Molecular Status of BRAF Mutation in Epithelial Ovarian Cancer: An Analysis of 57 Cases in the Northeast of Iran.

Background & Objective: Epithelial ovarian cancer (EOC) is the most prevalent type of ovarian cancer. Previous studies have elucidated different pathways for the progression of this malignancy. The mutation in the B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene, a member of the MAPK/ERK signaling pathway, plays a role in the development of EOC. The current study aimed to determine the frequency of the BRAF V600E mutation in ovarian serous and mucinous tumors, including borderline and carcinoma subtypes. Methods: A total of 57 formalin-fixed paraffin-embedded samples, including serous borderline tumors (SBTs), low-grade serous carcinomas (LGSCs), high-grade serous carcinomas (HGSCs), mucinous borderline tumors (MBTs), and mucinous carcinomas, and 57 normal ovarian tissues were collected. The BRAF V600E mutation was analyzed using polymerase chain reaction (PCR) and sequencing. Results: While 40% of the SBT harbor BRAF mutation, we found no BRAF mutation in the invasive serous carcinoma (P=0.017). Also, there was only 1 BRAF mutation in MBT and no mutation in mucinous carcinomas. In addition, we found no mutation in the control group. Conclusion: The BRAF mutation is most frequent in borderline tumors but not in invasive serous carcinomas. It seems that 2 different pathways exist for the development of ovarian epithelial neoplasms: one for borderline tumors and the other for high-grade invasive carcinomas. Our study supports this hypothesis. The BRAF mutation is rare in mucinous neoplasms.

Assessment of the Tumor-Stroma Ratio and Tumor-Infiltrating Lymphocytes in Colorectal Cancer: Inter-Observer Agreement Evaluation.

BACKGROUND: To implement the new marker in clinical practice, reliability assessment, validation, and standardization of utilization must be applied. This study evaluated the reliability of tumor-infiltrating lymphocytes (TILs) and tumor-stroma ratio (TSR) assessment through conventional microscopy by comparing observers' estimations. METHODS: Intratumoral and tumor-front stromal TILs, and TSR, were assessed by three pathologists using 86 CRC HE slides. TSR and TILs were categorized using one and four different proposed cutoff systems, respectively, and agreement was assessed using the intraclass coefficient (ICC) and Cohen's kappa statistics. Pairwise evaluation of agreement was performed using the Fleiss kappa statistic and the concordance rate and it was visualized by Bland-Altman plots. To investigate the association between biomarkers and patient data, Pearson's correlation analysis was applied. RESULTS: For the evaluation of intratumoral stromal TILs, ICC of 0.505 (95% CI: 0.35-0.64) was obtained, kappa values were in the range of 0.21 to 0.38, and concordance rates in the range of 0.61 to 0.72. For the evaluation of tumor-front TILs, ICC was 0.52 (95% CI: 0.32-0.67), the overall kappa value ranged from 0.24 to 0.30, and the concordance rate ranged from 0.66 to 0.72. For estimating the TSR, the ICC was 0.48 (95% CI: 0.35-0.60), the kappa value was 0.49 and the concordance rate was 0.76. We observed a significant correlation between tumor grade and the median of TSR (0.29 (95% CI: 0.032-0.51), p-value = 0.03). CONCLUSIONS: The agreement between pathologists in estimating these markers corresponds to poor-to-moderate agreement; implementing immune scores in daily practice requires more concentration in inter-observer agreements.

Comparison of two routes of administration of a cationic liposome formulation for a prophylactic DC vaccination in a murine melanoma model.

Dendritic cell (DC) vaccination can be achieved via straight loading of vaccine into DCs ex vivo or administration to DCs in vivo. However, there is no certain consensus on which approach is preferable, and each strategy has its advantages and disadvantages, which affect the efficacy and safety of vaccines. It will also be more complicated when a vaccine delivery system is included. In this study, the efficacy of ex vivo pulsed DC-based vaccine compared with in vivo subcutaneous administration of a cationic liposomes (CLs) formulation containing gp100 antigen (gp100-CLs) was evaluated in a murine melanoma model. In combination with an anti-PD-1 antibody, the ex vivo approach of gp100-CLs yielded a significant (P < 0.01) increase in the number of antigen-specific tumors infiltrated lymphocytes (TILs) with a significant upregulation of IFN-γ (P < 0.0001) and PD-1 (P < 0.0001) expression level. They also dampened the function of immunosuppressive regulatory T cells (Tregs) via significant downregulation of IL-10 and TGF-ß (P < 0.0001) expression level compared to in vivo approach in the tumor microenvironment (TME). Furthermore, prophylactic immunization with gp100-CLs pulsed DCs ex vivo delayed tumor growth and induced the survival benefit over in vivo immunization. Collectively, the ex vivo DC-based vaccination pulsed with gp100 encapsulated in liposomes synergizes with anti-PD-1 antibody and represents a preferable approach against melanoma.

Ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of liposomal antigen and CpG-ODN improved PD-1 blockade immunotherapy.

Lack of pre-existing tumor infiltrated T cells resulting in resistance to programmed cell death protein 1 (PD-1) blockade therapies can be solved by combining with anti-cancer vaccines and CpG-ODN in increasing T cell expansion and infiltration. Therefore, we prepared an ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of either liposomal or non-liposomal gp100 antigen (2.8 µg) plus CpG-ODN (800 ng) formulations and evaluated its anti-tumor activity in combination with anti-PD-1 therapy. Our results showed a combination of liposomal peptide plus CpG-ODN pulsed DC with anti-PD-1 antibody was more efficacious, as evidenced by a significant increase in Teff/Treg TILs with a marked fourfold elevation of IFN-γ expression level in the tumor site of treated mice which reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, this combination also led to a remarkable tumor remission and prolonged survival rate in melanoma-bearing mice compared to non-liposomal peptide plus CpG-ODN or single-treated liposomal peptide formulations. Our results provide essential insights to devise combining regimens to improve the efficacy of immune checkpoint blockers even by a low dose of peptide and CpG-ODN.

Human T-Cell Leukemia Virus Type 1 Changes Leukocyte Number and Oxidative Stress in the Lung and Blood of Female BALB/c Mice.

BACKGROUND: Human T-cell leukemia virus type 1(HTLV-1) infection is likely to induce nonneoplastic inflammatory pulmonary diseases. Therefore, an experimental study was conducted to evaluate the leukocytes' number alteration and oxidative stress in the lung and blood of HTLV-1-infected BALB/c mice, which could be of benefit for the recognition of HTLV-1 mechanism in the induction of pulmonary disorders. MATERIALS AND METHODS: Twenty female BALB/c mice were divided into two groups of control and HTLV-1-infected animals. The HTLV-1-infected group was inoculated with 106 MT-2 HTLV-1-infected cells. Two months later, the infection was confirmed using real-time polymerase chain reaction, and then lung pathological changes, total and differential inflammatory cell counts in the blood and bronchoalveolar lavage fluid (BALF), along with oxidative stress biomarker levels in the BALF and lung tissue were evaluated. RESULTS: In the HTLV-1-infected group, the peribronchitis score (P < 0.01), the number of total leukocytes, neutrophils, lymphocytes, and monocytes (P < 0.05) in the blood and BALF were increased. The number of eosinophils in the blood of the HTLV-1-infected group was higher than in the control group (P < 0.01), whereas the number of basophils of BALF was increased in the HTLV-1-infected group (P < 0.001). The lung and BALF oxidative stress results showed that the MDA level was increased, while the total thiol level and superoxide dismutase activity were decreased in the HTLV-1-infected group (P < 0.01). CONCLUSION: The HTLV-1 infection seems to induce pulmonary inflammatory reactions by recruiting leukocytes as well as inducing oxidative stress in the lung tissue.

Liposomal gp100 vaccine combined with CpG ODN sensitizes established B16F10 melanoma tumors to anti PD-1 therapy.

OBJECTIVES: Program death 1 (PD-1)/ program death-ligand 1 (PD-L1) pathways, as the main inhibitory checkpoints, induce immunosuppression in the tumor microenvironment (TME). Despite the importance of inhibitor checkpoint receptor (ICR) blockers, their outcomes have been limited by the low immune response rate and induced acquired resistance. Pre-existing tumor-specific T cells is related to the improvement of their therapeutic efficacy. In the present study, we show that the combination of liposomal gp100 nanovaccine with anti PD-1 monoclonal antibody (mAb) potentiates the therapeutic effect in the melanoma model. MATERIALS AND METHODS: In this study, we first decorate the cationic liposome with gp10025-33 self-antigen and then characterize it. Mice bearing B16F10 melanoma tumors were vaccinated with different formulations of gp100 peptide (free or liposomal form) with or without CpG ODN adjuvant in combination with anti PD-1 mAb. RESULTS: Therapeutic combination of liposomal nanovaccine and CpG with anti PD-1 mAb, demonstrated the increased number of tumor infiltrated lymphocytes (TILs) in TME with the highest IFN-γ production and cytotoxic activity, which led to remarkable tumor regression. CONCLUSION: Our results demonstrated the synergism between Lip-peptide+CpG nanovaccine and anti PD-1 regime, which improved the therapeutic efficacy of PD-1 checkpoint blocker in melanoma mice models.

Matrix metalloproteinase-9 (MMP-9) Expression in Non-Small Cell Lung Carcinoma and Its Association with Clinicopathologic Factors.

BACKGROUND & OBJECTIVE: Matrix metalloproteinases-9 (MMP-9) is one of the most important enzymes to breakdown extracellular matrix which plays a major role in tumor invasion and metastasis. This study aimed to determine tumor MMP-9 expression in non-small-cell lung carcinoma (NSCLC) and whether it is associated with histopathologic factors and has prognostic value to affect overall survival (OS). METHODS: The specimens of 92 patients with NSCLC diagnosis were included. Tumor sections were stained by immunohistochemistry method. Using scores for the percentage of cells positively stained and the intensity of staining, MMP-9 expression total score was classified as low-score (scores of 0 to 2), moderate-score (scores of 3 to 5), or high-score (scores of 6 or 7). OS was defined as the time interval since the diagnosis of NSCLC to the status at the last follow-up (dead or alive). The follow up period was up to 70 months. RESULTS: About 74% of undifferentiated specimens (grade III tumors) showed high scores for MMP-9 expression which was significantly higher than moderately differentiated tumors (25% had high scores for MMP-9 expression) and well differentiated ones which did not have high scores (P<0.001). A total of 74 patients (80.4%) died during the follow-up period. Of this, 36% had high scores for MMP-9 expression. In contrast, none of the patients who were alive at the last follow-up had high scores for MMP-9 expression (P<0.001). Median OS was significantly lower in high score group (6 months) compared to moderate score (9 months) and high score group (15 months) (P<0.001). CONCLUSION: MMP-9 expression may serve as a significant prognostic factor for mortality and overall survival in NSCLC. Undifferentiated tumors significantly express higher MMP-9 immunohistochemically.

Evaluation of CD133 and CD56/NCAM expression in Wilms tumor and their association with prognostic factors.

OBJECTIVES: To validate certain markers for cancer stem cell populations and their clinical importance in Wilms tumor (WT). MATERIALS AND METHODS: Immunohistochemical study for CD133 and CD56/NCAM was performed on forty-six cases of WT that were diagnosed between 1999 and 2015, and the association of these markers with survival and prognostic factors was analyzed. RESULTS: Thirty-four (73.9%) of WTs were positive for CD133 and thirty-nine (84.8%) were positive for CD56/NCAM. A significant positive correlation between CD133 and CD56/NCAM expression and the National Wilms Tumor Stage (NWTS) and death was found. Moreover, overall survival time was significantly correlated with CD133 and CD56/NCAM H-score, NWTS stage, and death. CONCLUSION: It seems that CD133 and CD56/NCAM expressions can be used as strong prognostic parameters for the survival of patients with WT and can be used to predict WT patients' stage. Moreover, their targeted therapies can abolish cancer stem cells in children with recurrent tumors.

Vaccination with dendritic cells pulsed ex vivo with gp100 peptide-decorated liposomes enhances the efficacy of anti PD-1 therapy in a mouse model of melanoma.

BACKGROUND: Targeting antigens to dendritic cells (DCs) via nanoparticles is a powerful strategy which improves the efficacy of ex vivo antigen-pulsed DC vaccines. METHODS: In this study, liposomes were first decorated with gp10025-33 self-antigen and then characterized. Then, DCs were pulsed ex vivo with liposomal gp100 and injected subcutaneously in mice bearing B16F10 established melanoma tumors in combination with anti-PD-1 therapy. RESULTS: Treatment with liposomal pulsed DC vaccine elicited the strongest anticancer immunity and enhanced intratumoral immune responses based on infiltration of gp100-specific CD4+ and CD8+ T cells to the tumor leading to significant tumor growth regression and prolonged survival rate. Treatment with liposomal pulsed DC vaccine also markedly enhanced specific cytotoxic T lymphocytes (CTL) responses with a significant higher titer of IFN-γ in the spleen. Moreover, a significant increase of PD-1 expressing CD8+ tumor infiltrating lymphocytes (TILs) was detected in tumors. CONCLUSION: Our results demonstrate an optimum dose of liposomal gp100 significantly increases the efficacy of anti-PD-1 therapy in mice and might be an effective strategy to overcome resistance to anti-PD-1 therapy.

Rosuvastatin suppresses cytokine production and lung inflammation in asthmatic, hyperlipidemic and asthmatic-hyperlipidemic rat models.

BACKGROUND: Given the role that T lymphocytes play on the pathogenesis of allergic asthma, drugs targeting Th2 and Th17 cells may be a hopeful therapeutic strategy. This study aimed to evaluate the effect of rosuvastatin treatment on cytokine production and lung inflammation in allergic asthma. METHODS: The animals were assigned into control (C), asthmatic (A), hyperlipidemic (H), asthmatic-hyperlipidemic (AH), rosuvastatin (40 mg/kg/day intraperitoneally, for 3 weeks)-treated asthmatic (AR), rosuvastatin-treated hyperlipidemic (HR) and rosuvastatin-treated asthmatic-hyperlipidemic (AHR) groups (n = 6 in each group). The levels of IL-4, IFN-γ and IL-17, total and differential WBC counts in bronchoalveolar lavage fluid (BALF), Th1/Th2 balance, and pathological changes were evaluated. RESULTS: The BALF level of IL-4 in A, H and AH groups, and IL-17A in A and AH groups were significantly higher than that in C group (p < 0.05 to p < 0.001). IFN-γ level and Th1/Th2 balance (IFN­Î³/IL-4 ratio) in A and AH groups were significantly decreased (p < 0.05 to p < 0.01). Inflammatory cells infiltration, muscle hypertrophy and emphysema were also observed in A and AH groups. The BALF levels of IL-4 in AR, HR and AHR groups, IFN-γ level in HR group, and IL-17A level in AR and AHR groups showed a significant improvement compared to that of A, H and AH groups (p < 0.05 to p < 0.001). Rosuvastatin treatment increased Th1/Th2 balance in all treated groups (p < 0.05 to p < 0.01), decreased total WBC counts, neutrophilia, eosinophilia and lung inflammation in AR and AHR groups, and improved muscle hypertrophy and emphysema in AHR group. CONCLUSIONS: Rosuvastatin treatment improved lung pathological changes by suppression of Th2 and Th17-mediated cytokines which was unrelated to its lipid-lowering activity. Therefore, rosuvastatin might be a candidate immunomodulatory drug for treatment of patients with allergic asthma.

Epstein-Barr Virus and Human Herpesvirus 8 in Idiopathic Pulmonary Fibrosis.

BACKGROUND & OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a chronic and uniformly fatal interstitial lung disease with incompletely understood pathogenesis. Several studies have given the evidence for and against viral cofactors in the pathogenesis of Idiopathic pulmonary fibrosis. In this study Epstein-Bar Virus (EBV) and Human Herpesvirus 8 (HHV-8) have been studied for a possible role in the pathogenesis of IPF. METHODS: Polymerase chain reaction (PCR) was employed for the detection of EBV and HHV-8 in 58 formalin-fixed paraffin-embedded lung tissue specimens (29 controls and 29 IPF specimens). RESULTS: EBV DNA was present in the lung tissue of 6 out of 29 (20.7%) IPF specimens compared with 1 out of 29 (3.4%) controls (P=0.102). The HHV-8 gene was identified in 3 out of 29 (10.3%) cases of IPF specimens. The control group showed no evidence of HHV-8 gene (P=0.227). CONCLUSION: Although multiple studies are strongly suggestive of a role for EBV and HHV-8 in the development of IPF, there was no statistically significant difference in the prevalence of EBV and HHV-8 DNA in the IPF specimens and controls in this study.

The Relationship Between Fibroblastic Growth Factor Receptor-1 (FGFR1) Gene Amplification in Triple Negative Breast Carcinomas and Clinicopathological Prognostic Factors.

BACKGROUND & OBJECTIVE: In Triple-Negative Breast Cancers (TNBCs), estrogen receptor (ER), progesterone receptor (PR) and HER2/neu genes are not expressed. Fibroblastic Growth Factor Receptor-1 (FGFR1) gene product is a protein that acts as a receptor of thyrosin kinase. It plays a role in the proliferation, differentiation, and migration of malignant cells. The objective was to evaluate the possible relation between FGFR1 over-expression and amplification in TNBCs and other clinicopathological variables. METHODS: In this cross sectional study, purposive sampling was used to collect eighty-four TNBC specimens from mastectomy specimens collected between 2013 and 2017. Tissue microarrays were evaluated for FGFR1 over-expression and amplification respectively by immunohistochemistry (IHC) staining and real time Polymerase Chain Reaction (PCR). The needed clinical and paraclinical information were obtained from patients' files. To analyze the correlation among prognostic factors, we used a wide range of different statistic methods, namely Chi-square test, independent t-test, Fisher's exact test, and ANOVA. RESULTS: FGFR1 over-expression was found in 15 of the 84 samples (17.9%). FGFR1 gene amplification was observed in 33.3% (28 of 84) of the samples. We found no association between FGFR1 and clinicopathological parameters, including tumor grade, stage, and patient survival (P>0.005). CONCLUSION: FGFR1 over-expression and amplification may not be related to clinicopathological parameters, namely age, stage, and grade of the cancer not to mention TNBC survival. Using FGFR1 as a prognostic factor in TNBCs requires further study.

Vascular Mimicry Expression in Invasive Ductal Carcinoma; A New Technique for Prospect of Aggressiveness.

BACKGROUND & OBJECTIVE: In vascular (vasculogenic) mimicry (VM), tumoral cells mimic the endothelial cells and form the extracellular matrix-rich tubular networks. It has been proposed that VM is more extensive in aggressive tumors. This study was designed to investigate the rate of VM expression in the stromal cells of invasive ductal carcinoma (IDC) and to find its relationship with other clinicopathological factors. METHODS: In this cross-sectional study, 120 patients with histopathologic diagnosis of IDC who received mastectomy were included. The VM expression was determined by immunohistochemistry (IHC). The clinicopathologic data including age, tumor size, histological grade, clinical stage, axillary lymph node metastasis, hormonal receptors, and survival were documented. RESULTS: The mean (±SD) age of the patients was 51 (±13.83) years old. The stromal VM expression was detected in 16 of 120 patients (13.3%). Twelve specimens (75%) of positive VM expression group had grade 3 which was higher than negative VM expression group (9 cases, 8.65%; P<0.001). The VM expression showed statistically significant relationship with higher histologic grade higher clinical stage (stage 3) of the tumor (62.5% vs. 87%; P=0.003), the presence of axillary lymph node metastasis (95.6% vs. 55.8%; P<0.001), and positive HER-2 (100% vs. 31.1%; P<0.001); but not estrogen receptor (ER) or progesterone receptor (PR). However, age, tumor size and mortality rate were not significantly different among the patients with and without VM expression. CONCLUSION: The stromal VM expression showed significant relationship with higher stage and grade of the tumor and the presence of nodal metastasis. The VM expression in IDC can be used as a marker for tumor aggressiveness.

Evaluation of Cyclooxygenase-2 Expression in Association with Clinical-Pathological Factors in Malignant Melanoma.

BACKGROUND AND OBJECTIVE: The primary goal of this study is to develop a rigorous understanding of the correlation between COX-2 expression and malignant melanoma prognostic factors. MATERIAL AND METHODS: In this cross-sectional study, we analyzed 60 cases of cutaneous malignant melanoma. The related stained slides were reviewed by two pathologists. The results were interpreted according to the COX2 staining index (SI), tumor thickness (Breslow, Clark), number of mitoses per 10 hpf, and melanoma types. Gender, lymph node involvement, metastasis, and survival were considered as evaluation factors as well. RESULTS: The expression of the COX-2 protein was evident in 98.4% of cases. A strong Staining Index(SI) was reported in 60% of all melanomas, moderate staining was detected in 20.8% and weak staining in 10%; 1.6% of studied cases showed no staining. Benign nevus specimens showed no staining for the COX-2 enzyme. CONCLUSION: We have demonstrated that COX-2 is strongly expressed in the majority of malignant melanomas and that the SI score of COX-2 is related to the number of mitoses, tumor thickness (based on Clark level and Breslow), melanoma sub-type, lymph node involvement, and metastases; No association was noted between the anatomic site, gender, and survival. COX-2 can be applied as a prognostic factor in malignant melanoma and a promising candidate for future target therapies.

Aminoguanidine affects systemic and lung inflammation induced by lipopolysaccharide in rats.

BACKGROUND: Nitric oxide is a mediator of potential importance in numerous physiological and inflammatory processes in the lung. Aminoguanidine (AG) has been shown to have anti-inflammation and radical scavenging properties. This study aimed to investigate the effects of AG, an iNOS inhibitor, on lipopolysaccharide (LPS)-induced systemic and lung inflammation in rats. METHODS: Male Wistar rats were divided into control, LPS (1 mg/kg/day i.p.), and LPS groups treated with AG 50, 100 or 150 mg/kg/day i.p. for five weeks. Total nitrite concentration, total and differential white blood cells (WBC) count, oxidative stress markers, and the levels of IL-4, IFN-γ, TGF-ß1, and PGE2 were assessed in the serum or bronchoalveolar lavage fluid (BALF). RESULTS: Administration of LPS decreased IL-4 level (p < 0.01) in BALF, total thiol content, superoxide dismutase (SOD) and catalase (CAT) activities (p < 0.001) in BALF and serum, and increased total nitrite, malondialdehyde (MDA), IFN-γ, TGF-ß1 and PGE2 (p < 0.001) concentrations in BALF. Pre-treatment with AG increased BALF level of IL-4 and total thiol as well as SOD and CAT activities (p < 0.05 to p < 0.001), but decreased BALF levels of total nitrite, MDA, IFN-γ, TGF-ß1, and PGE2 (p < 0.01 to p < 0.001). AG treatment decreased total WBC count, lymphocytes and macrophages in BALF (p < 0.01 to p < 0.001) and improved lung pathological changes including interstitial inflammation and lymphoid infiltration (p < 0.05 to p < 0.001). CONCLUSIONS: AG treatment reduced oxidant markers, inflammatory cytokines and lung pathological changes but increased antioxidants and anti-inflammatory cytokines. Therefore, AG may play a significant protective role against inflammation and oxidative stress that cause lung injury.

Overexpression of cancer-testis antigen melanoma-associated antigen A1 in lung cancer: A novel biomarker for prognosis, and a possible target for immunotherapy.

AIM: We investigated melanoma-associated antigen A1 (MAGE-A1) expression in lung cancer tissues and its correlation with prognostic factors. MATERIALS AND METHODS: In this cross-sectional study, samples from 101 patients with lung cancer were obtained between 2007 and 2014 and stained for MAGE-A1 by immunohistochemistry. Correlation with prognostic factors was assessed by t test, and χ 2 , and Pearson's tests. RESULTS: Eighty non-small-cell lung cancer (NSCLC) and 21 small-cell lung cancer specimens were stained for MAGE-A1. MAGE-A1 was detected more commonly in adenocarcinomas and was expressed more frequently in male and patients >60 years. CONCLUSIONS: MAGE-A1 was found in several lung cancer patients. MAGE-A1 was expressed more commonly in NSCLC, elderly, and men. Further investigations are needed to assess MAGE-A1 as potential cancer biomarkers.

Evaluation of photoshop based image analysis in cytologic diagnosis of pleural fluid in comparison with conventional modalities.

BACKGROUND: The aim of this study is to investigate and compare the results of digital image analysis in pleural effusion cytology samples with conventional modalities. MATERIALS AND METHODS: In this cross-sectional study, 53 pleural fluid cytology smears from Qaem hospital pathology department, located in Mashhad, Iran were investigated. Prior to digital analysis, all specimens were evaluated by two pathologists and categorized into three groups as: benign, suspicious, and malignant. Using an Olympus microscope and Olympus DP3 digital camera, digital images from cytology slides were captured. Appropriate images (n = 130) were separately imported to Adobe Photoshop CS5 and parameters including area and perimeter, circularity, Gray Value mean, integrated density, and nucleus to cytoplasm area ratio were analyzed. RESULTS: Gray Value mean, nucleus to cytoplasm area ratio, and circularity showed the best sensitivity and specificity rates as well as significant differences between all groups. Also, nucleus area and perimeter showed a significant relation between suspicious and malignant groups with benign group. Whereas, there was no such difference between suspicious and malignant groups. CONCLUSION: We concluded that digital image analysis is welcomed in the field of research on pleural fluid smears as it can provide quantitative data to apply various comparisons and reduce interobserver variation which could assist pathologists to achieve a more accurate diagnosis.

Evaluation of Subclinical Extension of Basal Cell Carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) is the most common malignancy with increasing incidence worldwide. The tumor invades surrounding tissues in an irregular pattern via subclinical and microscopic finger-like growths known as subclinical extension. Subclinical extension may be responsible for incomplete resection of the tumor. This study investigates the subclinical extension of BCC. METHODS: In a retrospective study for evaluation of subclinical extension of BCC, Patients' demographic data and characteristics (disease duration, location, size, and history of radiotherapy) were documented. Pathology samples were assessed in terms of histological type, subclinical extension, depth, and involvement of margins. RESULTS: The study was conducted on 102 pathological samples of 84 patients (49 males, 35 females) with BCC. The mean age was 65.4±12.55 years. Overall, 83% of pathology samples had subclinical extension. Subclinical extension had no correlation with lesion size (p=0.591; r=0.056), but had a direct correlation with lesion depth (p=0.033; r=0.220). Resection of the tumor with a margin of 5.5 mm eliminated the entire lesion and its subclinical extension area with a confidence rate of 95%. CONCLUSION: Based on this study, resection of BCC lesions with a margin of 5.5 mm will eradicate the whole lesion including the subclinical extension area with 95% confidence rate. Depth of the tumor, not its size or histologic subtype, affects the required margin of excision.

Kidney stone formation and antioxidant effects of Cynodon dactylon decoction in male Wistar rats.

OBJECTIVES: The antioxidant capacity impairs in kidney and urinary bladder of animals with stone disease. Herbal medicine can improve the antioxidant condition of renal tissue. Cynodon dactylon (C. dactylon) is a medicinal plant with antioxidative and diuretic properties and different preparations of this plant have shown promising effects in stone disease. Assessment of the whole plant decoction to prevent kidney stone disease as well as its antioxidant effects was the aim of this paper. MATERIALS AND METHODS: Fifty male Wistar rats were randomly divided into 5 experimental groups (n=10). One group was left without treatment and four groups received ethylene glycol (1% v/v) in drinking water for 6 weeks. Three doses of Cynodon dactylon aqueous decoction (12.5, 50 and 200 mg/kg BW) were added to the drinking water of groups 3-5. Finally, water intake, 24-hour urine volume, MDA, total thiol concentration and FRAP value were measured in the serum and kidney tissues. The CaOx depositions were evaluated by hematoxylin and eosin staining. RESULTS: Compared to the ethylene glycol-treated group, 200 mg/kg C. dactylon, lowered stone incidents, decreased urine volume, increased FRAP/g Cr (43%) and thiol content (p<0.05) with no significant alteration of water intake, MDA decreased significantly compared to C. dactylon 12.5 (p<0.01). Kidney weight increased and body weight decreased in ethylene glycol-treated group compared to the control group (p<0.05). CONCLUSION: A minimum dose of 200 mg/kg C. dactylon reduced stone formation and simultaneously increased total antioxidant power of serum and preserved MDA content and water.

Beneficial Effects of Selective Orexin-A Receptor Antagonist in 4-aminopyridine-induced Seizures in Male Rats.

BACKGROUND: Orexins are excitatory neuropeptides which stimulate the central regulatory pathways. Orexins increase the penicillin-induced epileptic activity in rats. Orexin-A increases in different types of seizures and its elevated level is the characteristic feature in the epileptic children during polysomnography. Recently, the orexin receptor blockage has been reported to increase seizure threshold in mice; however, effect of the selective orexin-A receptor antagonist (SB-334867) on 4-aminopyridine (4-AP)-induced seizures has not been investigated. MATERIALS AND METHODS: We used the intraperitoneal injection of 4-AP to induce seizure in male rats. Under urethane anesthesia, SB-334867 (50 and 100 nmol) was injected stereotaxically into the ventral hippocampal commissure. Using video recording, the effects of SB-334867 on electroencephalogram and tonic-clonic convulsions were compared to those that received diazepam or dimethyl sulfoxide (DMSO). RESULTS: SB-334867 significantly decreased the duration of spike trains compared to DMSO-treated rats (P < 0.001) and reduced the duration of convulsive seizures (P < 0.05). Seizure onset was increased significantly by SB-334867, 50 nmol, compared to DMSO (P < 0.05) and diazepam (P < 0.01) treated rats. CONCLUSION: Antagonism of orexin-A receptor by a low-dose SB-334867 showed protective effects in 4-AP-induced seizure-like activities in anesthetized rats.