Search results for: Immunohistochemistry

Authors: Xing Wang, Lin Feng, Lingling E., Hongchen Liu

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In patients with type 2 diabetes mellitus (DM) there is poorer quality osseointegration than in non-diabetic (n-DM) patients, and the success of dental implants is less. Recent studies have demonstrated that insulin could stimulate bone cells to produce and accelerate implant osseointegration in DM patients.This raised the question whether insulin could provide local bone anabolic effects in non-diabetic patients. In this study,48 SD rats were divided into four groups randomly: DM group, DM+insulin group, n-DM group, n-DM + insulin group. All rats were implanted the titanium implant near the epiphyseal end of tibia, then the DM + insulin and n-DM + insulin group received twice-daily subcutaneous injections of insulin (10U/day).Two,four and eight weeks after implantation, rats were killed in batches. Histomorphometry and immunohistochemistry were used to evaluate bone formation and osseointegration. The amount of newly formed bone, Implant–bone contact and the expression of OCN,RUNX2 in the DM+insulin, n-DM and n-DM+insulin group were significantly more than in the DM group (p<0.05). Compared with the n-DM group,the Implant–bone contact and expression of OCN,RUNX2 were significantly increased in n-DM+insulin group (p< 0.05). Taken together,these observations provide evidence that insulin has the potential to increase bone formation and osseointegration around implant not only in diabetic subjects but also in non-diabetic subject.

Keywords: insulin, diabetes mellitus, osseointegration, dental implants

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Authors: Boonyalitpun P., Pruckpattranon P., Thonghom A., Rotpenpian N.

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Osteoarthritis is a musculoskeletal and neuromuscular abnormality, masticatory muscle, and other tissue that causes pain and breaks down the articular surface of the temporomandibular joint (TMJ). The aim of this study is to investigate the change in the mandibular condyle, in terms of thickness and porosity, and osteoclast marker in the mandibular condyle of TMJ induced osteoarthritis mice (TMJ-OA mice). We investigated the bony changes in the TMJ structure of a complete Freund adjuvant (CFA)-injected TMJ in a mice model over 28 days. On day 28, we observed any change in the TMJ by a micro computed tomography scan (micro-CT scan) in the parameters of trabecular microarchitecture. Then we studied the thickness of the condyles by hematoxylin and eosin staining. Moreover, we calculated the area around the TMJ’s condylar head containing the osteoclast expression by TRAP (Tartrate-resistant acid phosphatase) immunohistochemistry staining. The result found that the parameter of a micro-CT scan was no different from microarchitecture in the TMJ compared with the control group; however, mandibular condyles of the TMJ-OA group was significantly thinner than the control groups, and the osteoclast expression significantly increased in the TMJ-OA group. Therefore, our findings suggest that CFA-induced TMJ-OA represents an expression of osteoclast mandibular condyle of the TMJ, which is the proposed mechanism for a TMJ-OA model.

Keywords: condyle, osteoarthritis, osteoclast, temporomandibular joint

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Authors: Rajeswari Raguraman, Sowmya Parameswaran, Krishnakumar Subramanian, Jagat Kanwar, Rupinder Kanwar

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Background: Tumor microenvironment has been implicated in several cancers to regulate cell growth, invasion and metastasis culminating in outcome of therapy. Tumor stroma consists of multiple cell types that are in constant cross-talk with the tumor cells to favour a pro-tumorigenic environment. Not much is known about the existence of tumor microenvironment in the pediatric intraocular malignancy, Retinoblastoma (RB). In the present study, we aim to understand the multiple stromal cellular subtypes and tumor stromal interactions expressed in RB tumors. Materials and Methods: Immunohistochemistry for stromal cell markers CD31, CD68, alpha-smooth muscle (α-SMA), vimentin and glial fibrillary acidic protein (GFAP) was performed on formalin fixed paraffin embedded tissues sections of RB (n=12). The differential expression of stromal target molecules; fibroblast activation protein (FAP), tenascin-C (TNC), osteopontin (SPP1), bone marrow stromal antigen 2 (BST2), stromal derived factor 2 and 4 (SDF2 and SDF4) in primary RB tumors (n=20) and normal retina (n=5) was studied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. The differential expression was correlated with the histopathological features of RB. The interaction between RB cell lines (Weri-Rb-1, NCC-RbC-51) and Bone marrow stromal cells (BMSC) was also studied using direct co-culture and indirect co-culture methods. The functional effect of the co-culture methods on the RB cells was evaluated by invasion and proliferation assays. Global gene expression was studied by using Affymetrix 3’ IVT microarray. Pathway prediction was performed using KEGG and the key molecules were validated using qRT-PCR. Results: The immunohistochemistry revealed the presence of several stromal cell types such as endothelial cells (CD31+;Vim+/-); macrophages (CD68+;Vim+/-); Fibroblasts (Vim+; CD31-;CD68- );myofibroblasts (α-SMA+/ Vim+) and invading retinal astrocytes/ differentiated retinal glia (GFAP+; Vim+). A characteristic distribution of these stromal cell types was observed in the tumor microenvironment, with endothelial cells predominantly seen in blood vessels and macrophages near actively proliferating tumor or necrotic areas. Retinal astrocytes and glia were predominant near the optic nerve regions in invasive tumors with sparse distribution in tumor foci. Fibroblasts were widely distributed with rare evidence of myofibroblasts in the tumor. Both gene and protein expression revealed statistically significant (P<0.05) up-regulation of FAP, TNC and BST2 in primary RB tumors compared to the normal retina. Co-culture of BMSC with RB cells promoted invasion and proliferation of RB cells in direct and indirect contact methods respectively. Direct co-culture of RB cell lines with BMSC resulted in gene expression changes in ECM-receptor interaction, focal adhesion, IL-8 and TGF-β signaling pathways associated with cancer. In contrast, various metabolic pathways such a glucose, fructose and amino acid metabolism were significantly altered under the indirect co-culture condition. Conclusion: The study suggests that the close interaction between RB cells and the stroma might be involved in RB tumor invasion and progression which is likely to be mediated by ECM-receptor interactions and secretory factors. Targeting the tumor stroma would be an attractive option for redesigning treatment strategies for RB.

Keywords: gene expression profiles, retinoblastoma, stromal cells, tumor microenvironment

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Authors: Vandana Singh, Subash Singh

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Introduction: Oral squamous cell carcinoma is the most common malignant tumor of the oral cavity. Normally the death of cell and the growth are active processes and depend not only on external factors but also on the expression of genes like Bcl-2, which activate and inhibit apoptosis. The term Bcl-2 is an acronym for B-cell lymphoma/ leukemia -2 genes. Objectives: An attempt was made to evaluate Bcl-2 oncoprotein expression in patients with oral precancer and cancer and to assess possible correlation between Bcl-2 oncoprotein expression and clinicopathological features of oral precancer and cancer. Material and Methods: This is a selective prospective clinical and immunohistochemical study. Clinicopathological examination is correlated with immunohistochemical findings. The immunolocalization of Bcl-2 protein is performed using the labeled streptavidin biotin (LSAB) method. To visualize the reaction, 3, 3-diaminobenzidine (DAB) is used. Results: Bcl-2 expression was positive in 11 [36.66 %, low Bcl-2 expression 3 (10.00 %), moderate Bcl-2 expression 7 (23.33 %), and high Bcl-2 expression 1 (3.33 %)] oral cancer cases and in 14 [87.50 %, low expression 8 (50 %), moderate expression 6 (37.50 %)] precancer cases. Conclusion: On the basis of the results of our study we conclude that positive Bcl-2 expression may be an indicator of poor prognosis in oral cancer and precancer. Relevance: It has been reported that there is deregulation of Bcl-2 expression during progression from oral epithelial dysplasia to squamous cell carcinoma. It can be used for revealing progression of epithelial dysplasia to malignancy and as a prognostic marker in oral precancer and cancer.

Keywords: BcL-2, immunohistochemistry, oral cancer, oral precancer

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Authors: Awf A. Al-Khan, Michael J. Day, Judith Nimmo, Mourad Tayebi, Stewart D. Ryan, Samantha J. Richardson, Janine A. Danks

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Osteosarcoma (OS) is the most common type of malignant primary bone tumour in dogs. In addition to their critical roles in bone formation and remodeling, parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) are involved in progression and metastasis of many types of tumours in humans. The aims of this study were to determine the localisation and expression levels of PTHrP and PTHR1 in canine OS tissues using immunohistochemistry and to investigate if this expression is correlated with survival time. Formalin-fixed, paraffin-embedded tissue samples from 44 dogs with known survival time that had been diagnosed with primary osteosarcoma were analysed for localisation of PTHrP and PTHR1. Findings showed that both PTHrP and PTHR1 were present in all OS samples. The dogs with high level of PTHR1 protein (16%) had decreased survival time (P<0.05) compared to dogs with less PTHR1 protein. PTHrP levels did not correlate with survival time (P>0.05). The results of this study indicate that the PTHR1 is expressed differently in canine OS tissues and this may be correlated with poor prognosis. This may mean that PTHR1 may be useful as a prognostic indicator in canine OS and could represent a good therapeutic target in OS.

Keywords: dog, expression, osteosarcoma, parathyroid hormone receptor 1 (PTHR1), parathyroid hormone-related protein (PTHrP), survival

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Authors: Dalia M. Abouelfadl, Noha N. Yassen, Marwa E. Shabana

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Background: The evolution of immune therapy motivated many to study the relation between immune response and progression of cancer. Little is known about expression of PD-L1 (a newly evolving immunotherapeutic drug) in papillary thyroid carcinoma (PTC) arising de-novo and PTC arising on top of autoimmune thyroiditis (Hashimoto's (HT) and lymphocytic thyroiditis (LT)). The aim of this work is to study the alteration of expression of PD-L1 in PTCs arising from de-novo or on top of HT OR LT using immunohistochemistry and image analyser system. Method: 100 paraffin blocks for PTC cases were collected retrospectively for staining using PD-L1 rabbit monoclonal antibody (BIOCARE-ACI 3171 A, C). The antibody expression is measured digitally using Image Analyzer Leica Qwin 3000, and the membranous and cytoplasmic expression of PD-L1 in tumor cells was considered positive. The results were correlated with tumor grade, size, and LN status. Results: The study samples consisted of 41 cases of PTC arising De novo, 36 cases on top of HT, and 23 on top of LT. Expression of PD-L1 was highest among the PTC-HL group (25 case-69%) followed by PTC-TL group (14 case-60.8%) then de-novo PTC (19 case-46%) with P Value < 0.05. PD-L1 expression correlated with nodal metastasis and was not relevant to tumor size or grade. Conclusion: The severity of the immune response in tumor microenvironment directly influences PTC prognosis. The anti PD-L1 Ab can be a very successful therapeutic agent for PTC arising on top of HT.

Keywords: carcinoma, Hashimoto's, lymphocytic, papillary, PD-L1, thyroiditis

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Authors: Debasish Pradhan, Shaktiprasad Pradhan, Rakesh Kumar Pradhan, Gitanjali Tripathy

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Suppressors of cytokine signalling (SOCS1), a newly indentified antiapoptotic molecule is a downstream effector of the receptor tyrosine kinase-Ras signalling pathway. The current study has uncovered that SOCS1 may have wide and imperative capacities, particularly because of its close correlation with malignant tumors. To investigate the impact of SOCS1 on MDR, we analyzed the expression of P-gp and SOCS1 by immunohistochemistry and found there was a positive correlation between them. At that point, we effectively interfered with RNA translation by the contamination of siRNA of SOCS1 into MCF7/ADM breast cancer cell lines through a lentivirus, and the expression of the target gene was significantly inhibited. After RNAi, the drug resistance was reduced altogether and the expression of MDR1 mRNA and P-gp in MCF7/ADM cell lines demonstrated a significant decrease. Likewise, the expression of P53 protein increased in a statistically significant manner (p ≤ 0.01) after RNAi exposure. Moreover, flow cytometry analysis uncovers that cell cycle and anti-apoptotic enhancing capacity of cells changed after RNAi treatment. These outcomes proposed SOCS1 may take part in breast cancer MDR by managing MDR1 and P53 expression, changing cell cycle and enhancing the anti-apoptotic ability.

Keywords: breast cancer, multidrug resistance, SOCS1 gene, MDR1 gene, RNA interference

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Authors: Imeobong Joseph Inyang, Aniekan-Augusta Okon Eyo, Abel William Essien

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Hepatitis B virus (HBV) is one of the known human carcinogens. The presence of HBsAg in liver tissues indicates active viral replication. More than 85% of Hepatocellular Carcinoma (HCC) cases occur in countries with increased rates of chronic HBV infection. An evaluation study to determine the relationship between positivity for HBsAg and development of HCC and its distribution between age and gender of subjects was done. Shikata Orcein and Haematoxylin and Eosin (H&E) staining techniques were performed on liver sections. A total of 50 liver tissue specimens comprising 38 biopsy and 12 post-mortem specimens were processed. Thirty-five of the 50 specimens were positive for HBsAg with Orcein stain whereas only 16 were positive with H&E stain, and these were also positive with Orcein stain, giving an HBsAg prevalence of 70.0% (35/50). The prevalence of HCC in the study was 56.0% (28/50), of which 21 (75.0%) cases were positive for HBsAg, 18 (64.3%) were males while 10 (35.7%) were females distributed within the age range of 20-70 years. The highest number of HBsAg positive HCC cases, 7/21 (33.3%) occurred in the age group 40-49 years. There was no relationship in the pattern of distribution of HCC between age and gender using the Pearson correlation coefficient (r = 0.0474; P < 0.05). HBV infection predisposed to HCC. Orcein technique was more specific and is therefore recommended for screening of liver tissues where facilities for immunohistochemistry are inaccessible.

Keywords: Hepatitis B. surface antigen, hepatocellular carcinoma, orcein, pathology

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Authors: Samantha Shallop, Hakinya Karra, Tytus Bernas, Gladys Shaw, Gretchen Neigh, Jeffrey Dupree, Mathula Thangarajh

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Abnormal neuronal homeostasis is considered a structural correlate of cognitive deficits in Duchenne Muscular Dystrophy. Neurons are highly polarized cells with multiple dendrites but a single axon. Trafficking of cellular organelles are highly regulated, with the cargo in the somatodendritic region of the neuron not permitted to enter the axonal compartment. We investigated the molecular mechanisms that regular organelle trafficking in neurons using a multimodal approach, including high-resolution structural illumination, proteomics, immunohistochemistry, and computational modeling. We investigated the expression of ankyrin-G, the master regulator controlling neuronal polarity. The expression of ankyrin G and the morphology of the axon initial segment was profoundly abnormal in the CA1 hippocampal neurons in the mdx52 animal model of DMD. Ankyrin-G colocalized with kinesin KIF5a, the anterograde protein transporter, with higher levels in older mdx52 mice than younger mdx52 mice. These results suggest that the functional trafficking from the somatodendritic compartment is abnormal. Our data suggests that dystrophin deficiency compromised neuronal homeostasis via ankyrin-G-based mechanisms.

Keywords: neurons, axonal transport, duchenne muscular dystrophy, organelle transport

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Authors: So Youn Park, Yi Sle Lee, Ki Whan Hong, Chi Dae Kim

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The role of metabolism in the pathogenesis of osteoarthritis is an emerging field. Metabolic alterations may be a role in osteoarthritis (OA) pathogenesis, and these changes influence joint destruction via several cytokine. Especially, in OA patients, levels of IL-1β are elevated in the synovial fluid, synovial membrane, subchondral bone, and cartilage. The IL-1β is activated by NLRP3 inflammasomes, and NLRP3 inflammasomes are cytosolic complexes that drive the production of other inflammatory cytokines, including IL-1β. In this study, we examined that SIRT1 suppresses IL-1β through inhibiting NLRP3 inflammasomes and SIRT1 ameliorates osteoarthritis. OA fibroblasts were isolated from synovium of OA patients. IL-1β and NLRP3 were detected in synovium of OA patients by immunohistochemistry. Lipopolysaccharides (LPS) stimulated the expression of active IL-1β mRNA in OA fibroblasts and combination of LPS, and adenosine triphosphate increased more the expression of active IL-1β in OA fibroblasts. The level of IL-1β was measured by western blot and ELISA assay. NLRP3 inflammasomes complex were measured by western blot. SIRT1 did not inhibit expression of NLRP3 inflammasome. So caspase-1, apoptotic speck-like protein containing a caspase recruitment domain (ASC) and NLRP3 protein were expressed in OA fibroblasts. But SIRT1 suppressed activation of IL-1β by inhibiting activity of caspase-1 via NLRP3 inflammasome in OA fibroblasts under LPS plus ATP stimulation. These results suggest that SIRT1 is a modulator of NLRP3 inflammasomes in OA fibroblasts and ameliorate IL-1β, so expression of SIRT1 in OA fibroblast may be a potential strategy for OA inflammation treatment.

Keywords: osteoarthritis, inflammasome, SIRT1, IL-1beta

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Authors: W. G. Kim, J. M. Chang, W. S. Kim

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Immunologically non-treated and acellularized porcine xenografts were implanted as an arterial graft in goats and comparatively analyzed for the explanted grafts with gross observation, as well as light microscopy and immunohistochemistry, following the predetermined periods. For immunologically non-treated xenografts, bilateral porcine carotid arteries were harvested, and after short-term freezing at -70°C, were implanted into goats. The preparation of acellularized xenograft vessels has been performed with Nacl-SDS solution and stored at the freezer until use. The goats were randomly assigned for three periods of observation (3, 6, and 12 months after implantation), four animals were observed at each of these times. Periodic ultrasonographic examinations were performed during observation period. Following the predetermined periods, the explanted grafts were analyzed. Among 12 animals, one goat died prematurely, and a total of 22 grafts were evaluated. Gross observations revealed non-thrombotic patent smooth lumens. Microscopic examinations of the explanted grafts showed satisfactory cellular reconstruction up to the 12-month observation period. The proportions of CD3 positive T lymphocytes among inflammatory cells infiltrations were very low. In conclusion, these findings, as a whole, suggest that porcine vessel xenografts can be clinically acceptably implanted in the goats as a form of small-diameter vascular graft, regardless of the acellularized xenograft or immunologically non-treated xenograft.

Keywords: xenograft, arterial graft, long-term survival animals, immunology

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Authors: Zhang Zhenzhen

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Valproate (VPA) is commonly used in the treatment of bipolar disorder and epilepsy. The mechanism is complicated, including its ability to inhibit histone deacetylases (HDACs). Here, we show that VPA attenuated VEGF gene expression and the morphological changes in choroidal neovascularization (CNV) induced by photocoagulation in retina. C57BL/6 mice were injected subcutaneously at 300mg/kg twice daily with VPA before insult. Vascular endothelial growth factor (VEGF)-A and VEGF-B were examined in the eyes of VPA-treated mice and in human retinal pigment epithelial cell lines (ARPE-19) exposed to VPA. In addition, CNV was induced by photocoagulation in mice injected with VPA, and the volume of CNV was compared by fluorescence-labeled choroidal flat mount. Morphological changes were analyzed on stained histological sections. Western blot analysis was used to determine protein levels of VEGF-A and VEGF-B, and acetylation of histone H3 in each group. VPA injected intraperitoneally attenuated the VEGF-A and VEGF-B expression in the retina, accompanied by the hyperacetylation of retina tissue, indicating that VPA acts directly on retina tissues through acetylation to reduce the expression of VEGF. VPA also attenuated the VEGF-A mRNA expression in the retinal pigment epithelium showed by immunohistochemistry. Moreover, the administration of VPA significantly attenuated photocoagulation-induced CNV in mice. These results demonstrate that VPA attenuated VEGF production in retina associated with choroidal neovascularization possibly via the HDAC inhibition.

Keywords: retina, acetylation, chorodial neovascularization, vascular endothelial growth factor

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Authors: M. Sunitha, B. Nithyavani, Mathew Yohannan, S. Thiruvieni Balajji, M. A. Rathi, C. Arul Raj, P. Ragavendran, V. K. Gopalkrishnan

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Establishment of an early and reliable biomarker for ovarian carcinogenesis whose expression can be monitored through noninvasive techniques will enable early diagnosis of cancer. Carbonic anhydrases (CA) isozymes IX and XII have been suggested to play a role in oncogenic processes. In von Hippel-Lindau (VHL)-defective tumors, the cell surface transmembrane carbonic anhydrase (CA) CA XI and CA XII genes are overexpressed because of the absence of pVHL. These enzymes are involved in causing a hypoxia condition, thereby providing an environment for metastasis. Aberrant expression of the facilitative glucose transporter GLUT I is found in a wide spectrum of epithelial malignancies. Studying the mRNA expression of CA IX, CA XII and Glut I isozymes in ovarian cancer cell lines (OAW-42 and PA-1) revealed the expression of these hypoxia genes. Immunohistochemical staining of carbonic anhydrases was also performed in 40 ovarian cancer tissues. CA IX and CA XII were expressed at 540 bp and 520 bp in OAW42, PA1 in ovarian cancer cell lines. GLUT-1 was expressed at 325bp in OAW 42, PA1 genes in ovarian cancer cell lines. Immunohistochemistry revealed high to moderate levels of expression of these enzymes. The immuostaining was seen predominantly on the cell surface membrane. The study concluded that these genes CA IX, CA XII and Glut I are expressed under hypoxic condition in tumor cells. From the present results expression of CA IX, XII and Glut I may represent potential targets in ovarian cancer therapy.

Keywords: ovarian cancer, carbonic anhydrase IX, XII, Glut I, tumor markers

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Authors: Salina Yahya Saddik

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The present study is designed to demonstrate the Ovarian Surface Epithelial cells (OSE) Estrogen Receptor α (ERα) and Progesterone Receptor (PR) during pregnancy and estrous cycle in rat. Moreover, determination of the levels of plasma progesterone, estradiol, FSH and LH were also made. The levels of plasma progesterone, estradiol, FSH and LH concentrations were determined on days 7 (n=5), 14 (n=5), and 21(n=5) of pregnancy in three groups of rats and during the estrous cycle (n=5) using ELISA kit. Immunohistochemical method for PR and ERα expression was also made on the ovary. During pregnancy, FSH and LH remained low except at term when LH levels began to increase from 16 ng/ml to 47 ng/ml. Progesterone levels significantly exceeded estradiol values in all pregnant rats with a peak value of 202 ng/ml on day 14. Elevated progesterone levels were associated negatively with LH and estradiol levels during pregnancy. The levels of estradiol surged significantly on day 21. Immunohistochemistry of the ovary showed low levels of OSE cells staining positive for ERα expression. ERα positive cells were absent on day 7 and 14 of pregnancy, only day 21 recorded a very low percentage of immunostaining (0.5%) within the nuclei of OSE cells. On the contrary, immunostaining of PR was not observed within the nuclei of OSE cells in all groups of study. In conclusions, these results may suggest that progesterone effect during pregnancy seems to be overriding the positive effect of estrogens on OSE cells. High progesterone levels may have a direct negative effect on gonadotropin production and thereby it might inhibit events leading to both follicular development and OSE proliferation. Understanding the factors affecting OSE proliferation may help elucidating the mechanism(s) of assisted diseases such as ovarian cancer.

Keywords: ovarian surface, pregnancy, gonadotropins, steroids

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Authors: Sujata Chaturvedi, Ishita Pant, Deepak Kumar Jha, Vinod Kumar Singh Gautam, Chandra Bhushan Tripathi

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Objective: To describe clinical, histopathological and immunohistochemical profile of glioblastoma in patients and to correlate these findings with patient survival. Material and methods: 30 cases of histopathologically diagnosed glioblastomas were included in this study. These cases were analysed in detail for certain clinical and histopathological parameters. Immunohistochemical staining for p53, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), mouse double minute 2 homolog (MDM2) and Ki67 was done and scores were calculated. Results of these findings were correlated with patient survival. Results: A retrospective analysis of the histopathology records and clinical case files was done in 30 cases of glioblastoma (WHO grade IV). The mean age of presentation was 50.6 years with a male predilection. The most common involved site was the frontal lobe. Amongst the clinical parameters, age of the patient and extent of surgical resection showed a significant correlation with the patient survival. Histopathological parameters showed no significant correlation with the patient survival, while amongst the immunohistochemical parameters expression of MDM2 showed a significant correlation with the patient survival. Conclusion: In this study incorporating clinical, histopathological and basic panel of immunohistochemistry, age of the patient, extent of the surgical resection and expression of MDM2 showed significant correlation with the patient survival.

Keywords: glioblastoma, p53, EGFR, VEGF, MDM2, Ki67

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Authors: Mohammed Abdelsabour-Khalaf, F. Yusuf , B Brand-Saberi

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Purpose: Applications of nanotechnology nowadays extended to include a wide range of scientific areas such electron micrscopy and gene expression. The aim of the current study was to investigate the developmental expression pattern of genes involved in human glomerulo-nephropathies associated with massive proteinuria and podocyte differentiation using the chicken mesonephros as a model system. Method: We performed in situ hybridization using chicken specific mRNA probes for genes expressed in the early nephron and slit diaphragm genes. The probes used were cNeph1, cNeph2, cSim1, cLmx1b, and cAtoh8. Chicken embryos from Hamburger Hamilton developmental stage HH19 (E3) to HH 34 (E9) were used for the in situ hybridization (ISH). ISH was performed on whole mount embryos which were sectioned by vibratome. Results: Our result show that Neph1, Neph2, Sim1. Lmx1b and Atoh8 genes are dynamically expressed during nephron morphogenesis and Neph1 and Atoh8 are also specifically expressed in the podocytes during late stages of differentiation. Conclusion: We conclude from our results that the genes implicated in congenital and acquired glomerulo-nephropathies like Neph1 and Neph2 are dynamically expressed during mesonephros development pointing towards a role in the formation of the filtration barrier and the differentiation of the mesonephric podocytes. Thus the avian mesonephros could serve as a model to study human kidney diseases.

Keywords: mesonephros, chicken embryo, gene expression, immunohistochemistry

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Authors: Chengcao Sun, Cuili Yang, Shujun Li, Ruilin Xue, Yongyong Xi, Liang Wang, Dejia Li

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Backgrounds: A few lines of evidence show that Sulforaphane (SFN) has anti-fibrosis effect in liver tissue via Nrf2-mediated inhibition of TGF-β/Smad signaling. However, its effects on muscular dystrophic fibrosis remain unknown. This work was undertaken to evaluate the effects of SFN on fibrosis in dystrophic muscle. Methods: 3-month-old male mdx mice were treated with SFN by gavage (2 mg/kg body weight per day) for 3 months. Gastrocnemius, tibial anterior and triceps brachii muscles were collected for related analysis. Fibrosis in skeletal muscles was analyzed by Sirius red staining. Histology and morphology of skeletal muscles were investigated by H&E staining. Moreover, the expressions of Nrf2, NQO1, HO-1, and TGF-β/Smad signaling pathway were detected by western blot, qRT-PCR, immunohistochemistry and immunofluorescence assays. Results: Our results demonstrated that SFN treatment significantly decreased and improved morphological features in mdx muscles. Moreover, SFN increased the expression of muscle phase II enzymes NQO1 and HO-1 and significantly decreased the expression of TGF-β1，p-smad2, p-smad3, α-SMA, fibronectin, collagen I, PAI-1, and TIMP-1 in Nrf2 dependent manner. Additionally, SFN significantly decreased the expression of CD45 and TNF-α. Conclusions: Collectively, these results show that SFN can ameliorate muscle fibrosis in mdx mice by Nrf2-induced inhibition of TGF-β/Smad signaling pathway, which indicate Nrf2 may be useful for the treatment of muscular dystrophy.

Keywords: sulforaphane, Nrf2, TGF-β/smad signaling, duchenne muscular dystrophy, fibrosis

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Authors: D. Adel, F. Giacomini, R. Gildenhaar, G. Berger, C. Gomes, U. Linow, M. Hardt, B. Peleskae, J. Günster, A. Houshmand, M. Stiller, A. Rack, K. Ghaffar, A. Gamal, M. El Mofty, C. Knabe

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The goal of this study was to develop 3D scaffolds from a silica containing calcium alkali orthophosphate utilizing two different fabrication processes, first a replica technique namely the Schwartzwalder Somers method (SSM), and second 3D printing, i.e. Rapid prototyping (RP). First, the mechanical and physical properties of the scaffolds (porosity, compressive strength, and solubility) was assessed and second their potential to facilitate homogenous colonization with osteogenic cells and extracellular bone matrix formation throughout the porous scaffold architecture. To this end murine and rat calavarie osteoblastic cells were dynamically seeded on both scaffold types under perfusion with concentrations of 3 million cells. The amount of cells and extracellular matrix as well as osteogenic marker expression was evaluated using hard tissue histology, immunohistochemistry, and histomorphometric analysis. Total porosities of both scaffolds were 86.9 % and 50% for SSM and RP respectively, Compressive strength values were 0.46 ± 0.2 MPa for SSM and 6.6± 0.8 MPa for RP. Regarding the cellular behavior, RP scaffolds displayed a higher cell and matrix percentage of 24.45%. Immunoscoring yielded strong osteocalcin expression of cells and matrix in RP scaffolds and a moderate expression in SSM scaffolds. 3D printed RP scaffolds displayed superior mechanical and biological properties compared to SSM. 3D printed scaffolds represent excellent candidates for bone tissue engineering.

Keywords: calcium alkali orthophosphate, extracellular matrix mineralization, osteoblast differentiation, rapid prototyping, scaffold

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Authors: Alka Yadav, Mayank Jain, Rajan Saxena, Niraj Kumari, Narendra Krishnani, Ashok Kumar

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Purpose: To study the mismatch repair (MMR) protein expression and its clinicopathological correlation in colorectal cancer patients in North India. Methods: A prospective study was conducted on histologically proven 52 (38 males and 14 females) patients with adenocarcinoma of colorectum. MMR protein loss was determined by using immunohistochemistry for MLH1, MSH2, PMS2 and MSH6. Results: 52 patients (38 males and 14 females) underwent resection for colorectal cancer with the median age of 52 years (16-81 years). 35% of the patients (n=18) were younger than 50 years of the age. 3 patients had associated history of malignancy in the family. 29 (56%) patients had right colon cancer, 9 (17%) left colon cancer and 14 (27%) rectal cancer. 2 patients each had synchronous and metachronous cancer. Histology revealed well-differentiated tumour in 16, moderately differentiated in 10 and poorly differentiated tumour in 26 patients. MMR protein loss was seen in 15 (29%) patients. Seven (46%) of these patients were less than 50 years of age. Combined loss of MSH2 and MSH6 was seen most commonly and it was found in 6 patients. 12 (80%) patients with MMR protein loss had tumour located proximal to the splenic flexure compared to 3 (20%) located distal to the splenic flexure. There was no difference in MMR protein loss based on patients' age, gender, degree of tumour differentiation, stage of the disease and tumour histological characteristics. Conclusions: This study revealed that there was less than 30% MMR protein loss in colorectal cancer patients. The loss was most commonly seen in right sided colon cancer than left. A larger study is further required to validate these findings.

Keywords: colorectal cancer, mismatch repair protein, immunohitochemistry, clinicopathological correlation

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Authors: S. Tebibel, R. L. Bouchouka, C. Mechati, S. Messaoudi

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The stomach cancer or gastric cancer is an aggressive cancer with a significant geographic disparity. The decrease in frequency is attributed to refrigeration, which has several beneficial consequences, increased consumption of fresh fruits and vegetables, reduced consumption of salt, which was widely used as a food preservative, and less contamination of food by carcinogenic compounds. The infection with Helicobacter pylori is responsible for progressive inflammatory changes in the gastric mucosa usually evolving into stomach cancer in 80% of cases. Methodology: This epidemiological and analytical study concerns 65 patients (46 men and 19 women) with gastric adenocarcinomas with an average age of 56.5 years and a male predominance with a sex ratio of 2.4. Results and Discussion: In this series, the clinical symptoms are dominated by epigastralgia (72.31%), vomiting (27,69%), and slimming (24,62%). The FOGD (Oeso-Gastro Duodenal Fibroscopy) performed in the 65 patients revealed a predominance of the antro-pyloric localization in 19 cases (i.e., 29.23%) and anulcerative budding appearance in 33 subjects (50,77%). Histologically, the moderately differentiated adenocarcinoma is found in 30.77% of patients, followed by well differentiated adenocarcinoma with 26.15% of patients. The immunohistochemical study revealed a positive labeling of half of the T cells by anti-CD3 AC, and a positive labeling of anti-CD20 AC in a diffuse and intense manner, with the presence of CD20-positive lymphoepithelial lesions compatible with CD20 a low grade MALT non-Hodgkin's lymphoma. Conclusion: This framework of analysis revealed some risk factors for gastric cancer, such as food, hygiene, Helicobacter pylori infection, smoking and family history.

Keywords: cancer, Helicobacter pylori, immunohistochemistry, stomach

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Authors: Mahmoodreza Tahamtan

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Background: Malignant surface epithelial ovarian tumors (SEOT) account for approximately 90% of primary ovarian cancer. Wilms tumor gene (WT1) product was defined as a tumor suppressor gene, but today it is considered capable of performing oncogenic functions. There seems to be differences in WT1 expression patterns among SEOT subtypes. We evaluate the immunohistochemical expression of WT1 protein among different histologic subtypes of SEOT. Materials and Methods: Immunohistochemistry for WT1 was done on 35 serous cystadenocarcinomas, 9 borderline serous tumors, 3 mucinous cystadenocarcinomas, 10 borderline mucinous tumors, 7 endometrioid ovarian carcinomas, 3 clear cell carcinomas, 1 malignant Brenner tumor, 2 metastatic adenocarcinomas, and 6 endometrial adenocarcinomas. A tumor was considered negative if < 1% of tumor cells were stained.Positive reactions were graded as follows:1+,1%-24%; 2+,25%-49%; 3+,50%-74%; 4+,75%-100%. Results: Of the 35 cases of ovarian serous cystadenocarcinoma, 30(85.7%) were diffusely positive (3+,4+),4 showed reactivity of < 50% of the tumor cells (1+,2+), and one were negative. All 9 borderline serous tumors showed immunoreactivity with WT1. All the mucinous tumors(n:13), endometrioid carcinomas (n: 7), clear cell carcinomas (n: 3), metastatic adenocarcinomas (n: 2) and primary endometrial carcinomas (n:6) were negative. The single malignant Brenner tumor showed a positive reaction for WT1(4+) Conclusion: WT1 is a good marker to distinguish primary ovarian serous carcinomas from other surface epithelial tumors (especially endometrioid subtype) and metastatic carcinomas (especially endometrial serous carcinoma), other than malignant mesothelioma. We cannot rely to the degree of expression inorder to separate high grade borderline serous tumors from low grade ones.

Keywords: WT1, ovary, epithelial tumors, malignant

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Authors: Yasmin M. Attia, Hanan S. El-Abhar, Mahmoud M. Al Marzabani, Samia A. Shouman

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Background: Tamoxifen (TAM) is the most commonly used hormone therapy for the treatment of early and metastatic breast cancer. Although it significantly decreases the tumor recurrence rate and provides an overall benefit, as much as 20–30% of women still relapse during or after long-term therapy. 3-Bromopyruvate (3-BP) is a promising agent with impressive antitumor effects in several models of animal tumors and cell lines. Aim: This study was designed to investigate the combined effect of (TAM) and (3-BP) in breast cancer cells and to explore their molecular interaction via assessment of apoptotic, angiogenic, and metastatic markers. Methods: In vitro cytotoxicity study was carried out for both compounds to determine the combination regimen producing a synergistic effect and mechanistic pathways were studied using RT-PCR and western techniques. Moreover, the anti-oncolytic and anti-angiogenic potentials were assessed in mice bearing solid Ehrlich carcinoma (SEC). Results: The combined treatment significantly increased the expressions and protein levels of caspase 7, 9, and 3 and decreased of angiogenic markers VEGF, HIF-1α, and HK2 compared to cells treated with either drug individually. However, there were no significant changes in MMP-2 and MMP-9 protein levels. Interestingly, the in vivo results supported the in vitro findings; there was a decrease in the tumor volume and VEFG using immunohistochemistry in the combination-treated groups compared to either TAM or 3-BP treated one. Conclusion: 3-BP synergizes the cytotoxic effect of TAM by increasing apoptosis and decreasing angiogenesis which makes this combination a promising regimen to be applied clinically.

Keywords: tamoxifen, 3-bromopyruvate, breast cancer, cytotoxicity, angiogenesis

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Authors: Chengcao Sun, Cuili Yang, Shujun Li, Ruilin Xue, Yongyong Xi, Liang Wang, Dejia Li

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Backgrounds: Inflammation is widely distributed in patients with Duchenne muscular dystrophy (DMD), and ultimately leads to progressive deterioration of muscle function with the co-effects of chronic muscle damage, oxidative stress, and reduced oxidative capacity. NF-E2-related factor 2 (Nrf2) plays a critical role in defending against inflammation in different tissues via activation of phase II enzymes, heme oxygenase-1 (HO-1). However, whether Nrf2/HO-1 pathway can attenuate muscle inflammation on DMD remains unknown. The purpose of this study was to determine the anti-inflammatory effects of Sulforaphane (SFN) on DMD. Methods: 4-week-old male mdx mice were treated with SFN by gavage (2 mg/kg body weight per day) for 4 weeks. Gastrocnemius, tibial anterior and triceps brachii muscles were collected for related analysis. Immune cell infiltration in skeletal muscles was analyzed by H&E staining and immuno-histochemistry. Moreover, the expressions of inflammatory cytokines,pro-inflammatory cytokines and Nrf2/HO-1 pathway were detected by western blot, qRT-PCR, immunohistochemistry and immunofluorescence assays. Results: Our results demonstrated that SFN treatment increased the expression of muscle phase II enzymes HO-1 in Nrf2 dependent manner. Inflammation in mdx skeletal muscles was reduced by SFN treatment as indicated by decreased immune cell infiltration and lower expressions of the inflammatory cytokines CD45, pro-inflammatory cytokines tumour necrosis factor-α and interleukin-6 in the skeletal muscles of mdx mice. Conclusions: Collectively, these results show that SFN can ameliorate muscle inflammation in mdx mice by Nrf2/HO-1 pathway, which indicates Nrf2/HO-1 pathway may represent a new therapeutic target for DMD.

Keywords: sulforaphane, Nrf2, HO-1, inflammation

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Authors: Sushila Jaiswal, Awadhesh Kumar Jaiswal

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Background: Primary melanocytic tumors of the central nervous system (CNS) are uncommon lesions and arise from the melanocytes located within the leptomeninges. Aim and objective: The aim of the study was to evaluate the clinical details, histomorphology of the primary melanocytic tumor of CNS. Method: The study was performed by the retrospective review of the case records of the primary melanocytic tumors of CNS diagnosed in our department. The formalin-fixed, paraffin embedded tissue blocks and tissue sections were retrieved and reviewed. Results: Seven cases (6 males, 1 female; age range- 16-40 years; mean age- 27 years) of primary melanocytic tumors of CNS were retrieved over last seven years. The tumor was intracranial (n=5; frontal – 1 case, parietal – 1 case, cerebello-pontine angle- 1 case, occipital -1 case, foramen magnum-1 case) and intra spinal (n=2; cervical – 2 cases). All patients presented with the neurological deficits related to the location of the tumor. Four cases were malignant melanoma; two were melanocytoma of intermediate grade and remaining one was melanocytoma. On histopathology, melanocytoma and melanoma both displayed sheets of well-differentiated melanocytes having round to oval nuclei with finely dispersed chromatin, occasional single eosinophilic nucleoli and a moderate amount of cytoplasm with abundant granular melanin pigment. The absence of mitosis and macronucleoli was noticed in melanocytoma while melanoma showed frequent mitosis and macronucleoli. On immunohistochemistry, both showed diffuse strong HMB45 and S-100 immunopositivity. Conclusion: Primary melanocytic tumors of CNS are rare and predominantly seen in males. It is important to differentiate melanoma from melanocytoma as prognosis of later is good.

Keywords: melanocytoma, melanoma, brain tumor, melanin

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Authors: Kamyar Moradi

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Background: Acute mesenteric ischemia is known as a life-threatening condition. Re-establishment of blood flow in this condition can lead to mesenteric ischemia reperfusion (MIR) injury, which is accompanied by inflammatory response. Still, clear blueprint of inflammatory mechanism underlying MIR injury has not been provided. Interestingly, Albendazole has exhibited notable effects on inflammation and cytokine production. In this study, we aimed to evaluate outcomes of MIR injury following pretreatment with Albendazole with respect to assessment of mesenteric inflammation and ischemia threshold. Methods: Male rats were randomly divided into sham operated, vehicle treated, Albendazole 100 mg/kg, and Albendazole 200 mg/kg groups. MIR injury was induced by occlusion of superior mesenteric artery for 30 minutes followed by 120 minutes of reperfusion. Samples were utilized for assessment of epithelial survival and villous height. Immunohistochemistry study revealed intestinal expression of TNF-α and HIF-1-α. Gene expression of NF-κB/TLR4/TNF-α/IL-6 was measured using RTPCR. Also, protein levels of inflammatory cytokines in serum and intestine were assessed by ELISA method. Results: Histopathological study demonstrated that pretreatment with Albendazole could ameliorate decline in villous height and epithelial survival following MIR injury. Also, systemic inflammation was suppressed after administration of Albendazole. Analysis of possible participating inflammatory pathway could demonstrate that intestinal expression of NF-κB/TLR4/TNF-α/IL-6 is significantly attenuated in treated groups. Eventually, IHC study illustrated concordant decline in mesenteric expression of HIF-1-α/TNF-α. Conclusion: Single dose pretreatment with Albendazole could ameliorate inflammatory response and enhance ischemia threshold following induction of MIR injury. Still, more studies would clarify existing causality in this phenomenon.

Keywords: albendazole, ischemia reperfusion injury, inflammation, mesenteric ischemia

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Authors: Ahmed Gaber Abdel Raheem, Nashwa Ahmed Mohamed

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Introduction: Sensorineural hearing loss (SNHL) is largely caused by the degeneration of the cochlea. Therapeutic options for SNHL are limited to hearing aids and cochlear implants. The cell transplantation approach to the regeneration of hair cells has gained considerable attention because stem cells are believed to accumulate in the damaged sites and have the potential for the repair of damaged tissues. The aim of the work: was to assess the use of bone marrow transplantation in repair of damaged inner ear hair cells in rats after the damage had been inflicted by Amikacin injection. Material and Methods: Thirty albino rats were used in this study. They were divided into three groups. Each group ten rats. Group I: used as control. Group II: Were given Amikacin- intratympanic injection till complete loss of hearing function. This could be assessed by Distortion product Otoacoustic Emission (DPOAEs) and / or auditory brain stem evoked potential (ABR). GroupIII: were given intra-peritoneal injection of bone marrow stem cell after complete loss of hearing caused by Amikacin. Clinical assessment was done using DPOAEs and / or auditory brain stem evoked potential (ABR), before and after bone marrow injection. Histological assessment of the inner ear was done by light and electron microscope. Also, Detection of stem cells in the inner ear by immunohistochemistry. Results: Histological examination of the specimens showed promising improvement in the structure of cochlea that may be responsible for the improvement of hearing function in rats detected by DPOAEs and / or ABR. Conclusion: Bone marrow stem cells transplantation might be useful for the treatment of SNHL.

Keywords: amikacin, hair cells, sensorineural hearing loss, stem cells

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Authors: Rania Hanafi Said, Rasha Mohamed Taha

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Background: By using nanoparticles as drug delivery, it may be possible to avoid the drawbacks of systemic antibiotic dosing, including bacterial antibiotic resistance. The goal of this study was to see how well tetracycline loaded on nanochitosan worked to treat gingival inflammation in albino rats caused by Porphyromonas gingivalis. The study analyzed immunohistochemically the localization of the pro-inflammatory cytokine Interleukin-1beta (IL-1β). Material and methods: In this study, fifty mature male albino rats weighing 150 to 180 grams each were used. They were randomly divided into five groups. We checked for weight changes in rats. Ten male albino rats were included in Group I, which served as a negative control group. Ten rats were included in Group II, where they were exposed once to Porphyromonas. Group III contained ten rats, which were treated the same as Group II plus daily injections of diluted tetracycline powder at the infection sites. Ten rats in Group IV received the same procedure as those in Group II before receiving daily injections of nanochitosan at the injection sites. Finally, Group V, which had ten rats. Following the same protocol as Group II, they received localized injections of tetracycline loaded on nanochitosan once daily. Rats' gingivae were extracted and prepared after they were anesthetized. The biopsies were examined histologically and immunohistochemically by light microscopy. Results: Groups I and V had a nearly normal histological appearance of gingival tissue. In Groups II, III, and IV, degeneration was seen because the epithelial cells were bigger, collagen fibers were pulling away from the lamina propria connective tissue, and the basement membranes had come to an end. There was no discernible difference between groups V and I when they were examined immunohistochemically. Conclusion: The use of nano chitosan as a tetracycline carrier is a novel technique to overcome the drug's rising level of resistance.

Keywords: Immunohistochemistry, Nanochitosan, porphyromonas gingivitis, Tetracycline

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Authors: Hussain Mustatab Wahedi, Sun You Kim

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Skin acts as a barrier against the harmful environmental factors. Integrity and timely recovery of the skin from injuries and harmful effects of radiations is thus very important. This study aimed to investigate the importance of Sirt1 in the recovery of skin from UVB-induced damage and cutaneous wounds by using natural and synthetic novel Sirt1 activators. Juglone, known as a natural Pin1 inhibitor, and NED416 a novel synthetic Sirt1 activator were checked for their ability to regulate the expression and activity of Sirt1 and hence photo-damage and wound healing in cultured skin cells (NHDF and HaCaT cells) and mouse model by using Sirt1 siRNA knockdown, cell migration assay, GST-Pulldown assay, western blot analysis, tube formation assay, and immunohistochemistry. Interestingly, Sirt1 knockdown inhibited skin cell migration in vitro. Juglone up regulated the expression of Sirt1 in both the cell lines under normal and UVB irradiated conditions, enhanced Sirt1 activity and increased the cell viability by reducing reactive oxygen species synthesis and apoptosis. Juglone promoted wound healing by increasing cell migration and angiogenesis through Cdc42/Rac1/PAK, MAPKs and Smad pathways in skin cells. NED416 upregulated Sirt1 expression in HaCaT and NHDF cells as well as increased Sirt1 activity. NED416 promoted the process of wound healing in early as well as later stages by increasing macrophage recruitment, skin cell migration, and angiogenesis through Cdc42/Rac1 and MAPKs pathways. So, both these compounds activated Sirt1 and promoted the process of wound healing thus pointing towards the possible role of Sirt1 in skin regeneration and wound healing.

Keywords: skin regeneration, wound healing, Sirt1, UVB light

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Authors: Yaya Gao, Jifeng Liu, Yafeng Liu

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Objectives: This study aimed to improve clinicians' understanding and diagnosis of the Mammary analogue secretory carcinoma of the salivary gland（MASC）. Methods: The clinical features of a MASC patient who was admitted to WestChina Hospital of Sichuan University in July 2020 were reviewed and analyzed. A 49-year-old woman with left upper neck pain for three months was admitted to the hospital. She underwent adenoma resection of the left submandibular gland 14 years ago and mucoepidermoid carcinoma resection surgery five years ago. Three months before admission, the patient developed pain in the left mandibular angle after "fatigue" and gradually developed radiation pain in the left ear, which could be relieved after rest. A mass of 1cm could be touched at the mandibular, with tenderness, poor mobility, and hard texture. No swelling, heat, pain, rupture, or pus was found on the surrounding skin. Color doppler ultrasonography of the salivary gland indicated a weak echo mass of 23*14*17mm in the left parotid gland. Results: Surgical excision was completed. Immunohistochemistry of the tumor samples after operation showed that P63（a few，+）, CK7（+）, S100（+）, DOG1（-）, Ki67（MIB-1）（+，5%），pan-TRK（+）, PAS(+) . ETV-6 gene translocation was detected in FISH in postoperative pathology, which indicated MASC. After this diagnosis, the patient sent the postoperative specimen of the second submandibular tumor to our hospital for consultation. The morphology of the two was similar. FISH detected ETV-6 gene translocation, so the second pathological diagnosis was revised to MASC. Conclusion: MASC of the salivary gland is a rare salivary gland tumor whose diagnosis depends on the result of the ETV6-NTRK3 fusion gene.

Keywords: mammary analogue secretory carcinoma, ETV6-NTRK3, salivary gland, misdiagnosed

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Authors: Wafa Toumi, Alessandro Ripalti, Luigi Ricciardiello, Dalila Gargouri, Jamel Kharrat, Abderraouf Cherif, Ahmed Bouhafa, Slim Jarboui, Mohamed Zili, Ridha Khelifa

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Background & aims: Colorectal cancer (CRC) is one of the most common malignancies throughout the world. Several risk factors, both genetic and environmental, including viral infections, have been linked to colorectal carcinogenesis. A few studies report the detection of human polyomavirus JC (JCV) DNA and transformation antigen (T-Ag) in a fraction of the colorectal tumors studied and suggest an association of this virus with CRC. In order to investigate whether such an association of JCV with CRC will hold in a different epidemiological setting, we looked for the presence of JCV DNA and T-Ag expression in a group of Tunisian CRC patients. Methods: Fresh colorectal mucosa biopsies were obtained from 17 healthy volunteers and from both colorectal tumors and adjacent normal tissues of 47 CRC patients. DNA was extracted from fresh biopsies or from formalin-fixed, paraffin-embedded tissue sections using the Invitrogen Purelink Genomic DNA mini Kit. A simple PCR and a nested PCR were used to amplify a region of the T-Ag gene. The obtained PCR products revealed a 154 bp and a 98 bp bands, respectively. Specificity was confirmed by sequencing of the PCR products. T-Ag expression was determined by immunohistochemical staining using a mouse monoclonal antibody (clone PAb416) directed against SV40 T-Ag that cross reacts with JCV T-Ag. Results: JCV DNA was found in 12 (25%) and 22 (46%) of the CRC tumors by simple PCR and by nested PCR, respectively. All paired adjacent normal mucosa biopsies were negative for viral DNA. Sequencing of the DNA amplicons obtained confirmed the authenticity of T-Ag sequences. Immunohistochemical staining showed nuclear T-Ag expression in all 22 JCV DNA- positive samples and in 3 additional tumor samples which appeared DNA-negative by PCR. Conclusions: These results suggest an association of JCV with a subpopulation of Tunisian colorectal tumors.

Keywords: colorectal cancer, immunohistochemistry, Polyomavirus JC, PCR

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