-6 (similar to 80%), IL-8 (similar to 76%) and TNF-alpha (similar to 69%). In contrast, quercetin had no effect on UV irradiation activation of three MAP kinases, ERK, JNK, or p38. Accordingly, induction of AP-1 target genes such as MMP-1 and MMP-3 by UV irradiation was not suppressed by quercetin. Conclusion: Our data indicate that the ability of quercetin to block UV irradiation-induced skin inflammation is mediated, at least in part, by its inhibitory effect on NF-kappa B activation and inflammatory cytokine production. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study

Reasons for performing study: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. Objectives: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. Methods: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. Results: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. Conclusions: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. Potential relevance: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.

Aseptic Loosening of Total Hip Arthroplasty: Preliminary Genetic Investigation

Femoral and acetabular loosening call be attributed different factors, but the Causes and mechanism of early failure are still obscure, The objective of this Study was to investigate the relationship between gene polymorphisms and early implant failure. Fifty-eight patients older than 50 years was recruited for analysis of MMP-1 promoter polymorphisms in early osseointegrated implant failure. The results showed in control group a frequency of 20.97% of 2G allele and 67.74% the genotype 1G/1G whereas, in the test group, a frequency of 83.33% of 2G allele and 66.66% the genotype 2G/2G. These results indicate that the polymorphism ill the promoter of the MMP-1 gene could be it risk factor for early implant failure of total hip arthroplasty.

Down-regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin-10 knockout mice

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.

Quercetin restores plasma nitrite and nitroso species levels in renovascular hypertension

Quercetin has antioxidants properties which may increase nitric oxide (NO) bioavailability. However, the effects of quercetin on NO status have been poorly studied. We evaluated whether quercetin improves the plasma levels of NO metabolites in two-kidney one-clip (2K1C) hypertensive rats and assessed its effect on endothelial function. Sham-operated and 2K1C rats were treated with quercetin (10 mg(-1) kg(-1) day(-1) by gavage) or vehicle for 3 weeks. Systolic blood pressure (SBP) was monitored weekly. Vascular responses to acetylcholine (Ach) and sodium nitroprusside (SNP) were assessed in hindquarter vascular bed. Plasma nitrate levels were assessed by Griess reagent and plasma nitrite and nitroso species (S, N-nitroso species) were assessed by ozone- based chemiluminescence. Aortic NADPH oxidase activity and superoxide production were evaluated. While quercetin had no effects in control normotensive rats (P > 0.05), it significantly reduced SBP in 2K1C rats (P < 0.05). At the end of treatment, plasma nitrate levels were similar in all experimental groups (P > 0.05). However, plasma nitrite and the nitroso species levels were significantly lower in 2K1C rats when compared with controls (P < 0.05). Quercetin treatment restored plasma nitrite and nitroso species levels to those found in the sham-vehicle group (P < 0.05). While quercetin treatment induced no significant changes in responses to SNP (P > 0.05), it restored the vascular responses to Ach. Quercetin significantly attenuated 2K1C-hypertension-induced increases in NADPH oxidase activity and vascular superoxide production (P < 0.05). These results suggest that the antihypertensive effects of quercetin were associated with increased NO formation and improved endothelial function, which probably result from its antioxidant effects.

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta Aims: Thoracic ascending aortic aneurysms (TAA) are characterized by elastic fibre breakdown and cystic medial degeneration within the aortic media, associated with progressive smooth muscle cell (SMC) rarefaction. The transforming growth factor (TGF)-beta/Smad2 signalling pathway is involved in this process. Because the pericellular fibrinolytic system activation is able to degrade adhesive proteins, activate matrix metalloproteinase (MMP), induce SMC disappearance and increase the bioavailability of TGF-beta, the aim was to investigate the plasminergic system in TAA. Methods and results: Ascending aortas [21 controls and 19 TAAs (of three different aetiologies)] were analysed. Immunohistochemistry showed accumulation of t-PA, u-PA and plasmin in TAAs, associated with residual SMCs. Overexpression of t-PA and u-PA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and zymography on TAA extracts and culture medium conditioned by TAA. Plasminogen was present on the SMC surface and inside cytoplasmic vesicles, but plasminogen mRNA was undetectable in the TAA medial layer. Plasmin-antiplasmin complexes were detected in TAA-conditioned medium and activation of the fibrinolytic system was associated with increased fibronectin turnover. Fibronectin-related material was detected immunohistochamically in dense clumps around SMCs and colocalized with latent TGF-beta binding protein-1. Conclusions: The fibrinolytic pathway could play a critical role in TAA progression, via direct or indirect impact on ECM and consecutive modulation of TGF-beta bioavailability.

Cysteine Cathepsins in Human Dentin-Pulp Complex

Introduction: Collagen-degrading matrix metalloproteinases (MMPs) are expressed by odontoblasts and present in dentin. We hypothesized that odontoblasts express other collagen-degrading enzymes such as cysteine cathepsins, and their activity would be present in dentin, because odontoblasts are known to express at least cathepsin D. Effect of transforming growth factor beta (TGF-beta) on cathepsin expression was also analyzed. Methods: Human odontoblasts and pulp tissue were cultured with and without TGF-beta, and cathepsin gene expression was analyzed with DNA microarrays. Dentin cathepsin and MMP activities were analyzed by degradation of respective specific fluorogenic substrates. Results: Both odontoblasts and pulp tissue demonstrated a wide range of cysteine cathepsin expression that gave minor responses to TGF-beta. Cathepsin and MMP activities were observed in all dentin samples, with significant negative correlations in their activities with tooth age. Conclusions: These results demonstrate for the first time the presence of cysteine cathepsins in dentin and suggest their role, along with MMPs, in dentin modification with aging. (J Endod 2010;36:475-481)

Effect of supplementation of soft drinks with green tea extract on their erosive potential against dentine

Background: Matrix metalloproteinase (MMP) inhibitors reduce dentine erosion. This in vitro study evaluated the effect of the supplementation of soft drinks with green tea extract, a natural inhibitor of MMPs, on their erosive potential against dentine. Methods: For each drink tested (Coca-Cola (TM), Kuat (TM) guarana, Sprite (TM) and light Coca-Cola (TM)), 40 dentine specimens were divided into two subgroups differing with respect to supplementation with green tea extract at 1.2% (OM24 (R), 100% Camellia sinensis leaf extract, containing 30 +/- 3% of catechin; Omnimedica, Switzerland) or not (control). Specimens were subjected to four pH cycles, alternating de-and remineralization in one day. For each cycle, samples were immersed in pure or supplemented drink (10 minutes, 30 mL per block) and in artificial saliva (60 minutes, 30 mL per block) at 37 degrees C, under agitation. Dentine alterations were determined by profilometry (mu m). Data were analysed by two-way ANOVA and Bonferroni`s test (p < 0.05). Results: A significant difference was observed among the drinks tested with Sprite (TM) leading to the highest surface loss and light Coca-Cola (TM) to the lowest. Supplementation with green tea extract reduced the surface loss by 15% to 40% but the difference was significant for Coca-Cola (TM) only. Conclusions: Supplementation of soft drinks with green tea extract might be a viable alternative to reduce their erosive potential against dentine.