The role of autophagy in odontogenesis, dental implant surgery, periapical and periodontal diseases.

Autophagy is a cellular process that is evolutionarily conserved, involving the sequestration of damaged organelles and proteins into autophagic vesicles, which subsequently fuse with lysosomes for degradation. Autophagy controls the development of many diseases by influencing apoptosis, inflammation, the immune response and different cellular processes. Autophagy plays a significant role in the aetiology of disorders associated with dentistry. Autophagy controls odontogenesis. Furthermore, it is implicated in the pathophysiology of pulpitis and periapical disorders. It enhances the survival, penetration and colonization of periodontal pathogenic bacteria into the host periodontal tissues and facilitates their escape from host defences. Autophagy plays a crucial role in mitigating exaggerated inflammatory reactions within the host's system during instances of infection and inflammation. Autophagy also plays a role in the relationship between periodontal disease and systemic diseases. Autophagy promotes wound healing and may enhance implant osseointegration. This study reviews autophagy's dento-alveolar effects, focusing on its role in odontogenesis, periapical diseases, periodontal diseases and dental implant surgery, providing valuable insights for dentists on tooth development and dental applications. A thorough examination of autophagy has the potential to discover novel and efficacious treatment targets within the field of dentistry.

Do Wortmannin and Thalidomide induce apoptosis by autophagy inhibition in 4T1 breast cancer cells in vitro and in vivo?

The aim of this study was to show the effects of autophagy inhibitor Wortmannin and antiangiogenic-proapoptotic Thalidomide on autophagy and apoptosis markers in 4T1 breast cancer cells in vitro and in vivo. The half-maximal inhibitory concentration (IC50) values of 4T1 cells for Wortmannin and Thalidomide were evaluated by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. After cancer formation in 28 BALB/C female mice, drugs were administered for seven days. Cells and tissue sections were evaluated for anti-phosphoinositide 3-kinase (PI3K), anti- the microtubule-associated protein 1 light chain3 (MAPLC3ß), anti-caspase 8, anti-caspase 9, and anti-caspase 3 immunoreactivities by immunohistochemical staining and apoptosis by Terminal Transferase dUTP Nick End Labeling (TUNEL) assay. Both PI3K and MAPLC3ß immunoreactivities decreased in all treatments when compared to control group except Thalidomide treatment in primary cancer tissue. The caspase 3, 8, and 9 immunoreactivities were increased in all treatment groups and TUNEL positive cells were the highest in the Wortmannin and Thalidomide group. Our findings suggest that autophagy is an important mechanism for 4T1 cells and both Wortmannin and Thalidomide treatments inhibit autophagy and induce apoptosis. In primary cancer tissues, autophagy was not effective as in vitro. The treatment of Wortmannin and Thalidomide increased the apoptotic cells in vivo independent from autophagy inhibition. Different results may be because of microenvironment. Further studies must be done to elucidate the effect of microenvironment.

Anti-VEGF treatment suppresses remodeling factors and restores epithelial barrier function through the E-cadherin/ß-catenin signaling axis in experimental asthma models.

Besides maintaining a physical barrier with adherens junctional (AJ) and tight junctional proteins, airway epithelial cells have important roles in modulating the inflammatory processes of allergic asthma. E-cadherin and ß-catenin are the key AJ proteins that are involved in airway remodeling. Various mediators such as transforming growth factor-ß (TGF-ß), epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulin-like growth factor (IGF), tumor necrosis factor-α (TNF-α) and angiogenic factors, such as vascular endothelial growth factor (VEGF), are released by the airway epithelium in allergic asthma. The signaling pathways activated by these growth factors trigger epithelial-mesenchymal transition (EMT), which contributes to fibrosis and subsequent downregulation of E-cadherin. The present study used a mouse asthma model to investigate the effects of anti-VEGF, anti-TNF and corticosteroid therapies on growth factor and E-cadherin/ß-catenin expression. The study used 38 male BALB/c mice, divided into 5 groups. A chronic mouse asthma model was created by treating 4 of the groups with inhaled and intraperitoneal ovalbumin (n= 8 per group). Saline, anti-TNF-α (etanercept), anti-VEGF (bevacizumab) or a corticosteroid (dexamethasone) were applied to each group by intraperitoneal injection. No medication was administered to the control group (n=6). Immunohistochemistry for E-cadherin, ß-catenin and growth factors was performed on lung tissues and protein expression levels assessed using H-scores. Statistically significant differences were observed in E-cadherin, ß-catenin, EGF, FG, and PFGF (P<0.001 for all) as well as the IGF H-scores between the five groups (P<0.005). Only anti-VEGF treatment caused E-cadherin and ß-catenin levels to increase to the level of non-asthmatic control groups (P>0.005). All treatment groups had reduced TGF-ß, PDGF and FGF H-scores in comparison with the untreated asthma group (P=0.001). The EGF and IGF levels were not significantly different between the untreated asthmatic and non-asthmatic controls. The results suggested that anti-VEGF and TNF-α inhibition treatments are effective in decreasing growth factors, in a similar manner to conventional corticosteroid treatments. Anti-VEGF and TNF inhibition therapy may be an effective treatment for remodeling in asthma while offering an alternative therapeutic option to steroid protective agents. The data suggested that anti-VEGF treatment offered greater restoration of the epithelial barrier than both anti-TNF-α and corticosteroid treatment.

Immunohistochemical determination of mTOR pathway molecules in ovaries and uterus in rat estrous cycle stages.

mTOR is a member of the PI3K/Akt/mTOR signaling pathway that participates in cell growth, proliferation, protein synthesis, transcription, angiogenesis, apoptosis and autophagy. mTOR and MAPK pahways are two important key signal pathways which are related to each other. We investigated the role of mTOR and other signaling molecules in rat ovaries and uteruses in stages of the estrous cycle. Young adult female rats were divided into four groups as proestrous, estrous, metestrous and diestrous according to vaginal smears. Immunohistochemical staining of mTORC1, IGF1, PI3K, pAKT1/2/3, ERK1 and pERK1/2 was performed and pAKT1/2/3 and ERK1 were also analyzed using western blotting on ovarian and uterine tissue samples. According to our results, PI3K/Akt/mTOR and ERK/pERK showed an increase in the rat ovulation period. When all the groups were evaluated the immunoreactivities for all of the antibodies were detected in the oocytes, granulosa and theca cells, corpus luteum and stroma of ovary and lamina propria, surface and glandular epithelium of uterus with the strongest observed with anti-ERK1 antibody and then with a decreasing trend with anti-mTORC1, anti-pAkt1/2/3, anti-IGF1, anti-PI3K and anti-pERK1/2 antibodies in the proestrus and estrus stages. Differently from other parts of the ovary, highest antibody expression in the corpus luteum was observed in the metestrous stage. Moreover, the existence of pAKT1/2/3 and ERK1 proteins was confirmed with the Western blotting technique. We suggest that mTOR and mTOR-related ERK signaling molecules may participate in the rat ovulation process.

Significant Changes in Trans-Epithelial Barrier Proteins of Adenoid Tissue with Atopic Status in Children.

OBJECTIVES: Adenoid tissue is important in local immune response and epithelial barrier dysfunction of this tissue may contribute to allergies. The aim of this study was to evaluate the relationship between the status of cross-epithelial barrier elements in adenoid tissue lymphoepithelium and inhalant allergen sensitization. MATERIALS AND METHODS: Children aged 5-15 years, who underwent adenotonsillectomy, participated in this study. All subjects underwent skin prick testing with environmental inhalant allergens. Occludin, ZO1, e-cadherin, ß-catenin, desmoglein, desmoplakin, and connexon-43 were stained immunohistochemically in the adenoid tissues obtained and scored by H-score. RESULTS: We enrolled 76 children, 14 among whom were sensitized to environmental allergens. Among the zonula occludens proteins, median H-scores for occludin, claudin, and ZO-1 were significantly lower in the atopic compared to the nonatopic group respectively (p<0.001). Similarly, median H-scores for e-cadherin and ß catenin proteins of the zonula adherens were significantly lower in the atopic group (p<0.001). Both desmoglein and desmoplakin H-scores were significantly lower in the atopic group [60 (50-100) vs 280 (260-300), p<0.001 and 105 (87.5-120) vs 280 (67.25-300), p<0.001 respectively]. Moreover, connexin-43 protein of the gap junction was significantly lower in the atopic group (p<0.001). CONCLUSION: Adenoid tissue, which is the initial point of contact of inhalant allergens demonstrates epithelial barrier junctional protein, changes in children with inhalant allergen sensitization without clinical allergic disease symptoms. Therefore, it may be concluded that epithelial barrier function plays an important role in the development of allergen sensitization versus tolerance.

Adjuvant effects of chemotherapeutics and Metformin on MFE-319 endometrial carcinoma cell line.

We aimed to investigate the cytotoxicity of Metformin, Cisplatin, and Paclitaxel on MFE-319 endometrial carcinoma cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and immunocytochemistry assays. Half maximal inhibitory concentration (IC50) doses of three drugs alone and in the dual combinations were applied to the cells. Immunocytochemical method was performed for the cell survival and for phosphatidylinositol 3-kinase (PI3K), phosphorylated extracellular regulated kinases (pErk)-1∕2, Akt-1, phosphorylated Akt (pAkt)-1∕2∕3 cell growth markers and angiogenic vascular endothelial growth factor (VEGF). Immunoreactivities were evaluated using H-score and analyzed using the one-way analysis of variance (ANOVA) test for statistics. It was found that these drugs caused a decrease in the immunoreactivities of these markers. Particularly, dual combination of Paclitaxel and Cisplatin decreased the immunoreactivities of PI3K, pErk-1∕2, Akt-1, and pAkt-1∕2∕3. Cisplatin and Paclitaxel were more effective than Metformin; on the other hand, Metformin has been shown to enhance the efficacy of these two drugs. In vitro or in vivo further studies are needed to investigate the efficacy of these three drugs via PI3K∕Akt signal pathway.

Cell Signaling Pathways Related to Epithelial Mesenchymal Transition in Cancer Metastasis.

The epithelial mesenchymal transition (EMT) is a highly complex and dynamic morphogenetic process in which epithelial cells change their cellular characteristics and transform to mesenchymal cells. It plays a key role in tissue remodeling, not only during embryonic development and stem cell biology but also during wound healing, fibrosis, and cancer progression. In EMT, under different extracellular cell signals and stimuli, epithelial cells undergo an orchestrated series of morphological, cellular, biochemical, and molecular changes. The mechanism of cancer cell metastasis is similar to embryonic development, like epithelial cells migrating and differentiating into all tissues of the embryo. In this period, epithelial cells differentiate into mesenchymal cells, migrate, and return to the epithelial cells when necessary. The important cell signaling pathways in embryonic development, such as growth factor receptor tyrosine kinases (FGF, HGF, TGF-α), TGF-ß, Wnt, NOTCH, MAPK, JAK/STAT and inflammatory cytokines such as NF-κB, TNF-α, IL6, cathepsin and HIF-1α. All play crucial roles in EMT during cancer cell invasion and metastasis. In cancer progression, similar cell signaling pathways and molecular activation occur in embryonic development; therefore, it is important to understand the molecular mechanisms of cell signaling pathways related to cancer metastasis and improve new diagnostic and therapeutic approaches for resistance to drugs.

Influence of omega-3 fatty acid on orthodontic tooth movement in rats: A biochemical, histological, immunohistochemical and gene expression study.

OBJECTIVE: The aim of this study was to investigate the effects of omega-3 fatty acids on orthodontic tooth movement. SETTING AND SAMPLE POPULATION: For this study, 56 12-week-old adult male Wistar albino rats from the Animal Laboratory at Adnan Menderes University, Faculty of Medicine, were used. MATERIAL AND METHODS: Rats were randomly divided into seven groups (n = 8 each): control group (without any treatment), tooth movement groups (three groups of animals with only tooth movement) and omega groups (three groups of animals with tooth movement and omega-3 administration). Omega-3 fatty acids were administered to the rats systemically during the tooth movement period. On the 3rd, 7th and 14th days after the orthodontic tooth movement, the rats were sacrificed and biochemical, histological, immunohistochemical andgene expression examinations were performed. RESULTS: On the 14th experimental day, the amount of tooth movement in the omega groups was significantly lower than the tooth movement groups (P = 0.012). Biochemical experimentsshowed that the omega groups had significantly lower total oxidant levels and higher total antioxidant levels compared to the tooth movement group on the 14th experimental day (P = 0.001). The levels of RANKL, IL-6 and IL-1ß in the omega groups were significantly lower than the tooth movement groups on all experimental days (P < 0.05). CONCLUSION: Systemic administration of omega-3 fatty acids showed antioxidant and antiinflammatory effects and decelerate the orthodontic tooth movement.

Auditory and Histopathological Effects of Topical Mercurochrome Treatment in Rats with Tympanic Membrane Perforation.

OBJECTIVES: Topical treatment is first choice in the treatment of uncomplicated chronic otitis media. It was intended to assess auditory and histopathological safety of ototopical use of mercurochrome solution in rats with induced tympanic membrane perforation. MATERIALS AND METHODS: The study was conducted on 21 female Wistar-Albino rats which were randomly assigned into 3 groups. In all rats, perforation was performed at right tympanic membrane. Distortion product otoacoustic emissions (DPOAEs) measurements were performed at frequencies of 2000, 3000 and 4000 Hz (with L1/L2: 70 /70 dB at 2f1-f2 frequency; f2/f1 ratio: 1:22) before recovery from anesthesia and signal-to-noise ratio (SNR) were recorded. Normal saline, 2% mercurochrome and gentamicin were given to group 1, 2 and 3 twice daily over a week, respectively. Rats were sacrificed after DPOAE measurements on day 14. Right temporal bone specimens were examined under light microscope after processing. RESULTS: Based on DPOAE results, there was no significant difference among groups before treatment. On day 14, significant differences were found in DPOAE measurements at 3000 and 4000 Hz, and in mean SNR values in 2% mercurochrome and gentamicin groups when compared to normal saline group while no significant difference was detected at 2000 Hz among groups. In addition, significant degeneration was detected in Corti organs, spiral ganglions and stria vascularis in groups 2 and 3. CONCLUSION: In this study, it was observed that mercurochrome use in external otitis and otitis media with tympanic membrane perforation could cause ototoxicity and concluded that the solution should be used cautiously.

A Study on the Anticarcinogenic Effects of Calcium Fructoborate.

Evidences about the preventive and therapeutic effects of boron compounds on cancer have been increasing in the last years. Although calcium fructoborate (CaFB) is used as a nutritional supplement, data about its preventive and therapeutic effects on neoplastic transformations are limited. In the present study, the various concentrations of CaFB were applied to the MDA-MB-231 metastatic breast cancer cell line. First, we examined the cytotoxic effect and IC50 value of CaFB by MTT assay. For the evaluation of the DNA damage, apoptosis and metastatic potential, expression levels of ATM, pATM, PARP, p53, p-p53, caspase-3, caspase-9, and VEGF were investigated by using immunoblotting and immunohistochemical methods. Cell viability was significantly reduced at 50 µM CaFB treatment. pATM, p-p53, and caspase-9 levels increased significantly in all groups; furthermore, there was approximately 12.5-, 2.4-, and 10.7-fold increase, respectively, for 100 µM CaFB treatment. ATM and p53 levels did not change with CaFB treatment, but PARP levels significantly 2.5-fold decreased. While VEGF immunoreactivity decreased in all groups, significant increase in caspase-3 immunoreactivity was observed only in the group treated with 50 µM CaFB (p < 0,001). Our results imply that CaFB may have therapeutic potential as well as preventive benefits in cancer.

Effects of Cyclooxygenase on the Urothelium of the Urinary Bladder of Mice Exposed to Pelvic Radiation.

OBJECTIVE: To determine the role of cyclooxygenase (COX) expression in the urothelium of the urinary bladder during radiation injury caused by pelvic radiotherapy for cancer therapy. STUDY DESIGN: Twenty-four male Swiss Albino mice were separated into 4 groups. The first group was the control group (Group 1) and the second, third, and fourth groups were euthanized after 24 hours (Group 2), 48 hours (Group 3), and 7 days (Group 4), respectively. A single-fractioned 10 Gy of ionizing radiation was applied to all mice's pelvic zone with Co-60. Bladders were removed completely from the pelvic region. Histochemical analysis using hematoxylin and eosin and immunohistochemical analysis using anti-COX-1 and COX-2 antibodies were performed on tissue samples. The immunoreactivities of the urinary bladder were quantified using H-score measurement, and statistical comparison was performed. RESULTS: In the immunohistochemical examination the COX-1 immunoreactivities were found to be higher in the urothelium of the bladder in the radiation exposed groups than in the normal control group (group 1) (p < 0.005). Additionally, high immunoreactivity of COX-2 molecule was established in groups 2, 3, and 4 of radiation groups as compared to group 1 (p < 0.005) in examination of the urothelium. COX-1 and COX-2 immunoreactivities in the submucosa were detected higher in group 4 than in the other groups (p < 0.005). CONCLUSION: COX-1 and COX-2 expressions in the urothelium and subepithelium of the urinary bladder were investigated in mice during the acute radiation response. The expression of COX-1 and COX-2 in the urothelium seems to prevent bladder damage from radiation, supplying differentiation and restoration of the urothelium.

The role of CAPE in PI3K/AKT/mTOR activation and oxidative stress on testis torsion.

Ischemia reperfusion injury arises from testicular torsion resulting in a loss of spermatogenesis and significant germ cell apoptosis. This study evaluates the prooxidant/antioxidant effects of caffeic acid phenethyl ester (CAPE) through PI3K/AKT/mTOR signal pathways on testis torsion. A total of (28) male Wistar rats were divided randomly into 4 groups (n=7 for each group):group A (sham) group,group B torsion/detorsion group, group C (saturation group, during four days of CAPE, one dose (10 µmol/kg, i.p)) and group D (a single dose of CAPE 2h after torsion and before detorsion). At the end of the study, unilateral orchiectomies were performed for measurements of MDA and 8OHdG levels, histopathologic and immunohistochemical and TUNEL apoptotic cell examination. Testicular torsion-detorsion led to a significant decrease in the mean values of the Johnsen's scores and a significant increase in the apoptotic cell values of group B. There were no significant differences between group D and group A. In addition, the MDA and 8OHdG levels increased significantly in group B. The MDA and 8OHdG values were lower in group D. However, the 8OHdG levels were higher in group C than the groups A and D. On the other hand, CAPE suppresses mTOR activation and reduces the apoptosis on ischemia/reperfusion damage in rat testis. These results demonstrate that CAPE suppresses mTOR activation and reduces the apoptosis on ischemia/reperfusion damage in rat testis.

JAK/STAT pathway interacts with intercellular cell adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) while prostate cancer stem cells form tumor spheroids.

PURPOSE: JAK/STAT is an evolutionarily conserved pathway and very important for second messenger system. This pathway is important in malignant transformation and accumulated evidence indicates that this pathway is involved in tumorigenesis and progression of several cancers. It was possible to assume that activation of JAK/STAT pathway is associated with increase in the expressions of ICAM/1 and VCAM-1. In this study we hypothesized that when cells were maintained as spheroids or monolayers, the structure of cancer stem cells (CSCs) could show differentiation when compared with non-CSCs. METHODS: DU-145 human prostate cancer cells were cultured using the Ege University molecular embryology laboratory medium supplemented with 10% fetal bovine serum. Clusters of differentiation 133 (CD133)(+high)/CD44(+high) prostate CSCs were isolated from the DU145 cell line by using BD FACSAria. CD133//CD44+ CSCs were cultured until confluent with 3% noble agar. The expression of these proteins in CSCs and non-CSCs was analyzed by immunohistochemistry. RESULTS: Different expression profiles were observed in the conventional two-dimensional (2D) and three-dimensional (3D) experimental model system when CSCs and non-CSCs were compared. Human prostate CSCs exhibited intense ICAM-1 and VCAM-1 immunoreaction when compared with non-CSCs. These findings were supported by the fact that VCAM-1 on the surface of cancer cells binds to its counterreceptor, the α4ß1 integrin (also known as very-late antigen, VLA-4), on metastasis-associated macrophages, triggering VCAM-1-mediated activation of the phosphoinositide 3-kinase growth and survival pathway in cancer cells. CONCLUSIONS: The results of this study showed that changes in JAK/STAT pathway are related with adhesion molecules and could affect cancer progression.

Vascular endothelial growth factor antagonism restores epithelial barrier dysfunction via affecting zonula occludens proteins.

Epithelial barrier dysfunction is important in the pathogenesis of asthma and allergic responses, and is therefore a therapeutic target. The aim of the present study was to investigate the effects of dexamethasone, a classic therapeutic agent, an anti-tumor necrosis factor agent (etanercept), which is used to treat difficult cases of asthma, and an anti-vascular endothelial growth factor (VEGF) agent (bevacizumab), which is an angiogenesis inhibitor, on zonula occludens (ZO) proteins in an experimental asthma model. The experimental model of asthma was developed using intraperitoneal (IP) and inhaled administration of ovalbumin in 38 BALB/c mice, which were divided into four groups. The control group (n=6) did not receive any treatment, while the four remaining groups (n=8 per group) received an IP injection of saline, etanercept, bevacizumab or dexamethasone, respectively. Occludin, claudin and junctional adhesion molecule (JAM) were immunohistochemically stained in the left middle lobe samples using an indirect avidin-peroxidase method, after which the staining was semiquantified with H-scores. Statistically significant differences were observed in the occludin, claudin and JAM H-scores among the four groups (P<0.001). In the untreated asthma, etanercept, bevacizumab and dexamethasone groups, the median H-scores for occludin were 93, 177, 280 and 198, respectively, while the H-scores for claudin were 82, 193.5, 274 and 202.5, respectively, and the median H-scores for JAM were 130, 210, 288 and 210, respectively. Pairwise comparisons revealed that all three ZO protein H-scores were significantly lower in the saline group when compared with each treatment group. However, the H-scores of the ZO proteins were not significantly different between the etanercept and dexamethasone groups. Furthermore, the bevacizumab group exhibited higher H-scores for all the proteins compared with the dexamethasone group. Therefore, antagonism of VEGF with bevacizumab restores the epithelial barrier to a greater extent when compared with dexamethasone treatment. This result may be promising for the development of novel therapeutic agents.

Significance of apoptosis related proteins on malignant transformation of ovarian tumors: A comparison between Bcl-2/Bax ratio and p53 immunoreactivity.

In this study, we compared the immunoreactivities of Bcl-2, Bax and p53 proteins in ovarian tumors and related the immunohistochemical findings to the histological type of the tumors. Formalin-fixed, paraffin wax-embedded tissue sections from 40 patients who had serous-mucinous borderline tumors and serous-mucinous adenocarcinoma of the ovary (n=10 each) were stained with hematoxylin-eosin (H&E). After histopathological examination, serial sections were stained immunohistochemically with primary antibodies to Bcl-2, Bax and p53 using an avidin-biotin-peroxidase method. A semi-quantitative grading system was used to compare the immunohistochemical staining intensities. The nuclear DNA fragmentation of apoptosis was determined using TUNEL method. As a result of immunohistochemical staining, increased immunoreactivity of Bcl-2 was observed in adenocarcinomas when compared to borderline tumors (P<0.001). Strong immunoreactivity of Bcl-2 and mild immunoreactivities of Bax and p53 were detected in ovarian adenocarcinomas. There were no significant statistical differences in the immunoreactivity of Bax among the histological type of ovarian tumors. Whereas a balance was observed between the immunoreactivities of Bcl-2 and Bax in the borderline cases, and this balance was strongly changed toward the anti-apoptotic Bcl-2 protein in patients with adenocarcinoma. TUNEL staining of sections indicated apoptotic cells in the serous borderline tumors were about 8-fold higher than in the serous adenocarcinoma. The results of this study on apoptosis-related factors might help to develop novel protective and therapeutic approaches, such as isoflavonoids and isothiocyanates, which were associated with decreased Bcl-2/Bax ratio, against the malignant epithelial ovarian tumors.

Expression profiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells.

Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.

Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group.

OBJECTIVE: To analyze the expression patterns of extracellular signal-regulated kinase (ERK1/2) and phosphorylated (p)-AKT in the tissues of non-pathologic endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer using indirect immunohistochemistry, and also to investigate the effect of ERK1/2 and p-AKT expression patterns on prognosis in endometrioid adenocancer. STUDY DESIGN: Immunolocalization of ERK1/2 and p-AKT was examined in six different types of endometrial tissues: proliferative endometrium (PE; n=10, 11.2%), secretuar endometrium (SE; n=10, 11.2%), simple hyperplasia (SH; n=15, 16.9%), complex hyperplasia (CH; n=3, 3.4%) and atypical complex hyperplasia (ACH; n=10, 11.2%), which were obtained from endometrial biopsies, curettage materials, and hysterectomy specimens and classified as the benign group; and both early stage endometrioid (n=21, 23.6%) and advanced stage endometrioid adenocancer (AC; n=20, 22.5%), which were obtained from complete surgical staging materials and classified as the malignant group. All specimens were fixed in 10% formalin and processed using routine paraffin protocols. Immunostaining intensities were evaluated as negative or weak (assigned as low expression) and moderate or strong (assigned as high expression). RESULTS: In the malignant group, 23 of 41 patients (56.1%) had high ERK1/2 and p-AKT expression, whereas only three of 48 patients in the benign group (6.3%) had high ERK1/2 and p-AKT expression (P<0.0001 and P<0.0001, respectively). p-AKT expression was significantly higher in women with positive lymph nodes (OR 9.0; 95% CI: 1.2-100.0; P=0.03). Higher expression of p-AKT was significantly associated with poor progression-free survival (PFS) and overall survival (OS). In contrast, ERK1/2 expression was not associated with PFS or OS.Conclusions ERK1/2 and p-AKT can be useful in the differential diagnosis of benign vs. malignant endometrial lesions, as well as early vs. advanced stage endometrioid endometrial adenocancer. Additionally, higher p-AKT expression could be used as a marker of poor prognosis in the management of patients with endometrioid endometrial adenocancer.

Effect of nicotine on RANKL and OPG and bone mineral density.

AIM: The signaling pathway OPG/RANK/RANKL is a key in maintaining the balance between the activity of osteoblasts and osteoclasts in order to prevent bone loss. In this study, our aim was to assess the effects of long-term nicotine exposure on plasma RANKL and OPG levels, tissue RANKL and OPG immunoreactivities, and bone mineral density (BMD) scores in rats. MATERIALS AND METHODS: Thirty-six Swiss Albino rats weighing 70 ± 10 g were divided into three groups. While the controls (n = 12) were only given normal drinking water, for low-dose nicotine (LDN) group (n = 12) 0.4 mg/kg/day; for high-dose nicotine (HDN) group (n = 12), 6.0 mg/kg/day nicotine was added to drinking water for a year. At the end of 12th month, BMD scores were measured using an X-ray absorptiometry and bone turnover was assessed by measuring plasma RANKL and OPG levels and RANKL and OPG immunoreactivities in tail vertebrae of the rats. RESULTS: There was no statistically significant difference in BMD scores of lumbar spine and femoral regions of the nicotine groups in comparison to controls. Plasma OPG levels were found to be significantly higher in HDN group, in comparison to the controls and LDN groups (p = .001) unlike plasma RANKL levels. Tissue RANKL and OPG immunoreactivities decreased significantly in the LDN and HDN groups (p < .001, p < .01, respectively). CONCLUSIONS: The results of this study show that nicotine is not primarily responsible for the decrease in BMD frequently seen in smokers. Measuring plasma RANKL and OPG levels did not reflect tissue immunoreactivities.

Inhibitory effect of N-acetyl cysteine and ascorbic acid on the development of myringosclerosis: an experimental study.

OBJECTIVES: This study investigated the effects of ascorbic acid and N-acetyl cysteine (NAC) antioxidants on the development of myringosclerosis (MS) in an experimental model. METHODS: Myringotomies were performed in the ears of 15 guinea pigs, and Spongostan pieces were placed on the perforated regions of the tympanic membrane. The subjects were divided randomly into three groups and treated with three different solutions on the Spongostan-group 1: (control, 0.9% saline), group 2 (ascorbic acid), and group 3 (NAC). On day 15 after treatment, specimens from the tympanic membranes were obtained and examined via light microscopy. Sclerosis and inflammation scores and the tympanic membrane thicknesses were evaluated. Immunohistochemical methods were used to evaluate the expression of VEGF, TGF-ß, iNOS, and IL1-ß in all groups. RESULTS: Lower sclerosis and inflammation scores and reduced tympanic membrane thicknesses were observed in groups treated with NAC or ascorbic acid compared with the control group. Immunohistochemical studies revealed significantly less expression of VEGF, TGF-ß, and iNOS in groups 2 and 3 compared with group 1. Additionally, IL1-ß expression was significantly less in group 3 than in group 1. Compared with group 1, group 2 animals exhibited reduced inflammation in the lamina propria, fewer active fibroblasts, less leukocyte infiltration, and decreased thickness of the vessels; group 3 animals exhibited decreased numbers of active fibroblasts and collagen fibers in the lamina propria. CONCLUSIONS: Inflammation scores, cellular infiltration, and expression of VEGF, TGF-ß, and iNOS were reduced by ascorbic acid and/or NAC treatments, thereby decreasing MS development. Decreased expression of IL1-ß was observed only in animals treated with NAC.

Effects of oral intake of cetirizine HCl and desloratadine molecules on the middle ear mucosa: an experimental animal study.

We have planned to demonstrate histopathologic effects of mid- or long-term oral use of desloratadine and cetirizine HCl molecules on middle ear mucosa of rats. Thirty-six rats were randomized equally into six groups. Desloratadine groups received once daily doses of 1 mg/ml desloratadine for 30 (D30 Group) or 60 (D60 Group) days. The Cetirizine study groups were given once daily doses of 1 mg/ml cetirizine for 30 (S30 Group) or 60 (S60 Group) days. Control groups were given 2 cc physiologic saline using orogastric gavage method through a 12 G gavage catheter for 30 (K30 Group) or 60 (K60) days. At the end of 30 days, D30, S30 and K30 Groups were sacrificed. Tissue samples harvested from groups were evaluated between 1 and 4 Grades for histological characteristics of middle ear canal, eardrum, middle ear epithelium and connective tissue, edema, vascular congestion and inflammatory cells. In the control group no pathological finding was encountered in rats sacrificed on 30 and 60 days. No statistical difference was observed when groups were compared on external ear epithelial tissue, external ear sebaceous gland, middle ear inflammation, and middle ear capillary dilatation both on 30 and 60 days. Tympanic membrane collagen was more evident in D30 and D60 groups when compared with C30 and C60 groups. Comparison of histopathological grading results between 30 and 60 days revealed no significant changes. In conclusion, oral intake of cetirizine and desloratadine preparations has effects of tympanic membrane collagen, degrees of edema and vascular congestion being more prominent with desloratadine molecule.