Biocompatibility of vertically aligned multi-walled carbon nanotube scaffolds for human breast cancer cell line MDA-MB-231.

The aim of the current study was to determine whether the MWCNT-based scaffold has a suitable structure for cell growth and provides a biocompatible environment for human MDA-MB-231 cell lines. MWCNT-based nanostructured scaffolds were produced by plasma-enhanced chemical vapor deposition (PECVD) technique. MDA-MB-231 cells were seeded on MWCNTs-textured silicon scaffolds and on pristine silicon surfaces. After 1 week of culturing, the scaffolds were prepared for SEM analysis and immunocytochemical staining was performed for the two groups (MWCNT scaffold and pristine silicon surface), using MMP-2, MMP-9, PI3K, AKT and NF-κB primary antibodies. SEM analyses showed that the MDA-MB-231 cells better adhered to the MWCNT-based nanostructured scaffold than the pristine silicon surface. Immunohistochemical activity of the MDA-MB-231 cells on both materials has similar staining with anti-AKT MMP-2, MMP-9 and NF-κB primary antibodies. Therefore, the results of the present study suggest that the MWCNT-based scaffolds enhanced cell adhesion to the scaffold and exhibited more biomimetic properties and physiological adaptation with the potential to be used for in vitro metastasis studies of BrCa cell lines.

Assessment of mTOR pathway molecules during implantation in rats.

Mammalian target of rapamycin (mTOR) is a member of the PI3K/Akt/mTOR signaling pathway that participates in cell growth, proliferation, protein synthesis, transcription, angiogenesis, apoptosis and autophagy. We investigated the role of mTOR and other signaling molecules in the rat uterus during implantation. Female pregnant rats were divided into three groups: embryonic days (ED) 4.5, 5.5 and 6.5 according to vaginal smears. Immunohistochemical staining of mTORC1, mTORC2, IGF1, PI3K, pAkt1/2/3, ERK1 and pERK1/2 was performed on formalin fixed, paraffin embedded uterine tissue samples. pAkt1/2/3 and ERK1 also were analyzed using western blotting. We found that PI3K/Akt/mTOR and ERK/pERK were increased during the implantation period. Different amounts of mTORC1, mTORC2, IGF1, PI3K, pAKT1/2/3, ERK1 and pERK1/2 were expressed in luminal epithelium, decidual cells, embryoblast and trophoblast cells during implantation. We suggest that mTOR and associated signaling molecules may participate in implantation.

The expression of Forkhead transcription factors in decidua and placenta in patients with missed abortion.

BACKGROUND: Forkhead transcription factors 3a (FOXO3a) has pleiotropic biological functions in the female reproductive tract. FOXO3a has a function in decidualization, in placental development, and also in inhibition of apoptosis. This study aims to investigate a possible role of FOXO3a in missed abortion. MATERIALS AND METHODS: Decidual and placental tissue samples were obtained from the women with unwanted pregnancy as the control group and with missed abortion as the patient group. Immunohistochemistry technique was utilized to compare FOXO3a expression of the decidual cells in uterine decidual stroma and cytotrophoblast-syncytiotrophoblast cells in placental villous stroma. Immunohistochemistry was evaluated semiquantitatively utilizing the H-score technique. Results: It was demonstrated that H-Scores of FOXO3a expression in both uterine decidual stroma were increased in the missed abortion group (255.83 +/- 12.41) than in the normal pregnancy group (133.33 +/- 17.43). It was also shown that there was no difference between non-decidual area of the endometrium of the normal pregnancy and the missed abortion group (30.33 +/- 4.32; 39.66 +/- 14.30, respectively) and placental villous stroma (13.00 +/- 1.89; 13.00 +/- 1.67, respectively). However, the immunoreactivity of cytotrophoblast and syncytiotrophoblast cells significantly increased in the missed abortion group (18.83+1.47; 322.00 +/- 6.06, respectively) than in the normal pregnancy group (11.00 +/- 1.26; 254.00 +/- 8.17, respectively) (p < 0.05). CONCLUSION: These data support the hypothesis that increased FOXO3a expression in missed abortion may prevent the discharge of dead fetus to maintain decidualization, prevention of oxidative stress, immunomodulation, and inhibition of apoptosis.

A comparison of the molecular distribution of proangiogenic factors in endometrium of missed abortions and of voluntary first trimester termination cases.

OBJECTIVE: The authors aimed to evaluate the angiogenic changes that occur in the cases with missed abortions compared with the voluntary termination of pregnancy as control group, with this controlled clinical study. MATERIALS AND METHODS: The study included fifteen healthy volunteer women with unwanted pregnancy less than 10th gestational week in an academic research environment. The patients were 19 women between 6th and 11th gestational weeks diagnosed with missed abortion as the patient group. Immunohistochemistry was utilized to examine temporal and spatial expression of vascular endothelial growth factor (VEGF) and their two receptors: VEGF-R1 (Flt-1) and VEGF-R2 (Flk-1/KDR), and Trombospondin-1, eNOS, iNOS, and HIF-1α in the both deciduas and placenta of the both groups. RESULTS: This study discovered the significant difference (p < 0.005) between the groups of controlled and missed abortion in the decidual and placental cell components, and has put forward that thrombospondin and iNOS have an impact on abortion through antiangiogenic effect in cases of missed abortions. CONCLUSIONS: The potential role of molecules affecting angiogenesis in the etiology of missed abortion has been evaluated and the authors aimed for this to be a guide for studies on further treatments and on the prevention of the development of missed abortions.

The role of hypoxia related angiogenesis in uterine smooth muscle tumors.

Mechanisms of hypoxia-related angiogenesis are important for uterine smooth muscle tumors. Factors that are related to angiogenesis during hypoxia include vascular endothelial growth factor (VEGF), hypoxia inducible factor 1α (HIF1α), T-cell intracellular antigen1 (TIA1), eukaryotic translation initiation factor 2α (eIF2α) and thrombospondin 1 (TSP1). We investigated immunoreactivities of VEGF, HIF1α, TIA1, eIF2α and TSP1 using an indirect immunoperoxidase method for formalin fixed, paraffin embedded tumors that had been diagnosed as leiomyoma (LMY), cellular leiomyoma (CLM) or leiomyosarcoma (LMS). TSP1 immunoreactivity was scored as moderate, mild or minimal, while VEGF, eIF2α and TIA1 immunoreactivities were scored as mild, moderate and strong in LMY, CLM and LMS samples, respectively. HIF1α immunoreactivity was scored as mild to minimal in LMY, CLM and LMS samples, but showed no statistically significant differences among samples. Although angiogenic factors showed strong immunohistochemical staining intensity in LMS, anti-angiogenic factors showed minimal immunohistochemical intensity. There was no difference in HIF-1α immunoreactivity compared to LMY, CLM and LMS samples. We suggest that HIF1α protein synthesis could be suppressed by eIF2α and TIA1. Furthermore, VEGF could be activated by pathways such as COX2, Ras, NF-ĸB or c-myc instead of HIF1α. Angiogenesis could trigger and accelerate tumor development; therefore, anti-angiogenic therapy could be useful for treatment of tumors.

Effect of GnRH antagonist therapy on the expression of MUC-1 and heparin binding growth factor expression in the endometrium of hyperstimulated rats.

PURPOSE: To determine the effect of GnRH-antagonist therapy on the expression of heparin binding-epidermal growth factor (HB-EGF) and MUC-1 glycoprotein in hyperstimulated rat ovaries. METHODS: 30 female Wistar rats were divided into three groups (control, FSH and FSH+cetrorelix). Control rats were given 0.2 ml oil/saline mixture for four days beginning from the day of estrus. In the second group, 30 IU/ml purified hFSH was injected SC for four days beginning from the day of estrus. The rats of the third group were injected 30 IU FSH for four days and 10 IU cetrorelix SC for three or four days. The rats were sacrificed and the staining intensity of HB-EGF and MUC-1 of the epithelial cells and stromal cells of the endometrium of the rats was calculated by H-score. RESULTS: Slight MUC-1 immunoreactivity was seen in the epithelial and decidual cells of the control and FSH groups. In the FSH+cetrorelix group, moderate MUC-1 immunostaining appeared in the epithelial and desidual cells. In rats in the control and FSH+cetrorelix groups, HB-EGF immunoreactivity in the epithelial cells and decidual cells was moderate. Strong immunoreactivity was seen in the FSH group. When the MUC-1 H-score values were compared statistically with the control and other groups, FSH+cetrorelix immunoreactivity in epithelial and decidual cells were significantly different from control and FSH groups. HB-EGF immunoreactivity of the epithelium and decidua was similar in the control and FSH+cetrorelix groups, but epithelial and decidual immunoreactivity of the FSH group was different from the other two groups. CONCLUSION: Our findings suggest that GnRH antagonists exert direct effects on the expression of HB-EGF and MUC-1 expression in the rat endometrium.

Effect of recombinant human activated protein C on apoptosis-related proteins.

The recombinant human activated protein C (rhAPC) has been reported to reduce mortality in patients with severe sepsis. An anti-apoptotic effect of rhAPC in sepsis is known, but the mechanism through which it acts on the apoptotic pathway is still unclear. Therefore, immunopositivity of the apoptosis-related proteins Bcl-2, an anti-apoptotic protein, c-myc, a proliferative protein, p-21 and p-53, two apoptotic proteins, was determined after rhAPC treatment in a mouse sepsis model. Sepsis was induced by Escherichia coli endotoxin injection. Increased neutrophil infiltration and immunoreactivity to p53 and p21 were observed in the group with sepsis and these immunoreactivities were decreased by rhAPC treatment. In the sepstic group; immunopositivity of Bcl-2 and c-myc was mild and moderate, respectively. In conclusion; p21- and p53-mediated apoptosis was increased in the sepsis model, and for the first time it has been shown that rhAPC decreases sepsis-induced apoptosis resulting from increased p21 and p53 proteins.

Increased expression of tissue vascular endothelial growth factor and foetal liver kinase-1 receptor in seasonal allergic rhinitis and relevance to asthma component.

BACKGROUND: There is a difference in the extent of remodelling in allergic rhinitis (AR) and asthma. This may be attributed to the difference in local tissue response to these mediators. OBJECTIVES: The aim of this study was to compare vascular endothelial growth factor (VEGF) and its receptor foetal liver kinase (Flk)-1 expression between seasonal AR patients with or without asthma and non-allergic controls as well as that between AR patients with and without asthma. METHODS: Thirteen subjects with seasonal AR and six non-allergic controls were included in the study. Allergic sensitization was demonstrated by a skin prick test. Inferior turbinate thiny biopsies were obtained from both groups. Monoclonal mouse antibodies were used to demonstrate VEGF and Flk-1. Nasal mucosal endothelial cells' staining intensity was graded semi-quantitatively and the histochemical score (HSCORE) was calculated. In all samples, VEGF- and Flk-1-labelled vessels were counted for the assessment of vascular surface density (VSD). RESULTS: The mean HSCORE for VEGF and anti-VEGF-based VSD were significantly higher in the patient group (P=0.001 and 0.002, respectively). The mean HSCORE for Flk-1 and anti-Flk-1-based VSD in the patient group were significantly higher than those in the control group (P=0.016 and 0.028, respectively). Differences between the mean HSCORE for VEGF and anti-VEGF-based VSD in patients with pure AR and AR and asthma were insignificant (P=0.16 and 0.39, respectively). The mean HSCORE for Flk-1 and anti-Flk-1-based VSD in patients with pure AR were significantly lower than those in patients with AR and asthma (P=0.004 and 0.018, respectively). CONCLUSION: Angiogenic factor VEGF and its receptor Flk-1 is increased in AR. A similar increase in VEGF in AR with and without asthma despite a higher Flk-1 in AR patients with asthma may be a possible explanation for the presence of angiogenesis in the airway wall in patients with asthma but not in those with pure AR.

The effect of oestradiol and neta on immunohistochemical staining of iNOS and eNOS in coronary arteries of ovariectomized rats.

AIM: The postmenopausal period is associated with increased risk for coronary atherosclerosis, and the effect of hormone replacement therapy in reducing this risk is controversial. Previous studies reported that nitric oxide synthetase (NOS) level might be important for the development of atherosclerosis, but no study has shown the interaction between hormone replacement therapy and endothelial NOS and inducible NOS intensity on coronary arteries yet. Our goal was to find out the immunostaining intensity of endothelial NOS and inducible NOS in ovariectomized rats which received oestradiol and norethisterone treatment. METHODS: We performed bilateral ovariectomy in 15, female, 90-day-old Wistar rats with an average weight of 250 grams. After waiting for 4 weeks for the menopausal state, they were divided into 3 groups to receive either placebo, 0.1 mg/day 17-beta-oestradiol (group E2), or 0.1 mg/day 17-beta-oestradiol + 0.1 mg/day norethisterone acetate (group E2-NETA) for 5 weeks. Another group included 5, normal, adult, female intact rats and served as controls. At the end of the treatment, all rats were sacrificed and coronary arteries were stained with inducible NOS and endothelial NOS polyclonal antibodies using streptavidin-biotin technique. RESULTS: The immunostaining of inducible NOS was prominent in perivascular connective tissue of the ovariectomized group but not in the control group. The inducible NOS immunostaining immunoreactivity was not detected in either treated groups. Immunostaining intensity of endothelial NOS did not differ in any 4 groups with similar staining. CONCLUSION: The present findings indicate that hormone replacement therapy down-regulates iNOS expression in coronary arteries of ovariectomized rats, and reduced iNOS may likely be involved in estrogen's beneficial effects.

Increased vascular surface density in ovarian endometriosis.

Our goal in this study was to investigate the presence of angiogenesis-related factors in endometriomas by evaluating their vascular surface densities. Thirty ovarian samples were included in the study. Of these, ten were histologically confirmed endometriomas, ten were ovarian specimens in the follicular phase and ten were ovarian specimens in the luteal phase, serving as controls. Histological specimens were immunostained for von Willebrand factor (vWF: factor VIII-related antigen) and CD34. The area with the highest microvessel density in endometriosis and in the normal ovary was evaluated by using an intercept grid. All microvessels in a specific field (x 100 magnification) were counted and vascular surface density was measured, as 164.01 +/- 21.26 vs. 125.15 +/- 11.28 and 117.44 +/- 9.27 by using vWF, and as 172.97 +/- 25.64 vs. 138.65 +/- 32.21 and 120.34 +/- 18.40 by using CD34 in endometriotic, follicular and luteal ovarian samples, respectively (p < 0.001). The mean vascular surface density was significantly higher in endometriosis than in the ovarian samples of the follicular phase or the luteal phase. No significant difference was seen between normal ovarian samples. Endometriosis was associated with angiogenic properties. Having demonstrated elevated angiogenic factors in endometriotic samples, we concluded that activation of angiogenesis might be a key factor in the pathogenesis of endometriosis.

Immunohistochemical staining of IGF-I, IGF-binding proteins-1 and -3, and transforming growth factor beta-3 in the umbilical cords of preeclamptic patients.

BACKGROUND: To detect the immunoreactivity of insulin-like growth factor-I, insulin-like growth factor-binding proteins-1 and -3 and transforming growth factor beta-3 in the umbilical cords of normal and preeclamptic patients. METHODS: Umbilical cords were obtained from 15 normal and 15 preeclamptic patients. Immunoreactivities were determined using either indirect immunofluorescence or immunoperoxidase techniques on formalin-fixed, paraffin-embedded sections. Staining intensity was graded by a semiquantitative scoring method. The results were compared by Mann-Whitney U-test. RESULTS: The umbilical cords were thinner and the vessels were hypoplastic in the preeclamptic group. Moderate staining intensity for insulin-like growth factor-I, insulin-like growth factor binding protein-1 and -3 and transforming growth factor-beta 3 was observed in normal patients. The preeclamptic group had mild and strong intensities for insulin-like growth factor-I and insulin-like growth factor binding protein-1, respectively, and intensity for insulin-like growth factor binding protein-3 did not change, but diffuse and increased intensity was observed for transforming growth factor-beta 3. CONCLUSION: Changes in the intensity of insulin-like growth factor-I and its major binding protein and the transformation of growth factor-beta 3 may play a role in the pathogenesis of preeclampsia by altering the structure and responsiveness of the umbilical cord.

Insulin: does it induce follicular arrest in the rat ovary?

The goal of this study was to investigate histological changes of the rat ovary treated with either insulin or insulin plus human chorionic gonadotropin (hCG). The study was conducted in Celal Bayar University, School of Medicine, Animal Research Laboratory. Eighteen adult female Wistar rats were divided into three groups to receive saline, or insulin, or insulin plus hCG for 4 weeks. At the end of treatment the rats were sacrificed and the ovaries were evaluated with hematoxylin and eosin. There was no abnormal change in rats treated with saline. A thickened capsule, stromal hypertrophy and stromal cell hyperplasia, and no developing follicles, were observed in the insulin-only group. A thin capsule, developing follicles and corpora lutea, and normal theca cells and stroma were observed in the insulin-plus-hCG group. We conclude that insulin may lead to histological changes similar to stromal hyperthecosis and polycystic ovary syndrome, and may be one of the factors causing follicular arrest.

Clomiphene citrate does not affect the secretion of alpha3, alphaV and beta1 integrin molecules during the implantation window in patients with unexplained infertility.

BACKGROUND: The expression of integrin molecules on the endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation. A prospective, controlled study was carried out to investigate the effect of clomiphene citrate (CC) on secretions of beta1, alpha3 and alphaV integrin molecules in the endometrium of patients with unexplained infertility during the implantation window. METHODS: A total of 40 endometrial samples was evaluated in both spontaneous (n = 13) and ensuing clomiphene-treated cycles (100 mg on days 5-9) and also from fertile women serving as controls (n = 14) during postovulatory 7th or 8th day of menstrual cycle. A semiquantitative grading system (H-score) was used to compare the immunohistochemical staining intensities. Endometrial thickness and serum oestradiol and progesterone concentrations were also measured on the day of sampling. RESULTS: Staining of alpha(v) but not beta1 and alpha3 integrins was significantly less intense in infertile cases than fertile control cases (1.42 +/- 0.12 versus 2.21 +/- 0.13 respectively, P = 0.012) and this was not restored to normal concentrations with treatment. CONCLUSIONS: Our study indicated that cc treatment significantly decreased the endometrial thickness and increased oestradiol and progesterone concentrations. However, secretion of alpha(v), beta1 and alpha3 integrin molecules, which might play a role in implantation, was not affected.

Adenoids and otitis media with effusion: a morphological study.

PURPOSE: Adenoidectomy, especially for the treatment of suppurative otitis media, has been used for a very long time. In this study, the role of adenoids in the origin of otitis media with effusion was investigated by using light microscopy, immunocytochemistry, enzyme chemistry, and electron microscopy. MATERIALS AND METHODS: A group of 28 children with otitis media with effusion (OME) was identified. Ages ranged from 3 to 12 years. A control group of 10 age-matched children without any middle ear and upper respiratory tract infection served as the basis for comparison. Specimens obtained at surgeries from both groups were divided into groups for light microscopy, immunocytochemistry, enzyme cytochemistry, and electron microscopy and then all were examined blindly. Also, quantitative analysis of antigen-presenting cells was performed blindly on 10 patients and 10 controls. RESULTS: There was an increase in the number of lymphocytes, mast cells, plasma cells, macrophages, dendritic cells, and M cells in the adenoids of patients with OME when compared with the normal cases. Stratified squamous epithelial areas, collagenous fibers, and fibrocytes were also increased in the patient group. Antigen-presenting functions of epithelial cells are shown by major histocompatibility complex (MHC) class II positivity of some ciliated-columnar epithelial cells in the patient group. CONCLUSION: Adenoid tissues of patients with OME in this study seem to be infectious foci, aggravating immune reactions, which might attack the middle ear through an ascending route.