Expression levels of novel cytokine IL-32 in periodontitis and its role in the suppression of IL-8 production by human gingival fibroblasts stimulated with Porphyromonas gingivalis

Background:IL-32 was recently found to be elevated in the tissue of rheumatoid arthritis and inflammatory bowel disease. Periodontitis is a chronic inflammatory disease caused by polymicrobial infections that result in soft tissue destruction and alveolar bone loss. Although IL-32 is also thought to be associated with periodontal disease, its expression and possible role in periodontal tissue remain unclear. Therefore, this study investigated the expression patterns of IL-32 in healthy and periodontally diseased gingival tissue. The expression of IL-32 in cultured human gingival fibroblasts (HGF) as well as effects of autocrine IL-32 on IL-8 production from HGF were also examined.Methods:Periodontal tissue was collected from both healthy volunteers and periodontitis patients, and immunofluorescent staining was performed in order to determine the production of IL-32. Using real-time PCR and ELISA, mRNA expression and protein production of IL-32 in HGF, stimulated by Porphyromonas gingivalis (Pg), were also investigated.Results:Contrary to our expectation, the production of IL-32 in the periodontitis patients was significantly lower than in the healthy volunteers. According to immunofluorescent microscopy, positive staining for IL-32 was detected in prickle and basal cell layers in the epithelium as well as fibroblastic cells in connective tissue. Addition of fixed Pg in vitro was found to suppress the otherwise constitutive expression of IL-32 mRNA and protein in HGF. However, recombinant IL-32 in vitro inhibited the expression of IL-8 mRNA by HGF stimulated with Pg. Interestingly, anti-IL-32 neutralizing antibody upregulated the IL-8 mRNA expression in non-stimulated HGF, indicating that constitutive expression of IL-32 in HGF suppressed IL-8 mRNA expression in the absence of bacterial stimulation.Conclusion:These results indicate that IL-32 is constitutively produced by HGF which can be suppressed by Pg and may play a role in the downregulation of inflammatory responses, such as IL-8 production, in periodontal tissue

Efficacy of antibacterial peptides against peptide-resistant mrsa is restored by permeabilization of bacteria membranes

Clinical application of antimicrobial peptides (AMPs), as with conventional antibiotics, may be compromised by the development of bacterial resistance. This study investigated AMP resistance in methicillin resistant Staphylococcus aureus, including aspects related to the resilience of the resistant bacteria toward the peptides, the stability of resistance when selection pressures are removed, and whether resistance can be overcome by using the peptides with other membrane-permeabilising agents. Genotypically variant strains of S. aureus became equally resistant to the antibacterial peptides melittin and bac8c when grown in sub-lethal concentrations. Subculture of a melittin-resistant strain without melittin for 8 days lowered the minimal lethal concentration of the peptide from 170 µg ml-1 to 30 µg ml-1. Growth for 24 h in 12 µg ml-1 melittin restored the MLC to 100 µg ml-1. Flow cytometry analysis of cationic fluorophore binding to melittin-naïve and melittin-resistant bacteria revealed that resistance coincided with decreased binding of cationic molecules, suggesting a reduction in nett negative charge on the membrane. Melittin was haemolytic at low concentrations but the truncated analog of melittin, mel12-26, was confirmed to lack haemolytic activity. Although a previous report found that mel12-26 retained full bactericidal activity, we found it to lack significant activity when added to culture medium. However, electroporation in the presence of 50 µg ml-1 of mel12-26, killed 99.3% of the bacteria. Similarly, using a low concentration of the non-ionic detergent Triton X-100 to permeabilize bacteria to mel12-26 markedly increased its bactericidal activity. The observation that bactericidal activity of the non-membranolytic peptide mel12-26 was enhanced when the bacterial membrane was permeablized by detergents or electroporation, suggests that its principal mechanism in reducing bacterial survival may be through interaction with intracellular organelles or processes. Additionally, our results showed that the haemolytic peptide bac8c, had increased antibacterial activity at non-haemolytic concentrations when used with membrane-permeabilizing surfactants

Diseño de sistema de alcantarillado sanitario y ampliación del sistema de agua potable del Barrio Villa Vallarta en la Ciudad de Managua

El presente estudio monográfico denominado "DISEÑO DEL SISTEMA DE ALCANTARILLADO SANITARIO Y AMPLIACIÓN DEL SISTEMA DE AGUA POTABLE EN EL BARRIO VILLA VALLARTA'', tiene como propósito mejorar las condiciones higiénico-sanitarias de la población, djsminuir el índice de mortalidad infantil y promover cambios de comportamientos en los miembros de las familias beneficiadas, para que mejoren su nivel de vida. EI Barrio Villa Vallarta se encuentra ubicado en la parie Nor-Este de la ciudad de Managua, localizándose en la zona alta del Acueducto de Managua; específicamente está ubicado en el Distrito No. Vl de la municipalidad de Managua. Se caracteriza por ser una zona de alta densidad debido que en dicho Barrio se encuentran construcciones sencillas y lotes con dimensiones y áreas homogéneas, de aproximadamente | 05 m2. Esta conformado por 393 viviendas,13 manzanas, 9 calles y 3 avenidas, alcanzando un área total de aproximadamente 67, 327.81 m2, incluyendo calles, avenidas y un área comunal de 1,575.45 m2 La calles miden aproximadamente 750.45 m2 Para formular el estudio monográfico, primeramente se realizó un diagnóstico situacional a través de ntrevistas y encuesta dirigidas. a funcionarios, líderes comunales y familias del Barrio. Posteriormente se efectuará el estudio topográfico y el estudio de suelo para conocer las características del terreno y la clasificación o tipo de suelo que predomina en el Barrio. Además se realizó la valoración del Sistema de Alcantarillado Sanitario y Sistema de Agua Potable existente

Aesthetic consideration in patient management of severe periodontitis aggravated by oral dexamethasone

Background: The treatment of severe periodontitis must consider immune responses and local conditions, including the aesthetical aspects. The tooth loss in the anterior area can lead to a psychological issue in some patients, and the daily use of dexamethasone contributes greatly to the severity of aesthetical aspects. Periodontal treatments must be cautious of these aesthetical effects. Purpose: To report the aesthetical consideration in patient management of severe periodontitis aggravated by oral dexamethasone. Case: The 44-year-old female patient reported having tooth mobility in the upper right and left central incisor and lower right and left posterior. Due to the condition, the central anterior needed to be extracted. The patient had seafood allergies and consumed oral dexamethasone periodically to prevent allergic reactions for two years. Case management: The initial periodontal therapy was designed prior to the tooth extraction, socket preservation, and immediate denture on teeth 11 and 21. A metal frame combined with an acrylic denture was designed to support the tooth splint and replace the teeth on the mandible. The patient was treated with 20 mg of sub-antimicrobial-dose doxycycline twice a day for three months, and vitamin E was prescribed once a day. Since dexamethasone may contribute to immune response and osteoclastogenesis, dexamethasone was replaced by cetirizine. Conclusion: The treatment of severe chronic periodontitis must consider immune responses, local conditions, and aesthetical aspects. In this case, the use of dexamethasone might worsen the periodontal breakdown. However, the periodontal treatment, use of host modulation therapy, and replacement of dexamethasone with cetirizine are expected to improve these conditions

Understanding the roles of gingival beta-defensins

Gingival epithelium produces β-defensins, small cationic peptides, as part of its contribution to the innate host defense against the bacterial challenge that is constantly present in the oral cavity. Besides their functions in healthy gingival tissues, β-defensins are involved in the initiation and progression, as well as restriction of periodontal tissue destruction, by acting as antimicrobial, chemotactic, and anti-inflammatory agents. In this article, we review the common knowledge about β-defensins, coming from in vivo and in vitro monolayer studies, and present new aspects, based on the experience on three-dimensional organotypic culture models, to the important role of gingival β-defensins in homeostasis of the periodontium

Aggregatibacter actinomycetemcomitans Omp29 Is Associated with Bacterial Entry to Gingival Epithelial Cells by F-Actin Rearrangement

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29+/OmpA− E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29−/OmpA− E. coli. While the entry of Aa and Omp29+/OmpA− E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway

Oral Antimicrobial Peptides and Biological Control of Caries

The presence of antimicrobial peptides (AMPs) in saliva may be a biological factor that contributes to susceptibility or resistance to caries. This manuscript will review AMPs in saliva, consider their antimicrobial and immunomodulatory functions, and evaluate their potential role in the oral cavity for protection of the tooth surface as well as the oral mucosa. These AMPs are made in salivary gland and duct cells and have broad antimicrobial activity. Alpha-defensins and LL37 are also released by neutrophils into the gingival crevicular fluid. Both sources may account for their presence in saliva. A recent study in middle school children aimed to determine a possible correlation between caries prevalence in children and salivary concentrations of the antimicrobial peptides human beta-defensin-3 (hBD-3), the cathelicidin, LL37, and the alpha-defensins. The levels of these AMPs were highly variable in the population. While levels of LL37 and hBD-3 did not correlate with caries experience, the mean alpha-defensin level was significantly higher in children with no caries than in children with caries (p < 0.005). We conclude that several types of AMPs that may have a role in oral health are present in unstimulated saliva. Low salivary levels of alpha-defensin may represent a biological factor that contributes to caries susceptibility. Our observation could lead to new ways to prevent caries and to a new tool for caries risk assessment

Optineurin regulates osteoblastogenesis through STAT1

A sophisticated and delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts regulates bone metabolism. Optineurin (OPTN) is a gene involved in primary open-angle glaucoma and amyotrophic lateral sclerosis. Although its function has been widely studied in ophthalmology and neurology, recent reports have shown its possible involvement in bone metabolism through negative regulation of osteoclast differentiation. However, little is known about the role of OPTN in osteoblast function. Here, we demonstrated that OPTN controls not only osteoclast but also osteoblast differentiation. Different parameters involved in osteoblastogenesis and osteoclastogenesis were assessed in Optn−/- mice. The results showed that osteoblasts from Optn−/- mice had impaired alkaline phosphatase activity, defective mineralized nodules, and inability to support osteoclast differentiation. Moreover, OPTN could bind to signal transducer and activator of transcription 1 (STAT1) and regulate runt-related transcription factor 2 (RUNX2) nuclear localization by modulating STAT1 levels in osteoblasts. These data suggest that OPTN is involved in bone metabolism not only by regulating osteoclast function but also by regulating osteoblast function by mediating RUNX2 nuclear translocation via STAT1

The role of released ATP in killing Candida albicans and other extracellular microbial pathogens by cationic peptides

A unifying theme common to the action of many cationic peptides that display lethal activities against microbial pathogens is their specific action at microbial membranes that results in selective loss of ions and small nucleotides chiefly ATP. One model cationic peptide that induces non-lytic release of ATP from the fungal pathogen Candida albicans is salivary histatin 5 (Hst 5). The major characteristic of Hst 5-induced ATP release is that it occurs rapidly while cells are still metabolically active and have polarized membranes, thus precluding cell lysis as the means of release of ATP. Other cationic peptides that induce selective release of ATP from target microbes are lactoferricin, human neutrophil defensins, bactenecin, and cathelicidin peptides. The role of released extracellular ATP induced by cationic peptides is not known, but localized increases in extracellular ATP concentration may serve to potentiate cell killing, facilitate further peptide uptake, or function as an additional signal to activate the host innate immune system at the site of infection

Fluctuating expression of miR-584 in primary and high-grade gastric cancer

BACKGROUND: Gastric cancer is the fifth most common cancer worldwide. Along with environmental factors, such as Helicobacter pylori (H. pylori) infection, genetic changes play important roles in gastric tumor formations. miR-584 is a less well-characterized microRNA (miRNA), with apparent activity in human cancers. However, miR-584 expression pattern in gastric cancer development has remained unclear. This study aims to analyze the expression of miR-584 in gastric cancer samples and investigates the associations between this miRNA and H. pylori infection and clinical characteristics. METHODS: The expression level of miR-584 was studied in primary gastric cancers versus healthy control gastric mucosa samples using the RT-qPCR method. The clinical data were analyzed statistically in terms of miR-584 expression. In silico studies were employed to study miR-584 more broadly in order to assess its expression and find new potential target genes. RESULTS: Both experimental and in silico studies showed up-regulation of miR-584 in patients with gastric cancer. This up-regulation seems to be induced by H. pylori infection since the infected samples showed increased levels of miR-584 expression. Deeper analyses revealed that miR-584 undergoes a dramatic down-regulation in late stages, invasive and lymph node-metastatic gastric tumors. Bioinformatics studies demonstrated that miR-584 has a substantial role in cancer pathways and has the potential to target STAT1 transcripts. Consistent with the inverse correlation between TCGA RNA-seq data of miR-584 and STAT1 transcripts, the qPCR analysis showed a significant negative correlation between these two RNAs in a set of clinical samples. CONCLUSION: miR-584 undergoes up-regulation in the stage of primary tumor formation; however, becomes down-regulated upon the progression of gastric cancer. These findings suggest the potential of miR-584 as a diagnostic or prognostic biomarker in gastric cancer