Recruitment, augmentation and apoptosis of rat osteoclasts in 1,25-(OH)2D3 response to short-term treatment with 1,25-dihydroxyvitamin D3in vivo

Background Although much is known about the regulation of osteoclast (OC) formation and activity, little is known about OC senescence. In particular, the fate of of OC seen after 1,25-(OH)2D3 administration in vivo is unclear. There is evidence that the normal fate of OC is to undergo apoptosis (programmed cell death). We have investigated the effect of short-term application of high dose 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on OC apoptosis in an experimental rat model. Methods OC recruitment, augmentation and apoptosis was visualised and quantitated by staining histochemically for tartrate resistant acid phosphatase (TRAP), double staining for TRAP/ED1 or TRAP/DAPI, in situ DNA fragmentation end labelling and histomorphometric analysis. Results Short-term treatment with high-dose 1,25-(OH)2D3 increased the recruitment of OC precursors in the bone marrow resulting in a short-lived increase in OC numbers. This was rapidly followed by an increase in the number of apoptotic OC and their subsequent removal. The response of OC to 1,25-(OH)2D3 treatment was dose and site dependent; higher doses producing stronger, more rapid responses and the response in the tibiae being consistently stronger and more rapid than in the vertebrae. Conclusions This study demonstrates that (1) after recruitment, OC are removed from the resorption site by apoptosis (2) the combined use of TRAP and ED1 can be used to identify OC and their precursors in vivo (3) double staining for TRAP and DAPI or in situ DNA fragmentation end labelling can be used to identify apoptotic OC in vivo

Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0–6 Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24–72 h exposure to 250–750 μM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24 h for all cell lines. Exposure to 750 μM meloxicam for 24 h increased the fraction of cells in the radiosensitive G2/M cell cycle phase in D384 (18–27%) and U251 (17–41%) cells. 750 μM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells

Finite volume analysis of temperature effects induced by active MRI implants with cylindrical symmetry: 1. Properly working devices

BACKGROUND: Active Magnetic Resonance Imaging implants are constructed as resonators tuned to the Larmor frequency of a magnetic resonance system with a specific field strength. The resonating circuit may be embedded into or added to the normal metallic implant structure. The resonators build inductively coupled wireless transmit and receive coils and can amplify the signal, normally decreased by eddy currents, inside metallic structures without affecting the rest of the spin ensemble. During magnetic resonance imaging the resonators generate heat, which is additional to the usual one described by the specific absorption rate. This induces temperature increases of the tissue around the circuit paths and inside the lumen of an active implant and may negatively influence patient safety. METHODS: This investigation provides an overview of the supplementary power absorbed by active implants with a cylindrical geometry, corresponding to vessel implants such as stents, stent grafts or vena cava filters. The knowledge of the overall absorbed power is used in a finite volume analysis to estimate temperature maps around different implant structures inside homogeneous tissue under worst-case assumptions. The "worst-case scenario" assumes thermal heat conduction without blood perfusion inside the tissue around the implant and mostly without any cooling due to blood flow inside vessels. RESULTS: The additional power loss of a resonator is proportional to the volume and the quality factor, as well as the field strength of the MRI system and the specific absorption rate of the applied sequence. For properly working devices the finite volume analysis showed only tolerable heating during MRI investigations in most cases. Only resonators transforming a few hundred mW into heat may reach temperature increases over 5 K. This requires resonators with volumes of several ten cubic centimeters, short inductor circuit paths with only a few 10 cm and a quality factor above ten. Using MR sequences, for which the MRI system manufacturer declares the highest specific absorption rate of 4 W/kg, vascular implants with a realistic construction, size and quality factor do not show temperature increases over a critical value of 5 K. CONCLUSION: The results show dangerous heating for the assumed "worst-case scenario" only for constructions not acceptable for vascular implants. Realistic devices are safe with respect to temperature increases. However, this investigation discusses only properly working devices. Ruptures or partial ruptures of the wires carrying the electric current of the resonance circuits or other defects can set up a power source inside an extremely small volume. The temperature maps around such possible "hot spots" should be analyzed in an additional investigation

Hemócitos de Bradybaena similaris e Megalobulimus abbreviatus (Gastropoda, Stylommatophora)

Hemocytes act in the defense system against invading organisms, foreign particles aiding the recognition of what is own to the body of gastropods and what is not. Information and studies on the hemocytes in species of mollusks healthy (no infections), especially in Bradybaena similaris (Fèrussac, 1821) and Megalobulimus abbreviatus (Bequaert, 1948) are scarce. Therefore, this work aims at characterization and quantification of hemocytes present in the hemolymph of these two species. In this work three cell types were identified in the hemolymph of both species: round cells, hyalinocytes and granulocytes. The three types of hemocytes were measured, and the average of total diameter and the nucleus for each was calculated. On B. similaris, the average diameter of round cells was 10.7 μm, of hyalinocytes was 20 μm and of granulocytes was 25.4 μm. On M. abbreviatus, the average diameter of round cells was 11.7 μm, of hyalinocytes was 21.5 μm and of granulocytes was 30.5 μm. Although the hyalinocytes have similar averages between B. similaris and M. abbreviatus, the cells were demonstrated significant differences in their total diameter and size of the nucleus (p<0.0001). The average density of cells per ml without distinction of cellular type was 197,813 cells/ml for M. abbreviatus, and 416,333 cells/ml for B. similaris. The most frequent hemocytes in M. abbreviatus and B. similaris were hyalinocytes, unlike other gastropods.Os hemócitos atuam no sistema de defesa contra organismos invasores e partículas estranhas, auxiliando o reconhecimento do que é próprio do corpo dos grastrópodes e o que não é. São escassas as informações e estudos sobre os hemócitos em espécies de moluscos saudáveis (sem infecções), principalmente em Bradybaena similaris (Fèrussac, 1821) and Megalobulimus abbreviatus (Bequaert, 1948). Portanto, este trabalho tem como objetivos a caracterização e quantificação dos hemócitos presentes na hemolinfa destas duas espécies. Neste trabalho, foram identificados três tipos celulares na hemolinfa de ambas espécies: as células redondas, hialinócitos e granulócitos. Os três tipos de hemócitos foram medidos e foi calculada a média do diâmetro total e do núcleo para cada um deles. Para B. similaris, o diâmetro médio das células redondas foi de 10,7 μm, dos hialinócitos foi de 20 μm e dos granulócitos de 25,4 μm. Para M. abbreviatus, o diâmetro médio foi de 11,7 μm para as células redondas, de 21,5 μm para os hialinócitos e de 30,5 μm para os granulócitos. Embora os hialinócitos possuam médias parecidas entre B. similaris e M. abbreviatus, foram detectadas diferenças significativas do diâmetro celular total e diâmetro do núcleo (p<0,0001) dessas células entre as espécies estudadas. A densidade média de células por ml, sem distinção de tipo celular foi de 197.813 células/ml para M. abbreviatus, e de 416.333 células/ml para B. similaris. Diferentemente de outros gastrópodes, os hemócitos mais frequentes em M. abbreviatus e em B. similaris foram os hialinócitos

Epigenetic change in e-cardherin and COX-2 to predict chronic periodontitis

<p>Abstract</p> <p>Background</p> <p>DNA methylation of certain genes frequently occurs in neoplastic cells. Although the cause remains unknown, many genes have been identified with such atypical methylation in neoplastic cells. The hypermethylation of E-Cadherin and Cyclooxygenase 2 (COX-2) in chronic inflammation such as chronic periodontitis may demonstrate mild lesion/mutation epigenetic level. This study compares the hypermethylation status of E-Cadherin and COX-2 genes which are often found in breast cancer patients with that in chronic periodontitis.</p> <p>Methods</p> <p>Total DNA was extracted from the blood samples of 108 systemically healthy non-periodontitis subjects, and the gingival tissues and blood samples of 110 chronic periodontitis patient as well as neoplastic tissues of 106 breast cancer patients. Methylation-specific PCR for E-Cadherin and COX-2 was performed on these samples and the PCR products were analyzed on 2% agarose gel.</p> <p>Results</p> <p>Hypermethylation of E-Cadherin and COX-2 was observed in 38% and 35% of the breast cancer samples, respectively. In chronic periodontitis patients the detection rate was 25% and 19% respectively, and none was found in the systemically healthy non-periodontitis control subjects. The hypermethylation status was shown to be correlated among the three groups with statistical significance (p < 0.0001). The methylation of CpG islands in E-Cadherin and COX-2 genes in periodontitis patients occurs more frequently in periodontitis patients than in the control subjects, but occurs less frequently than in the breast cancer patients.</p> <p>Conclusions</p> <p>This set of data shows that the epigenetic change in E-Cadherin and Cyclooxygenase-2 is associated with chronic periodontitis. The epigenetic changes presented in chronic inflammation patients might demonstrate an irreversible destruction in the tissues or organs similar to the effects of cancer. Chronic periodontitis to some extent might be associated with DNA hypermethylation which is related to cancer risk factors.</p

Role of the Endogenous Antioxidant System in the Protection of Schistosoma mansoni Primary Sporocysts against Exogenous Oxidative Stress

Antioxidants produced by the parasite Schistosoma mansoni are believed to be involved in the maintenance of cellular redox balance, thus contributing to larval survival in their intermediate snail host, Biomphalaria glabrata. Here, we focused on specific antioxidant enzymes, including glutathione-S-transferases 26 and 28 (GST26 and 28), glutathione peroxidase (GPx), peroxiredoxin 1 and 2 (Prx1 and 2) and Cu/Zn superoxide dismutase (SOD), known to be involved in cellular redox reactions, in an attempt to evaluate their endogenous antioxidant function in the early-developing primary sporocyst stage of S. mansoni. Previously we demonstrated a specific and consistent RNA interference (RNAi)-mediated knockdown of GST26 and 28, Prx1 and 2, and GPx transcripts, and an unexpected elevation of SOD transcripts in sporocysts treated with gene-specific double-stranded (ds)RNA. In the present followup study, in vitro transforming sporocysts were exposed to dsRNAs for GST26 and 28, combined Prx1/2, GPx, SOD or green-fluorescent protein (GFP, control) for 7 days in culture, followed by assessment of the effects of specific dsRNA treatments on protein levels using semi-quantitative Western blot analysis (GST26, Prx1/2 only), and larval susceptibility to exogenous oxidative stress in in vitro killing assays. Significant decreases (80% and 50%) in immunoreactive GST26 and Prx1/2, respectively, were observed in sporocysts treated with specific dsRNA, compared to control larvae treated with GFP dsRNA. Sporocysts cultured with dsRNAs for GST26, GST28, Prx1/2 and GPx, but not SOD dsRNA, were significantly increased in their susceptibility to H2O2 oxidative stress (60–80% mortalities at 48 hr) compared to GFP dsRNA controls (∼18% mortality). H2O2-mediated killing was abrogated by bovine catalase, further supporting a protective role for endogenous sporocyst antioxidants. Finally, in vitro killing of S. mansoni sporocysts by hemocytes of susceptible NMRI B. glabrata snails was increased in larvae treated with Prx1/2, GST26 and GST28 dsRNA, compared to those treated with GFP or SOD dsRNAs. Results of these experiments strongly support the hypothesis that endogenous expression and regulation of larval antioxidant enzymes serve a direct role in protection against external oxidative stress, including immune-mediated cytotoxic reactions. Moreover, these findings illustrate the efficacy of a RNAi-type approach in investigating gene function in larval schistosomes

The lectin concanavalin-A signals MT1-MMP catalytic independent induction of COX-2 through an IKKγ/NF-κB-dependent pathway

The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) which expression is increased in brain cancer. Given that MT1-MMP correlated to high expression of cyclooxygenase (COX)-2 in gliomas with increasing histological grade, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP’s catalytic function. ConA- and MT1-MMP-mediated intracellular signaling of COX-2 was also confirmed in wild-type and in Nuclear Factor-kappaB (NF-κB) p65−/− mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-κB1 (p50)−/− and in I kappaB kinase (IKK) γ−/− mutant MEF cells. Collectively, our results highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling. Concise summary Concanavalin-A (ConA) mimics biological lectin/carbohydrate interactions that regulate the proinflammatory phenotype of cancer cells through yet undefined signaling. Here we highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of cyclooxygenase-2, and that could be responsible for the therapy resistance phenotype of glioblastoma cells

Absence of the MGMT protein as well as methylation of the MGMT promoter predict the sensitivity for temozolomide

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) can cause resistance to the alkylating drug temozolomide (TMZ). The purpose of this study was to determine the relationship between the MGMT status, determined by means of several techniques and methods, and the cytotoxic response to TMZ in 11 glioblastoma multiforme (GBM) cell lines and 5 human tumour cell lines of other origins. Cell survival was analysed by clonogenic assay. The MGMT protein levels were assessed by western blot analysis. The MGMT promoter methylation levels were determined using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and quantitative real-time methylation-specific PCR (qMSP). On the basis of the results of these techniques, six GBM cell lines were selected and subjected to bisulphite sequencing. The MGMT protein was detected in all TMZ-resistant cell lines, whereas no MGMT protein could be detected in cell lines that were TMZ sensitive. The MS-MLPA results were able to predict TMZ sensitivity in 9 out of 16 cell lines (56%). The qMSP results matched well with TMZ sensitivity in 11 out of 12 (92%) glioma cell lines. In addition, methylation as detected by bisulphite sequencing seemed to be predictive of TMZ sensitivity in all six cell lines analysed (100%). The MGMT protein expression more than MGMT promoter methylation status predicts the response to TMZ in human tumour cell line