Madridge Journal of Internal and Emergency Medicine

ISSN: 2638-1621

International Translational and Regenerative Medicine Conference

April 25-27, 2018, Rome, Italy
Poster Session Abstracts
DOI: 10.18689/2638-1621.a1.003

Inhibition of Senescence and Promotion of the Proliferation of Chondrocytes from Articular Cartilage by CsA and FK506 Involves Inhibition of p38MAPK

Deug Y Shin1,2, Seok-Won Kang1,2 and Jungbin Kim1

1Department of Microbiology, Dankook University College of Medicine, Korea
2Cancer Research Institute, Seoul National University College of Medicine, Korea

CyclosporineA (CsA) and tacrolimus (FK506) are the most important immunosuppressive compounds that block the activation of helper T-cells. In this study, we investigated the effects of CsA and FK506 on growth and senescence of articular chondrocytes. Chondrocytes from young rabbit cartilage entered senescence after 8.6 ± 0.8 population doublings (PDs), while chondrocytes treated with CsA and FK506 entered senescence after 12.3 ± 1.4 and 13.7 ±0.6 PDs, respectively. Furthermore, chondrocytes from the cartilage of old rabbits were senescent after 2.6 ± 0.9 PDs, whereas those treated with CsA and FK506 were senescent after 8.2±1.8 and 6.9 ± 1.6 PDs, respectively. These compounds also inhibited senescence induction of chondrocytes in a high-cell density pellet culture system. We previously reported that p38MAPK plays a critical role in the onset of senescence in chondrocyte. This study revealed that the phosphorylation of p38MAPK was inhibited by either CsA or FK506. The early onset of senescence in chondrocyte harboring MKK6E, which is a constitutively-active form of MKK6 and increases p38MAPK phosphorylation, was blocked by CsA. These results suggest that CsA and FK506 increase the proliferation and inhibit the senescence of articular chondrocytes through inactivation of p38MAPK.

Keywords: Senescence, p38MAPK, CsA, FK506, chondrocyte.

Dr. Shin received his Phd in 1989 at the University of Tokyo, and worked as a Postdoc fellow and a research associate at NCI, USA. He was nominated as a chief of National Research Lab and a PM of National Cancer-Aging Research Program, and served as a Chair for Korean Assoaiciation of Genrontology and now serves as a professor and director of Aging-Cancer Center of Dankook University He provided first evidence that p53 can induce senescence in human tumor and suggested a novel cancer therapy to induce senescence in human tumors.

Decoding Ca2+ Signaling in Regulation of Transcription Factor Activation and Cell Cycle Progression using Optogenetics

Wen-Tai Chiu*, Ya-Han Chang, Chi-Sian Yu and Yi-Shyun Lai

Department of Biomedical Engineering, National Cheng Kung University, Taiwan

The ability of a simple ion such as Ca2+ to play an highly versatile intracellular signal results from the facility that cells have to shape Ca2+ signals in the dimensions of space, time and amplitude. Thus, spatial and temporal changes in intracellular Ca2+ concentration is important to determinate the cell fate. Optogenetics is the combination of genetics and optics to control well-defined events within specific cells of living tissue in real time. Optogenetic stimulation approach that uses channelrhodopsin-2 (ChR2) related proteins has been developed for providing more precise and targeted stimulation effect on cells in vivo and in vitro. We aimed to pose the formerly unaddressed fundamental question of how Ca2+ signals regulate transcription factor activation by manipulating intracellular Ca2+ through using optogenetic strategies. In this study, We found that (1) elevation of intracellular Ca2+ levels that correspond with the power, frequency and duty cycle of light illumination in a dose-dependent manner; (2) activation of Ca2+-dependent transcription factors is depends on Ca2+ oscillations with different frequency and amplitude; (3) cells in the various phases of the cell cycle showed different responses to Ca2+-mediated cell cycle progression. In summary, the delicate regulation and precise control of transcription factor activation and cell cycle progression can be achieved under the optogenetic platform. Parameters associated with light illumination will be optimized in the future. It will enable us to be confident in applying this advanced technique to (1) manipulate the expression patterns of genes by desire; and (2) control the growth and differentiation of cells. Such a process would be a potential platform in study of cell development and cancer.

Dr. Wen-Tai Chiu received the Bachelorʼs, Masterʼs and Ph.D. degrees from National Cheng Kung University, Taiwan in 1997, 1999 and 2007, respectively. He has been a postdoctoral fellow at the University of Texas MD Anderson Cancer Center from 2008 to 2010. Dr. Chiu is currently an associate professor in the Department of Biomedical Engineering at National Cheng Kung University. His research interests lie in the area of Ca2+ signaling and molecular imaging of cancers. Much of his work has been on improving the understanding, design, and performance of Ca2+ in focal adhesion dynamics, cell migration, metastasis and chemoresistance.

Gene Discovery in a Neurogenetic Cohort

Wilhemina Koomson*, Adife Ercan-Sencicek, Katsuhito Yasuno and Murat Gunel

Yale University, USA

We analyzed 1,505 patients with neurological/neurodevelopmental disorders of varying severity including brain structural malformations from a network of 28 clinical centers in Turkey, specifically ascertaining consanguineous cases. Next generation sequencing technologies, specifically whole-exome sequencing (WES), led to identification of disease causing variants in these cases. Our study considered various modes of inheritance, focusing on recessive forms of the disease as well as de novo variation, especially for non-consanguineous cases. We have identified several pathogenic mutations falling within a Homozygous by descent (HBD) segment in consanguineous families. With an improved filtering strategy we were able to identify multiple independent coding mutations or copy number variations (CNV), suggesting novel disease-causing genes have been identified. We prioritized these genes, as well as other strong candidate genes, based on known biological function, molecular interaction and Weighted Gene Co-expression Network Analysis (WGCNA). We identify gene co-expression module profiles of the new candidate genes, correlating them with the spatial and temporal expression patterns (during brain development) of known phenotype concordant disease genes.

Wilhemina Koomson is a 3rd year PhD candidate in the Department of Genetics at Yale University. Her research incorporates bioinformatics in studying Neurological and Neurodevelopmental disorders. She completed her undergraduate work at Princeton University majoring in Molecular Biology.

The Cytotoxic Effect of Pleurostylia Capensis Turcz (LOES)

N Cebekhulu1 and SCKM Motaung2

1Department of Biomedical Sciences, Mangosuthu University of Technology, South Africa
2Department of Biomedical Sciences, Tshwane University of Technology, South Africa

Background: Bone fractures non-unions and delayed unions remain a persistent orthopaedic challenge. The current treatments have proven to have constraints due to numerous adverse side effects. Traditional medicinal plants have a reputable outcome in the treatment of many diseases. Pleurostylia capensis Turcz (Loes) is a rich source of bioactive metabolites known to be anti-viral, antibacterial, antiparasitic and antineoplastic. It is utilised to treat arthritis, fractures, epilepsy and other diseases for centuries. It is a rich source of bioactive metabolites.

Objectives: To evaluate the cytotoxic activities of leaves and bark extracts Pleurostylia capensis against mouse skeletal (C2C12) cells invitro using the 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.

Method: C2C12 cells were cultured as monolayers in medium supplemented with 10 % foetal bovine serum, and antibiotic cocktail. Subsequently, the cells were treated with the aqueous bark and root extracts of P. capensis at different concentrations. The MTT CellTiter 96® non-radioactive cell proliferation assay was conducted at day 2, 4, and 8 post- incubation.

Results: The MTT assay results showed that P. capensis significantly increased the viability of the cells. The plant crude extracts enhanced cell proliferation and the percentage of viability with the highest peaks at Day 4, specifically on the positive control and concentrations 30µg/ml and 50 µg/ml.

Conclusion: The results obtained revealed significant proliferation, overall metabolic activity, division, turnover and viability of cells. These findings warrant further studies to isolate novel compounds which will serve to inform future scientific research towards the development of safe drug formulations for bone fracture repair.

Keywords: Medicinal plants; bone fractures, cell culture, MTT assay.

Dr. Nokukhanya Cebekhulu is a board certified Medical Technologist specialised in Clinical Pathology with the Health Professions Council of South Africa. She was a Masters candidate at the Tshwane University of Technology in South Africa and is currently employed as a junior faculty in Biomedical Technology at the Mangosuthu University of Technology, Durban South Africa. Her research interests are in the indigenous knowledge systems of South Africa, specifically medicinal plants and their cytotoxic validation and potential translation and application in tissue engineering and bone fracture regeneration.

Prognostic and Predictive Values of Cell Cycle Proteins Centrosomal Protein 5 (CEPP 5) and Cyclin D1 Expression Inepithelial Ovarian Carcinoma (EOC)

Ola A Harb1, Rham Z Ahmed2, Safa A Balata3 and Loay M Gertallah4

1Pathology Department, Zagazig University, Egypt
2Medical Oncology Department, Zagazig University, Egypt
3Clinical oncology& nuclear medicine Department, Zagazig University, Egypt
4General surgery Department, Zagazig University, Egypt

Background: Disturbances in the expressions of centrosomal proteins (CEPs) and regulatory proteins that control G1-Sphase transition, like cyclins could participate in dysregulation of cell cycle control that has been incriminated in the pathogenesis of several malignancies. Centrosomal protein 55 (CEP55) has an important role in participation in the final stage of cell division, and cell cycle progression. CEP55 and Cyclin D1 expressions were detected in several tumors but their prognostic and predictive roles in epithelial ovarian carcinoma (EOC) are still studied.

Aim of the study was to explore tissue expressions of CEPP55 and Cyclin D 1 in EOC correlating their expression with pathological, clinical and prognostic parameters.

Methods: CEP55 & Cyclin D1 expressions were evaluated in tissue biopsies that are retrieved from 60 cases of epithelial ovarian carcinoma using immunohistochemistry, patients that were followed up for 3 years. The relationship between their level of expressions and degree of differentiation, spread of the tumor, disease recurrence, response to therapy and survival were studied.

Results: CEP55 expression in EOC was positively correlated with loss of differentiation of the tumor, presence of L.N (p<0.001), and distant metastases (p=0.012) & advanced stage of the tumor (p=0.007), cyclin D1 expression in EOC was positively correlated with loss of differentiation & advanced stage of the tumor, presence of L.N (p<0.001), and distant metastases (p=0.009). CEPP 55 & Cyclin D1 were positively correlated with each other.

Low CEPP 55 & Cyclin D1 expressions were strongly correlated with optimal surgical eradication of the tumor, increased 3-year overall survival (OS) and low incidence of tumor recurrence after therapy (P <0.001).

Conclusion: High levels of expression of CEPP 55 & Cyclin D1and are markers of poor prognosis in EOC patients.

Keywords: CEP55, Cyclin D1 epithelial ovarian carcinoma, immunohistochemistry; prognosis

Dr. Ola A Harb, MD; completed her Pregraduate Medical Education (December 2005) in M.B.B.CH., with Total grade-Excellent from Zagazig University, Egypt. She obtained her Postgraduate/M.Sc (May, 2010) & M. D. (January 2015) in pathology from Zagazig University, Egypt. Dr. Ola is presently working as a Lecturer, at Department of pathology, Faculty of Medicine, Zagazig University, Egypt.

L1CAM Up-Regulation in Association with p53 Over-Expression and E-Cadherin Down-Regulation can Help in Detection of Non-Endometrioid Foci, Risk Stratification, Relapse and Outcome of Endometrial Carcinoma Patients

Ola A Harb1, Ola A Megahed1, BasantSh El Shafaay2 and Loay M Gertallah3

1Department of Pathology, Zagazig University, Egypt
2Department of Clinical Oncology and nuclear medicine, Zagazig University, Egypt
3Department of general surgery, Zagazig University, Egypt

Background: The recent guidelines for endometrial carcinoma (EC) management classify patients into; low-, intermediate-, high to intermediate and high-risk groups. Few numbers of patients in the category of low-risk disease were found to have relapse of the tumor that is not explained up till now. Moreover, it is essential to assess prognosis of other risk groups of EC. Also, the detection of patients that the adjuvant therapy will be more beneficial to them than other patients that will not require additional management after surgery is an essential aim of the recent researches about EC. So, recent studied has focused on detection of prognostic biomarkers that might improve risk stratification and detect which patients will need neoadjuvant chemotherapy after surgery. L1-cell adhesion molecule (L1CAM) is membrane glycoprotein member of immunoglobulin superfamily. P53 is a nuclear transcription factor and was found to be an established tumor suppressor. E-cadherin is an integral cell adhesion molecule and it is a component of adherens junction.

The aim of our study to investigate the prognostic significance of L1CAM, P53 and E-cadherin expression in both early-stage, low-, intermediate-risk and high-risk EC, correlate their expression with clinicopathological criteria, tumor progression, recurrence, risk stratification of EC patients and with identification of patients that will need neoadjuvant chemotherapy after surgery.

Methods: Expressions of L1CAM, P53 and E-cadherin were evaluated in 60 paraffin blocks EC patients that were followed up for 5 years. The relationship between their level of expressions, clinicopathological criteria and prognosis of patients was analyzed.

Results: Positive expression of L1CAM was positively correlated with higher grade of the tumor (p = 0.043), presence of L.N metastases (p = 0.039), presence of, LVSI (p = 0.022), higher risk groups (p = 0.021), and presence of distant metastases (p=0.039). High expression of P53 was positively correlated with higher & advanced FIGO stage of the tumor, presence of L.N metastases, LVSI, higher risk groups and presence of distant metastases (p<0.001). Positive expression f E-cadherin was negatively correlated with higher grade of the tumor (p = 0.019), presence of L.N metastases (p = 0.010), presence of LVSI (p = 0.018) and presence of distant metastases (p=0.013). In multi variant analysis L1CAM expression is the most significant indicator of poor DFS & OS rates (p<0.001).

We found a direct relationship between L1CAM, P53 an inverse relationship between L1CAM and E-cadherin, and an inverse relationship between P53 and E-cadherin (P<0.001).

Conclusion: L1CAM & P53 over expression in addition to loss of E-cadherin expression in EC are associated with non-endometrioid subtype of EC, worse clinicopathological parameters, poor prognosis and could help in identification of patients in need for neoadjuvant chemotherapy after surgery

Keywords: L1CAM, P53, E-cadherin, endometrial carcinoma, immunohistochemistry, risk stratification

Dr. Ola A Harb, MD; completed her Pregraduate Medical Education (December 2005) in M.B.B.CH., with Total grade-Excellent from Zagazig University, Egypt. She obtained her Postgraduate/M.Sc (May, 2010) & M. D. (January 2015) in pathology from Zagazig University, Egypt. Dr. Ola is presently working as a Lecturer, at Department of pathology, Faculty of Medicine, Zagazig University, Egypt.

Clinical Outcome of Autologous Bone Marrow Aspirates Concentrate (BMAC) Injection in Degenerative Arthritis of the Knee -36months Follow Up-

Young Dae Jeon1 and Jae Do Kim2

1Department of Orthopaedic Surgery, Military Manpower Administration, Korea
2Kimʼs Stem Orthopaedic Clinic, Korea

Purpose: As a treatment method of degenerative arthritis of knee, this study evaluated the clinical efficacy of the intra-articular injection of autologous bone marrow aspirates concentrate (BMAC) with adipose tissue.

Materials and Methods: Between April 2011 and May 2012, 41 patients (75 knees) who were diagnosed as a degenerative knee arthritis and underwent the BMAC injection with adipose tissue were included in this study. Mean age was 60.7 years old (ranged 53-80). Kellgren-Lawrence grade was used for assessing radiologic degree of osteoarthritis; there were each 12, 24, 33, and 6 cases of grade I, II, III, and IV. At preoperative and postoperative 6, 12, 36 months pain score using visual analogue scale (VAS) and functional scales were used for evaluation.

Results: After the procedure, mean VAS score was decreased from 8.7 preoperatively to 7.16, 4.4 and 4.3 postoperatively 6, 12, and 36 months. And functional scores were also improved; International Knee Documentation Committee score (from 37.7 preoperatively to 66.3, 69.3, 70.5 postoperatively), SF-36 health score (from 31.5 to 45.6, 47.7, 52.5), knee and osteoarthritis outcome score (from 43.1 to 64.9, 70.6, 72), Lysholm Knee Questionnaire (from 37.3 to 68.6, 71, 72.5), were all increased after the procedure. 9 patients with K-L grade III, IV underwent total knee arthroplasty after two years.

When classified according to K-L grade, the improvement of VAS score in grade IV group was 8.2 preoperatively to 5.3, 5.7, 5.6 postoperatively, which was significantly poorer than those of grade I-III groups. In the knee functional scales, similar pattern was checked.

Conclusions: BMAC injection significantly improved both knee pain and functions in the patients with degenerative arthritis of knee. Also, the injection would be more effective in early to moderate phases.

Keywords: Knee, Arthritis, Bone marrow aspirates concentrate (BMAC), Intra-articular injection

Dr. Young Dae Jeon, Department of Orthopaedic Surgery, Chief Doctor of the Division of Physical Examination for Draft, Military Manpower Administration, South Korea. He received the Master degree from Kosin Gospel University, South Korea in 2014. He worked as an orthopaedic surgeon at Kosin university gospel hospital from 2012 to 2016. His areas of research focus include: stem cell therapy in osteoarthritis of the knee, minimal invasive surgery spine surgery, arthroscopic rotator cuff repair, PRP therapy, BMAC therapy.