The in vitro effect of VEGF receptor inhibition on primary alveolar osteoblast nodule formation.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a master regulator and is required for the effective coupling of angiogenesis and osteogenesis supporting both skeletal development and postnatal bone repair. A direct role for VEGF in intramembranous-derived osteoblast growth and differentiation is not clear. We investigated the expression of primary alveolar osteoblast VEGF receptors and the subsequent effects on mineralization and nodule formation in vitro following VEGFR inhibition. METHODS: Primary human alveolar osteoblasts (HAOBs) were cultured in the presence of VEGF receptor inhibitors, exogenous VEGF or the bisphosphonate, zoledronic acid. VEGF, VEGFR1 and VEGFR2 mRNA expression and nodule formation following 21 days of culture. VEGFR1 protein expression was examined using immunofluorescence after 48 h. RESULTS: The HAOBs expressed high levels of VEGF and VEGFR1 protein but VEGFR2 was not detected. The VEGFR1/2 inhibitors, ZM306416 and KRN633, lead to a dose-dependent decrease in mineralization. Treatment with zoledronic acid showed no difference in HAOB VEGF receptor expression. CONCLUSION: VEGF/VEGFR1 pathway appears to be important for intramembranous-derived osteoblast differentiation and maturation in vitro.

Nonsteroidal anti-inflammatory drugs reduce the time to recovery of gut function after elective colorectal surgery: a systematic review and meta-analysis.

AIM: Postoperative ileus causes significant patient morbidity after abdominal surgery. Some evidence suggests nonsteroidal anti-inflammatory drugs (NSAIDs) may reduce time to gut recovery, but there has not been a meta-analysis to assess their efficacy. This systematic review and meta-analysis aimed to determine the benefit of NSAIDs for recovery of postoperative gut function in patients undergoing elective colorectal surgery. METHOD: MEDLINE, EMBASE, CENTRAL and reference lists were searched with no date or language restrictions. Randomized controlled trials comparing the use of NSAIDs with placebo in the perioperative or postoperative period were identified. Included studies reported outcomes relevant to gut function: time to pass flatus or stool and time to tolerate an oral diet. The mean difference in time from surgery until passage of flatus, stool and tolerance of diet were meta-analysed using a random-effects model in RevMan 5.3. RESULTS: This study identified 992 relevant articles. Five randomized controlled trials on patients undergoing elective colorectal surgery met our inclusion criteria and were meta-analysed. Compared with placebo, NSAIDs significantly improved the time to pass flatus (mean difference -9.44 h, 95% CI: -17.22, -1.65, I2 = 70%, P = 0.02), time to pass stool (mean difference -12.09 h, 95% CI: -17.16, -7.02, I2 = 0%, P < 0.001) and time to tolerate a diet (mean difference -11.95 h, 95% CI: -18.66, -5.24, I2 = 0%, P < 0.001). CONCLUSION: NSAIDs significantly improve time to gut recovery after elective colorectal surgery. Current evidence is not adequate to identify whether selective or nonselective drugs should be recommended. Further high-power studies using selective drugs are required.

Expression of IL33 and IL35 in oral lichen planus.

Oral lichen planus (OLP) is a complex immunological disorder, mediated in part by the release of cytokines by activated T-cells. Recently, the role of novel cytokines including IL33 and IL35 has been described in various chronic inflammatory diseases. IL33, a member of the IL-1 superfamily of cytokines, functions as an 'alarmin' released after cell necrosis to alert the immune system to tissue damage or stress. IL35, a member of IL12 cytokine family, is produced by regulatory T-cells and suppresses the immune response. The expression of IL33 and IL35 is yet to be investigated in OLP. The aim of this study was to determine the presence and topographical distribution of IL33 and IL35 in OLP using immunohistochemistry and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). For IHC, formalin-fixed paraffin-embedded archival specimens of OLP (n = 10) and a non-specific inflammatory (NSI) control group (n = 9) were used. A double-labelling immunofluorescence technique was used to determine the expression of IL33 and IL35 on CD3+ T-cells. In addition, 12 fresh tissue samples (OLP n = 6 and NSI controls n = 6) were used to determine the gene expression of IL33 and EBI3 (one chain of the dimeric IL35). Quantitative and qualitative analysis was performed with statistical significance set at p < 0.05. IHC showed positive immunostaining with IL33 and IL35 in both OLP and NSI. Comparison of the numbers of IL33+ and IL35+ cells in OLP and NSI did not show any significant difference. In OLP, there were significantly more IL33+ cells in the deeper connective tissue region than at the epithelial-connective tissue interface. Interestingly, all IL35+ cells observed in both OLP and NSI tissues showed ovoid/plasmacytoid morphology. Double-labelling immunofluorescence showed that IL33 and IL35 expression was not localized within CD3+ T-cells. The gene expression experiments showed significantly higher expression of EBI3 (fold regulation 14.02) in OLP when compared to the inflammatory controls. IL33 gene expression was not different between the groups. However, within the OLP tissues, there was a significantly higher expression of IL33 than EBI3. Our data demonstrate the expression of IL33 and IL35 in OLP lesions. Further studies are needed to understand the functional role of these cytokines in OLP pathogenesis.

FoxP3+ regulatory T cells, interleukin 17 and mast cells in chronic inflammatory periodontal disease.

BACKGROUND AND OBJECTIVE: T cells are known to play a pivotal role in periodontal disease; however, less is known about the T-helper subsets of regulatory T cells (Tregs) and Th17 cells. The aim of this study was to investigate the cell types expressing FoxP3 and interleukin (IL)-17A within periodontal disease tissues and to determine gene and protein expression profiles associated with periodontitis. MATERIAL AND METHODS: A total of 10 healthy/gingivitis and 10 chronic periodontitis tissues were investigated. Immunohistochemistry and immunofluorescence techniques were used to identify the FoxP3 and IL17-positive cells and to determine the cell types respectively. Gene expression was determined using semi-quantitative polymerase chain reaction array technology that allowed the analysis of 84 pathway-focused genes known to be associated with Tregs and Th17 cells. Transforming growth factor (TGF)-ß1, IL10 and IL17A protein levels were determined using enzyme-linked immunosorbent assay. RESULTS: Double immunofluorescence labeling revealed that all FoxP3+ cells were CD4+ , while IL17+ cells were neither CD4+ nor CD8+ but were tryptase+ , suggestive of mast cells. More FoxP3+ cells than IL17+ cells were found in all the tissues examined and overall there were few IL17+ cells. Statistically significant increases in gene expression were found for STAT5A, STAT3, SOCS1, TGFß1 and IL10 in the chronic periodontitis specimens predominantly infiltrated with B cells and plasma cells when compared with healthy/gingivitis specimens predominantly infiltrated with T cells. Protein analysis demonstrated higher levels of the TGFß1 and IL10 cytokines in periodontitis tissues and in B-cell and plasma cell predominant gingival tissues than in healthy/gingivitis tissues and T-cell predominant gingival tissues. IL17A gene and protein expression was not detected in any of the tissues. CONCLUSION: Based on the findings of this study, we suggest that the source of low levels of IL17A in periodontal tissues is mast cells not Th17 cells and that Tregs may have a more prominent role in the pathogenesis of periodontal disease than Th17 cells.

Feasibility of the salivary transcriptome as a novel biomarker in determining disease susceptibility.

BACKGROUND AND OBJECTIVE: The salivary transcriptome may present as a readily available and non-invasive source of potential biomarkers. The development of chronic periodontitis is determined by individual patient susceptibility; hence, the aim of this study was to determine the potential of the salivary transcriptome as a biomarker of disease susceptibility using chronic periodontitis as an example. MATERIAL AND METHODS: Using an Oragene® RNA kit, the total RNA was purified from the saliva of 10 patients with chronic periodontitis and 10 patients without chronic periodontitis. The quantity and quality of the total RNA was determined, and a measure of gene expression via cDNA was undertaken using the Affymetrix microarray system. The microarray profiling result was further validated by real-time quantitative polymerase chain reaction. RESULTS: Spectrophotometric analysis showed the total RNA purified from each participant ranged from 0.92 µg/500 µL to 62.85 µg/500 µL. There was great variability in the quantity of total RNA obtained from the 2 groups in the study with a mean of 10.21 ± 12.71 µg/500 µL for the periodontitis group and 15.97 ± 23.47 µg/500 µL for the control group. Further the RNA purity (based on the A260 /A280 ratio) for the majority of participants (9 periodontitis and 6 controls) were within the acceptable limits for downstream analysis (2.0 ± 0.1). The study samples, showed 2 distinct bands at 23S (3800 bp) and 16S (1500 bp) characteristic of bacterial rRNA. Preliminary microarray analysis was performed for 4 samples (P2, P6, H5 and H9). The percentage of genes present in each of the 4 samples was not consistent with about 1.8%-18.7% of genes being detected. Quantitative real-time polymerase chain reaction confirmed that the total RNA purified from each sample was mainly bacterial RNA (Uni 16S) with minimal human mRNA. CONCLUSION: This study showed that minimal amounts of human RNA were able to be isolated from the saliva of patients with periodontitis as well as controls. Further work is required to enhance the extraction process of human mRNA from saliva if the salivary transcriptome is to be used in determining individual patient susceptibility.

Effects of environmental tobacco smoke on the oral health of preschool children.

AIMS: This study investigated the association between the prevalence of oral health problems (caries, gingivitis, mucosal pigmentation and enamel defects in one to 5 year-old children exposed and not exposed to environmental tobacco smoke before and/or after birth. Exposure to environmental tobacco smoke (ETS) in childhood may have significant health effects. METHODS: A structured questionnaire was used to collect data on a child's current and previous illnesses, oral health behaviours, dietary habits, parental smoking behaviours and parents' dental history. The intraoral examination recorded dental caries (dmfs), enamel defects, gingival health, melanin pigmentation and soft tissue health. Stimulated saliva was collected. Total sIgA levels were quantified using indirect competitive ELISA with a SalimetricsTM kit. RESULTS: The 44 children (aged 15-69 months) recruited were divided into two groups: ETS and non-ETS (control). There were 22 children in each: 16 who were exposed to ETS during and after gestation were identified as the ETSB subgroup. Participants exposed to ETS were more likely to have had upper respiratory tract and middle ear infections during the neonatal period and had higher mean dmft, mean dmfs, mean percent of surfaces with demarcated opacities and mean GI than the non-ETS participants. The children exposed to ETS before and after birth had the highest occurrence of enamel opacities showed a higher risk for dental caries even though more children in this group used the recommended fluoride toothpaste (1000 ppm fluoride). Mothers who smoked either never breastfed their children or breastfed their children for less than the recommended period of 6 months. Children exposed to ETS were shown to have higher mean total sIgA (µg/ml) than the children in the control group. CONCLUSIONS: Associations between ETS exposure before and after gestation and oral health, including salivary changes in young children were shown in the present study. Dental health professionals should include a question about household smoking in children's dental histories, which would allow opportunities to discuss the impact of smoking on child oral health. Longitudinal oral health studies should include a history of maternal smoking during pregnancy and afterwards.

Limited evidence of abnormal intra-colonic pressure profiles in diverticular disease - a systematic review.

AIM: Abnormal colonic pressure profiles and high intraluminal pressures are postulated to contribute to the formation of sigmoid colon diverticulosis and the pathophysiology of diverticular disease. This study aimed to review evidence for abnormal colonic pressure profiles in diverticulosis. METHOD: All published studies investigating colonic pressure in patients with diverticulosis were searched in three databases (Medline, Embase, Scopus). No language restrictions were applied. Any manometry studies in which patients with diverticulosis were compared with controls were included. The Newcastle-Ottawa Quality Assessment Scale (NOS) for case-control studies was used as a measure of risk of bias. A cut-off of five or more points on the NOS (fair quality in terms of risk of bias) was chosen for inclusion in the meta-analysis. RESULTS: Ten studies (published 1962-2005) met the inclusion criteria. The studies followed a wide variety of protocols and all used low-resolution manometry (sensor spacing range 7.5-15 cm). Six studies compared intra-sigmoid pressure, with five of six showing higher pressure in diverticulosis vs controls, but only two reached statistical significance. A meta-analysis was not performed as only two studies were above the cut-off and these did not have comparable outcomes. CONCLUSION: This systematic review of manometry data shows that evidence for abnormal pressure in the sigmoid colon in patients with diverticulosis is weak. Existing studies utilized inconsistent methodology, showed heterogeneous results and are of limited quality. Higher quality studies using modern manometric techniques and standardized reporting methods are needed to clarify the role of colonic pressure in diverticulosis.

Regulatory T-cells and IL17A(+) cells infiltrate oral lichen planus lesions.

Oral lichen planus (OLP) is a complex immunological disorder, mediated in part by the release of cytokines from activated T-cells. Of late, two closely related T-helper (Th) cell subsets; regulatory T-cells (Tregs; FoxP3(+)) and Th17 cells (IL17(+)) have been described in various chronic inflammatory diseases. The aim of this study was to determine the expression of FoxP3 and IL17 in OLP using immunohistochemistry (IHC) and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). For IHC, formalin fixed, paraffin embedded archival specimens, an OLP group (n=10) and a non-specific inflammatory (NSI) control group (n=9) were used. In addition, 12 fresh tissue samples were used to determine gene expression of FoxP3 and IL17. Significantly more FoxP3(+) cells were present in OLP than in NSI. IL17(+) cells were significantly more frequent in the control tissues than in OLP. The gene expression experiments revealed a significantly higher expression of FoxP3 in OLP when compared to the controls. IL17 gene expression was not different between the groups. Double labelling immunofluorescence indicated co-localisation of IL17 with tryptase(+) mast cells. These findings suggest FoxP3(+) Tregs have a more prominent role in the pathogenesis of OLP when compared to IL17(+)cells.

Development of the Awareness of Cultural Safety Scale: A pilot study with midwifery and nursing academics.

BACKGROUND: Rates of academic success of Indigenous students compared to other students continues to be significantly lower in many first world countries. Professional development activities for academics can be used to promote teaching, learning and support approaches that value Indigenous worldviews. However, there are few valid and reliable tools that measure the effect of academic development strategies on awareness of cultural safety. OBJECTIVES: To develop and validate a self-report tool that aims to measure nursing and midwifery academics' awareness of cultural safety. METHODS: This study followed a staged model for tool development. This included: generation of items, content validity testing and expert Indigenous cultural review, administration of items to a convenience sample of academics, and psychometric testing. An online survey consisting of demographic questions, Awareness of Cultural Safety Scale (ACSS), and awareness of racism items was completed by academics undertaking a professional development program on cultural safety. FINDINGS: Ratings by experts revealed good content validity with an index score of 0.86. The 12-item scale demonstrated good internal reliability (Cronbach's alpha of 0.87). An evaluation of construct validity through factor analysis generated three factors with sound internal reliability: Factor 1 (Cultural Application, Cronbach's alpha=.85), Factor 2 (Cultural Support, Cronbach's alpha=.70) and Factor 3 (Cultural Acknowledgement, Cronbach's alpha=.85). The mean total scale score was 46.85 (SD 7.05, range 31-59 out of a possible 60). There was a significant correlation between scores on the Awareness of Cultural Safety Scale and awareness of racism scores (r=.461, p=.002). CONCLUSION: Awareness of cultural safety is underpinned by principles of respect, relationships, and responsibility. Results indicated the ACSS was valid and reliable. Completion of the scale aimed to foster purposeful consideration by nursing and midwifery academics about their perceptions and approaches to teaching in order to improve Indigenous student success.

Culturally capable and culturally safe: Caseload care for Indigenous women by Indigenous midwifery students.

BACKGROUND: Evidence is emerging of the benefits to students of providing continuity of midwifery care as a learning strategy in midwifery education, however little is known about the value of this strategy for midwifery students. AIM: To explore Indigenous students' perceptions of providing continuity of midwifery care to Indigenous women whilst undertaking a Bachelor of Midwifery. METHODS: Indigenous Bachelor of Midwifery students' experiences of providing continuity of midwifery care to Indigenous childbearing women were explored within an Indigenous research approach using a narrative inquiry framework. Participants were three Indigenous midwifery students who provided continuity of care to Indigenous women. FINDINGS: Three interconnected themes; facilitating connection, being connected, and journeying with the woman. These themes contribute to the overarching finding that the experience of providing continuity of care for Indigenous women creates a sense of personal affirmation, purpose and a validation of cultural identity in Indigenous students. DISCUSSION AND CONCLUSIONS: Midwifery philosophy aligns strongly with the Indigenous health philosophy and this provides a learning platform for Indigenous student midwives. Privileging Indigenous culture within midwifery education programs assists students develop a sense of purpose and affirms them in their emerging professional role and within their community. The findings from this study illustrate the demand for, and pertinence of, continuity of care midwifery experiences with Indigenous women as fundamental to increasing the Indigenous midwifery workforce in Australia. Australian universities should provide this experience for Indigenous student midwives.

Vitamin D supplementation during pregnancy and infancy reduces aeroallergen sensitization: a randomized controlled trial.

BACKGROUND: Vitamin D has immune-modulating effects. We determined whether vitamin D supplementation during pregnancy and infancy prevents aeroallergen sensitization and primary care respiratory illness presentations. METHODS: A randomized, double-blind, placebo-controlled parallel-group trial. We assigned pregnant women, from 27-week gestation to birth, and then their infants, from birth to 6 months, to placebo or one of two dosages of daily oral vitamin D. Woman/infant pairs were randomized to: placebo/placebo, 1000 IU/400 IU or 2000 IU/800 IU. When the children were 18 months old, we measured serum-specific IgE antibodies and identified acute primary care visits described by the doctor to be due to a cold, otitis media, an upper respiratory infection, croup, asthma, bronchitis, bronchiolitis, a wheezy lower respiratory infection or fever and cough. RESULTS: Specific IgE was measured on 185 of 260 (71%) enrolled children. The proportion of children sensitized differed by study group for four mite antigens: Dermatophagoides farinae (Der-f1, Der-f2) and Dermatophagoides pteronyssinus (Der-p1, Der-p2). With results presented for placebo, lower dose, and higher dose vitamin D, respectively (all P < 0.05): Der-f1 (18%, 10%, 2%), Der-f2 (14%, 3%, 2%), Der-p1 (19%, 14%, 3%) and Der-p2 (12%, 2%, 3%). There were study group differences in the proportion of children with primary care visits described by the doctor as being for asthma (11%, 0%, 4%, P = 0.002), but not for the other respiratory diagnoses. CONCLUSIONS: Vitamin D supplementation during pregnancy and infancy reduces the proportion of children sensitized to mites at age 18 months. Preliminary data indicate a possible effect on primary care visits where asthma is diagnosed.

Effects of zoledronic acid and geranylgeraniol on the cellular behaviour and gene expression of primary human alveolar osteoblasts.

INTRODUCTION: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious complication of bisphosphonate therapy. The mechanism underlying BRONJ pathogenesis is poorly understood. OBJECTIVES: To determine the effects of zoledronic acid (ZA) and geranylgeraniol (GGOH) on the mevalonate pathway (MVP) in osteoblasts generated from the human mandibular alveolar bone in terms of cell viability/proliferation, migration, apoptosis and gene expression. MATERIALS AND METHODS: Primary human osteoblasts (HOBs) isolated from the mandibular alveolar bone were phenotyped. HOBs were cultured with or without ZA and GGOH for up to 72 h. Cellular behaviour was examined using a CellTiter-Blue® viability assay, an Ibidi culture-insert migration assay, an Apo-ONE® Homogeneous Caspase-3/7 apoptosis assay and transmission electron microscopy (TEM). Quantitative real-time reverse transcriptase polymerase chain reaction (qRT2-PCR) was used to determine the simultaneous expression of 168 osteogenic and angiogenic genes modulated in the presence of ZA and GGOH. RESULTS: ZA decreased cell viability and migration and induced apoptosis in HOBs. TEM revealed signs of apoptosis in ZA-treated HOBs. However, the co-addition of GGOH ameliorated the effect of ZA and partially restored the cells to the control state. Twenty-eight genes in the osteogenic array and 27 genes in the angiogenic array were significantly regulated in the presence of ZA compared with those in the controls at one or more time points. CONCLUSION: The cytotoxic effect of ZA on HOBs and its reversal by the addition of GGOH suggests that the effect of ZA on HOBs is mediated via the MVP. CLINICAL RELEVANCE: The results suggest that GGOH could be used as a possible therapeutic/preventive strategy for BRONJ.

Periodontopathogen levels following the use of an Er:YAG laser in the treatment of chronic periodontitis.

BACKGROUND: Inflammatory periodontal diseases are initiated by microbial biofilms. The reduction of the biofilm is important in the management of the disease. This study compares periodontopathogen levels following the treatment of chronic periodontitis using Er:YAG laser (ERL) debridement and mechanical scaling and root planing (SRP). METHODS: Using a split-mouth design, two quadrants were randomly allocated for treatment. Two hundred and fifty-two subgingival plaque samples were collected from 21 patients, before treatment (baseline) and at 6 and 12 weeks post-therapy. Multiplex qPCR was used to determine relative levels of Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythensis (Tf), and Aggregatibacter actinomycetemcomitans (Aa). RESULTS: Tf and Pg were significantly reduced post-treatment for both ERL and SRP. ERL treatment resulted in a reduction of Td at 12 weeks. Following SRP treatment Aa was significantly reduced at 12 weeks. No statistically significant difference was seen when treatments were compared at 6 and 12 weeks. CONCLUSIONS: A comparable reduction in the level of the four periodontal pathogens assayed was achieved with Er:YAG laser debridement and mechanical scaling and root planing.

Integrated systematic review on educational strategies that promote academic success and resilience in undergraduate indigenous students.

BACKGROUND: Despite numerous recommendations by governments, researchers, and education policymakers the recruitment, retention and success of undergraduate indigenous students in higher education is not commensurate of the wider student population. There is minimal evidence of valuing indigenous worldviews and perspectives in curricula, and effectiveness of educational strategies to strengthen indigenous student success rates in completing undergraduate studies. OBJECTIVES: To conduct an integrative systematic review of educational strategies to promote academic success and resilience in undergraduate indigenous students. METHODS: Major databases of Scopus, ProQuest, Informit and Web of Science were searched. Inclusion criteria were peer reviewed research articles from scholarly journals that referenced indigenous, aboriginal, First Nation or Maori students in undergraduate programs in higher education. The search was limited to English language and studies conducted from 1995 to 2014. RESULTS: The search yielded 156 research papers which reduced to 16 papers that met the inclusion criteria. The included papers were critiqued from a standpoint theory approach that reflects feminism, cultural respect, and humanism. Much of the literature describes issues, and provides qualitative analyses of experiences, but empirical evaluations of interventions are rare. CONCLUSIONS: There was a gap in current research evaluating strategies to improve indigenous student success and resilience. Key strategies for indigenous student success are multi-faceted, layered support, underpinned by the principles of respect, relationships, and responsibility. Implications for nursing and midwifery education, research and health care practice are outlined.

The bisphosphonate zoledronic acid regulates key angiogenesis-related genes in primary human gingival fibroblasts.

BACKGROUND: Osteonecrosis of the jaws is recognised as a serious complication for patients receiving bisphosphonates. The anti-angiogenic effects of bisphosphonates have been implicated in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this study was to determine the effects of zoledronic acid on cultured human gingival fibroblasts in relation to the modulation of genes associated with angiogenic regulation. METHODS: Primary cultures of fibroblasts were developed from gingival tissues excised during crown-lengthening surgery from three patients. Cells were cultured with and without 30µM zoledronic acid for 6, 12 and 24h and cellular proliferation and migration investigated using CellTiter-Blue and scratch wound assays, respectively. Gene expression was determined using semi-quantitative PCR array technology that allowed the analysis of 84 pathway-focused genes known to be important in the regulation of angiogenesis. RESULTS: Zoledronic acid increased the proliferation of the gingival fibroblasts in a dose dependent manner with 12 and 24h of exposure. Scratch wounding of the human gingival fibroblasts and treatment with increasing doses and time exposure to zoledronic acid (ZA) inhibited their migration. Statistically significant increases in gene expression were found for RHOB, VEGFA, CD55 and BMP2 (p≤0.05) in response to 30µM zoledronic acid. CCL2 and IL6 genes were significantly downregulated (p≤0.05). CONCLUSIONS: The regulation of the prenylated protein RHOB in this study was consistent with the known effects of zoledronic acid on the mevalonate pathway. The down regulation of CCL2 and IL6 and the upregulation of CD55 may be associated with suppression of inflammation. An increase in VEGFA and BMP2 gene expression suggests that fibroblasts respond to zoledronic acid by producing a proangiogenic environment.

Zoledronic acid and geranylgeraniol regulate cellular behaviour and angiogenic gene expression in human gingival fibroblasts.

The mevalonate pathway (MVP) and the anti-angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon-alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co-addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic-acid-treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid-like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P < 0.05, FR > ± 2) up-regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon-alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic-acid-treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway.

Forkhead box P3-positive regulatory T-cells and interleukin 17-positive T-helper 17 cells in chronic inflammatory periodontal disease.

BACKGROUND AND OBJECTIVE: The role of two recently identified and closely related T-helper cell subsets - regulatory T-cells [Tregs; forkhead box P3-positive (FOXP3(+) )] and Th17 cells [interleukin-17-positive (IL-17(+) )] - in periodontal disease is yet to be determined. Tregs are essential in maintaining peripheral tolerance and regulating the immune response. Th17 cells play a critical role in several autoimmune diseases, inflammation and host defence. The aim of this study was to determine the presence of FOXP3(+) Tregs and IL-17(+) cells, and their possible spatial interaction, in diseased periodontal tissues. MATERIAL AND METHODS: Twenty-nine archival tissues with nonspecific gingival inflammation were grouped based on the intensity (minimally or intensely inflamed) and nature (T-cell predominant or B- and plasma-cell predominant) of the inflammatory infiltrate. Using double-labelling immunohistochemistry, the concomitant presence of FOXP3(+) and IL-17(+) cells was determined and their spatial relationship was established. In addition, the proportions of FOXP3(+) and IL-17(+) cells were compared between the groups. RESULTS: Of the 29 gingival specimens investigated, 17 were intensely inflamed (≥ 1000 inflammatory cells per 0.12 mm(2) ) and 12 were minimally inflamed (≤ 600 cells per 0.12 mm(2) ). Based on the percentage of CD19(+) B-cells and plasma cells collectively and CD3(+) T-cells, gingival tissues were also grouped into B- and plasma-cell-predominant gingival tissues (n = 21; 50.7% total B- and plasma cells vs. 19.1% T cells; p < 0.001) and T-cell-predominant gingival tissues (n = 8; 61.0% T-cells vs. 15.2% B- and plasma cells; p = 0.007). More FOXP3(+) cells than IL-17(+) cells were observed in all archival gingival tissues examined. A trend towards an increased number of FOXP3(+) cells was observed for intensely inflamed gingival tissues (6.7%) and for B- and plasma-cell-predominant tissues (6.4%) compared with minimally inflamed gingival tissues (4.6%) and T-cell-predominant gingival tissues (4.5%). However, no statistically significant difference in the mean percentage of FOXP3(+) cells between the groups was observed. Interestingly, FOXP3(+) cells were significantly correlated with the B- and plasma-cell/T-cell ratio in B- and plasma-cell-predominant tissues (r = 0.713, p < 0.001). Overall, there were very few IL-17(+) cells (< 1%). All IL-17(+) cells identified in this study had an ovoid/plasmacytoid morphology and were larger in size compared with adjacent inflammatory cells. IL-17(+) and FOXP3(+) cells were not adjacent to each other in any of the areas examined, suggesting that FOXP3(+) Tregs do not directly interact with IL-17(+) cells in diseased gingival tissues. IL-17(+) /FOXP3(+) cells were not detected in the tissues examined. CONCLUSION: These results show that FOXP3(+) cells are more prominent than IL-17(+) cells in periodontal disease processes, which may suggest a predominant role for FOXP3(+) cells in periodontal disease. Further studies are required to characterize these cells more precisely and to understand, in more detail, their roles in the pathophysiology of periodontal disease.

Inhibition of Yersinia pestis DNA adenine methyltransferase in vitro by a stibonic acid compound: identification of a potential novel class of antimicrobial agents.

BACKGROUND AND PURPOSE: Multiple antibiotic resistant strains of plague are emerging, driving a need for the development of novel antibiotics effective against Yersinia pestis. DNA adenine methylation regulates numerous fundamental processes in bacteria and alteration of DNA adenine methlytransferase (Dam) expression is attenuating for several pathogens, including Y. pestis. The lack of a functionally similar enzyme in humans makes Dam a suitable target for development of novel therapeutics for plague. EXPERIMENTAL APPROACH: Compounds were evaluated for their ability to inhibit Dam activity in a high-throughput screening assay. DNA was isolated from Yersinia grown in the presence of lead compounds and restricted to determine the effect of inhibitors on DNA methylation. Transcriptional analysis was undertaken to determine the effect of an active inhibitor on virulence-associated phenotypes. KEY RESULTS: We have identified a series of aryl stibonic acids which inhibit Dam in vitro. The most active, 4-stibonobenzenesulfonic acid, exhibited a competitive mode of inhibition with respect to DNA and a K(i) of 6.46 nM. One compound was found to inhibit DNA methylation in cultured Y. pestis. The effects of this inhibition on the physiology of the cell were widespread, and included altered expression of known virulence traits, including iron acquisition and Type III secretion. CONCLUSIONS AND IMPLICATIONS: We have identified a novel class of potent Dam inhibitors. Treatment of bacterial cell cultures with these inhibitors resulted in a decrease in DNA methylation. Expression of virulence factors was affected, suggesting these inhibitors may attenuate bacterial infectivity and function as antibiotics.

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group.

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture.

BACKGROUND AND OBJECTIVE: Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion-related genes by human periodontal ligament cells. MATERIAL AND METHODS: Cultured periodontal ligament cells were subjected to a cyclic in-plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6-24 h in a Flexercell FX-4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase-3 and -7 activity; and (iii) the expression of 84 genes encoding adhesion-related molecules using real-time RT-PCR microarrays. RESULTS: Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase-3 and -7 activity at 6 and 12 h. Seventy-three genes were detected at critical threshold values < 35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α-chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76-fold induction of SPP1 at 12 h to a 2.49-fold downregulation of COL11A1 at 24 h. CONCLUSION: The study has identified several mechanoresponsive adhesion-related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis 'executioner' caspases.