Kemal Ozbilgin

Nasal mucosal expression of nitric oxide synthases in patients with allergic rhinitis and its relation to asthma

BACKGROUND Nitric oxide (NO) has contradictory roles in the pathophysiology of allergic inflammation in both allergic rhinitis (AR) and asthma. Small amounts of NO produced by constitutive NO synthase (NOS) is anti-inflammatory, whereas large amounts produced by inducible NOS (iNOS) are proinflammatory. OBJECTIVE To investigate the difference in constitutive endothelial NOS (eNOS) and iNOS expression in nonallergic and allergic mucosa and the possible relation of this to the coexistence of asthma in seasonal AR. METHODS Seventeen patients (10 women and 7 men) with seasonal AR and 9 nonallergic patients (5 women and 4 men) with nasal septum deviation were enrolled. Inferior turbinate nasal biopsy specimens were obtained in all. Levels of eNOS and iNOS expressed as immunohistochemical scores (HSCOREs) were determined immunohistochemically from the specimens. RESULTS The mean +/- SD HSCOREs for eNOS in patients with seasonal AR were not significantly different from those of the nonallergic controls (1.85 +/- 0.78 vs 1.63 +/- 0.54; P = .12). On the other hand, the mean +/- SD HSCOREs for iNOS were significantly higher in patients with seasonal AR (1.75 +/- 0.75 vs 0.71 +/- 0.6; P = .004). Furthermore, although eNOS expression was not different between seasonal AR patients with and without asthma, the mean +/- SD HSCOREs for iNOS were significantly higher in the patients with asthma (1.93 +/- 0.78 vs 1.65 +/- 0.55; P = .01). CONCLUSION Increased expression of iNOS might have a role in the development of allergic inflammation in upper and lower airways and in comorbidity of AR and asthma.

Regulatory-T, T-helper 1, and T-helper 2 cell differentiation in nasal mucosa of allergic rhinitis with olive pollen sensitivity.

Background: Allergic rhinitis (AR) is a disease in which T-helper (Th)2 response is predominant and its pathogenic mechanism is still poorly understood. Aim: To evaluate the possible role of Th1, Th2 and regulatory-T (Treg) cells in the pathogenesis of AR. Methods: This case-control study enrolled 41 patients with seasonal AR (10–62 years old), sensitive to olive pollens, and 15 healthy controls (18–60 years old). Nasal biopsy was performed and specimens of nasal lavage fluid were obtained from all participants. The levels of interleukin (IL)-4, IL-10, interferon (IFN)-γ and transforming growth factor-β (TGF-β) were measured in nasal lavage fluid specimens. The expression of FOXP3, GATA-3 and T-bet was measured by immunohistochemical methods in the nasal biopsy specimens. Results: The levels of IFN-γ in the group with AR were significantly lower than those in the control group (p = 0.008). The levels of IL-4, IL-10 and TGF-β did not differ between the two groups. The expression of FOXP3 and T-bet in patients with AR was significantly lower than that in the control group (both p = 0.001). Expression of GATA-3 in the nasal mucosa was similar between the groups (p = 0.2). The ratios of T-bet/GATA-3 and FOXP3/GATA-3 in the AR group were significantly lower than those in the control group (p = 0.001). Conclusion: Insufficient Treg and Th1 cells may be associated with the allergic inflammation that may be attributed to the Th2 immune response in patients suffering from AR who are sensitive to olive pollen.

Effects of 5-fluorouracil and gemcitabine on a breast cancer cell line (MCF-7) via the JAK/STAT pathway.

Aberrant activation of the JAK/STAT pathway may predispose to malignancy as a consequence of the deregulation of cell proliferation, differentiation or apoptosis such as in cancer of the blood, head and neck, and breast. In our study we aimed to investigate the effects of 5-fluorouracil (5-FU) and gemcitabine on a breast cancer cell line (MCF-7 cells) via the JAK/STAT pathway. Distribution of JAK1, JAK2, JAK3 and STAT2, STAT3, STAT4, STAT5 were evaluated on MCF-7 cells following gemcitabine and 5-FU treatment and in the absence of drug treatment by an indirect immunohistochemical method. It was observed that JAK1, JAK3, STAT5 and particularly STAT2 activation were more effective than the other JAK/STATs in breast cancer progression. Following treatment with 5-FU, JAK1 and STAT5 immunoreactivities were decreased in MCF-7 cells in comparison with both gemcitabine-treated and non-treated groups. These results suggest that the JAK/STAT pathway plays an important role in breast cancer pathogenesis and may be more affected after 5-FU treatment rather than gemcitabine. Drugs which block STAT5 may provide a novel therapeutic approach for the treatment of breast cancer.

Comparative effects of risedronate, atorvastatin, estrogen and SERMs on bone mass and strength in ovariectomized rats

OBJECTIVE The aim of this study was to investigate bone protective effects of risedronate, atorvastatin, raloxifene and clomiphene citrate in ovariectomized rats. METHODS Our study was conducted on 63 rats at Experimental Research Center of Celal Bayar University. Six-month-old rats were divided into seven groups. There were five drug administered ovariectomized groups, one ovariectomized control group without drug administration and one non-ovariectomized control group without drug administration. Eight weeks postovariectomy, rats were treated with the bisphosphonate risedronate sodium, the statin atorvastatin, the estrogen 17beta-estradiol and the selective estrogen receptor modulators (SERMs) raloxifene hydrochloride and clomiphene citrate by gavage daily for 8 weeks. At the end of the study, rats were killed under anesthesia. For densitometric evaluation, left femurs and tibiae were removed. Left femurs were also used to measure bone volume. Right femurs were used for three-point bending test. RESULTS Compared to ovariectomized group, femur cortex volume increased significantly in non-ovariectomized group (p=0.016). Compared to non-ovariectomized group, distal femoral metaphyseal and femur midshaft bone mineral density values were significantly lower in ovariectomized group (p=0.047). In ovariectomy+atorvastatin group, whole femur and femur midshaft bone mineral density and three-point bending test maximal load values were significantly higher than ovariectomized group (p=0.049, 0.05, and 0.018). When compared to the ovariectomized group, no significant difference was found with respect to femoral maximum load values in groups treated with risedronate, estrogen, raloxifene and clomiphene (p=0.602, 0.602, 0.75, and 0.927). In ovariectomy+risedronate group, femur midshaft bone mineral density values were significantly higher than the values in ovariectomized group (p=0.023). When compared to ovariectomized group, no significant difference was found with respect to femur midshaft bone mineral density values in groups treated with estrogen, raloxifene and clomiphene (p=0.306, 0.808, and 0.095). CONCLUSIONS While risedronate sodium prevented the decrease in bone mineral density in ovariectomized rats, atorvastatin maintained mechanical characteristics of bone and also prevented the decrease in bone mineral density as risedronate sodium.

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 (× 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.

A histological analysis of the protective effect of ischemic preconditioning in the rat retina.

Purpose. Ischemic preconditioning (IP) protects the retina from the damaging effect of subsequent ischemia in vivo. We aimed to investigate the histological alterations induced by the protective effect of IP to the retina. Methods. The eyes of the rats were rendered ischemic by intra-ocular pressure (IOP) elevation. IP procedure consisted of producing ischemia for 5 minutes. Sham operation was similar to IP procedure except the pressure elevation. The operational eyes of sham and IP group underwent 60 minutes of ischemia 24 hours after the first procedure. The eyes contralateral to the experimental eyes made up the control group. The eyes were histologically analysed one week after the ischemia. Results. The total retinal thickness of the sham group was significantly less than total retinal thickness of the control group (p < 0.001). There was not a significant difference between control and IP group regarding the total retinal thickness (p > 0.05). The thickness of the inner retinal layers of the sham group were significantly less than corresponding retinal layers of the control group (p < 0.001). The inner plexiform layer (IPL) and inner nuclear layer (INL) thickness values of the sham group were significantly less than same layers of the IP group (p < 0.001). Ganglion cell layer (GCL) thickness of the IP group was significantly less than GCL thickness of the control group (p < 0.001). IPL thickness of the IP group was significantly less than that of control groups (p < 0.05). The GCL and total retinal thickness of the IP group were significantly more than thickness of the corresponding layers of the sham group (p < 0.05). Conclusion. IP considerably protects inner retinal layers from subsequent ischemic damage in a high IOP ischemic model. This endogenous process could further be utilized to tailor specific neuroprotective strategies for retinal cells.

Effect of raloxifene and atorvastatin in atherosclerotic process in ovariectomized rats

Aim:  The goal of this study was to investigate the combined effects of raloxifene and atorvastatin in aged ovariectomized rats during endothelial dysfunction and atherosclerotic process.

Effects of allergen-specific immunotherapy on functions of helper and regulatory T cells in patients with seasonal allergic rhinitis.

BACKGROUND Seasonal allergic rhinitis (SAR) is characterized by a helper T (Th)2 cell-mediated immune response at the target site. There is a relative Th1 and/or regulatory T (Treg) cell insufficiency in patients with SAR. It has been demonstrated that there is a change in the balance between these cells after allergen-specific immunotherapy (SIT), which is a curative treatment modality for this disease. However, there are few studies that evaluate the number and function of these cells in the inflammatory area after SIT treatment. OBJECTIVE We aimed to investigate the distribution of Th1, Th2 and Treg cells in nasal biopsies and lavage fluid (NLF) specimens from patients with SAR, before and after SIT. METHODS Twenty-four, symptomatic SAR patients sensitized to Olea europeae, were enrolled in the study prior to treatment. Fifteen, non-allergic subjects with nasal septum deviation, who needed surgical treatment, served as the control group. NLF and inferior turbinate biopsies were obtained from both groups during the pollen season. Conventional, subcutaneous SIT with Olea europeae extract was initiated in patients with SAR. One year after the first biopsy, biopsies and NLF specimens were again obtained for reevaluation. All biopsies were evaluated for Th1, Th2 and Treg cell counts by means of their transcription factors (T-bet, GATA-3 and FoxP3) using an immunohistochemical analysis method. Additionally, all NLF specimens were evaluated for the functions of these cells, by means of their specific cytokines, using an ELISA method. RESULTS When the basal status of those patients with SAR was evaluated based on transcription factors, prior to treatment, Th1 and Treg cells were found to be fewer than in non-allergic controls (p=0.001 for both T-bet and FoxP3). It was demonstrated that numbers of GATA-3-carrying cells, which are a marker for Th2, were not significantly different between the groups (p=0.276), but evaluation of the Th1/Th2 ratio revealed a relative Th2 dominance in patients with SAR prior to treatment. When evaluated on the basis of cytokine levels, it was observed that Th1-originated IFN-γ was lower in patients with SAR compared to the control group, both before and after treatment (p=0.012 for both comparisons), Th2-originated IL-4 levels were not significantly different between the groups either before or after treatment (p=0.649, p=0.855; respectively). Th2- and Treg cell-originated IL-10 levels were higher in patients with SAR before treatment (p=0.033), but this difference was not statistically signifant following treatment compared with controls (p=0.174). Treg cell-originated TGF-β levels were slightly lower in patients with SAR compared to the controls, although the difference was not statistically significant (p=0.178, p=0.296; respectively). None of the above mentioned cytokine levels changed significantly as a result of SIT. CONCLUSION The results of our study indicate that although clinical findings improve after one year of SIT, this duration may not be sufficient to detect changes in cytokine patterns and transcription factors. Further studies that evaluate outcome over a longer duration of treatment would provide valuable information.

Effect of ovarian stimulation with human menopausal gonadotropin and recombinant follicle stimulating hormone on the expression of integrins alpha(3), beta(1) in the rat endometrium during the implantation period

OBJECTIVE To investigate the effect of exogenous ovarian stimulation with human menopausal gonadotropin (hMG) and recombinant follicle stimulating hormone (rFSH) on the expression of integrins alpha(3), beta(1) in the rat endometrium during implantation. STUDY DESIGN Following three successive normal estrous cycles the animals were divided into five groups: Group I (n=10, control group) received no medication; Group II (n=10) received 10 units of hMG; Group III (n=10) received 20 units of hMG; Group IV (n=10) received 10 units of rFSH; Group V (n=10) received 20 units of rFSH at midday of middiestrous. The rats were then mated with fertile males. The animals were sacrificed on the day of implantation. The uterine horns were placed in fixative and paraffin blocks of the tissue were cut in 5 microm sections. The tissues were stained with primary antibodies; monoclonal anti-integrin alpha(3) and monoclonal anti-integrin beta(1) using immunohistochemical methods. The staining intensities of alpha(3) and beta(1) integrins were calculated separately for epithelium and stroma in each group. RESULTS Staining intensities of alpha(3) and beta(1) integrins in both the epithelium and the stroma were significantly lower in the treatment groups than the control group (p<0.05). CONCLUSION Ovarian stimulation by low and high doses of HMG and rFSH may have an effect on endometrial receptivity, possibly via a decrease in expression of integrins in the endometrium during the implantation period.

The role of hypoxia related angiogenesis in uterine smooth muscle tumors

Abstract 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.