The Indonesian Biomedical Journal (Prodia Education and Research Institute)
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Conditioned Media of Human Umbilical Cord Blood Mesenchymal Stem Cell-derived Secretome Induced Apoptosis and Inhibited Growth of HeLa Cells
BACKGROUND: Secreted factors contained in conditioned media (CM) of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) known as secretome, was suspected to have important roles in regulating cells. This study was conducted to investigate the role of CM-hUCB-MSCs-derived secretome in apoptosis and growth of HeLa cells.METHODS: HeLa cells were treated with secretome in various concentrations (0, 0.2, 2 and 20%) for 24 and 48 hours. Trypan blue exclusion assay was performed to detect cell viability. Meanwhile sub-G1 apoptotic assay was performed to detect apoptotic cells. The transition of mitochondrial transmembrane potential (TMP), which occurs in the apoptotic process, was analyzed by mitochondrial membrane potential (ΔΨM) assay. Both sub-G1 and ΔΨM assays were performed using FACSCanto flow cytometer. Statistical analyses were conducted using IBM SPSS Statistics to detect significance level at p<0.05.RESULTS: Secretome significantly induced cell death starting at concentration of 0.2% within a 24-hour period (p<0.05). Secretome significantly induced cell death in concentration and time dependent manner (p<0.05). The cell death was then confirmed as apoptosis through sub-G1 analysis. Due to the underlying apoptotic mechanism, we found distinct decrease of TMP, indicating an increase in mitochondrial membrane permeability of HeLa cells. In addition, we found that HeLa cell growth was inhibited partially by secretome.CONCLUSION: Taken together, we conclude that CMhUCB-MSCs-derived secretome significantly induced apoptosis of HeLa cells in a concentration and time dependent manner through mitochondrial apoptotic pathway. The secretome might also play important role in inhibiting HeLa cell growth.KEYWORDS: umbilical cord blood, mesenchymal stem cell, secretome, apoptosis, growth, cance
Hypertrophic Obesity and Subcutaneous Adipose Tissue Dysfunction
BACKGROUND: Over the past 50 years, scientists have recognized that not all adipose tissue is alike, and that health risk is associated with the location as well as the amount of body fat. Different depots are sufficiently distinct with respect to fatty-acid storage and release as to probably play unique roles in human physiology. Whether fat redistribution causes metabolic disease or whether it is a marker of underlying processes that are primarily responsible is an open question.CONTENT: The limited expandability of the subcutaneous adipose tissue leads to inappropriate adipose cell expansion (hypertrophic obesity) with local inflammation and a dysregulated and insulin-resistant adipose tissue. The inability to store excess fat in the subcutaneous adipose tissue is a likely key mechanism for promoting ectopic fat accumulation in tissues and areas where fat can be stored, including the intra-abdominal and visceral areas, in the liver, epi/pericardial area, around vessels, in the myocardium, and in the skeletal muscles. Many studies have implicated ectopic fat accumulation and the associated lipotoxicity as the major determinant of the metabolic complications of obesity driving systemic insulin resistance, inflammation, hepatic glucose production, and dyslipidemia.SUMMARY: In summary, hypertrophic obesity is due to an impaired ability to recruit and differentiate available adipose precursor cells in the subcutaneous adipose tissue. Thus, the subcutaneous adipose tissue may be particular in its limited ability in certain individuals to undergo adipogenesis during weight increase. Inability to promote subcutaneous adipogenesis under periods of affluence would favor lipid overlow and ectopic fat accumulation with negative metabolic consequences.KEYWORDS: obesity, adipogenesis, subcutaneous adipose tissue, visceral adipose tissue, adipocyte dysfunctio
Which Biology Marker Can be a Predictor for Higher Syntax Score?
BACKGROUND: Syntax score is a lesion-based angiographic scoring system, originally devised to grade the anatomic complexity of coronary artery disease (CAD) and thereby facilitate consensus in the study of a diagnostic angiogram between surgeons and interventional cardiologists. Suitable biology marker to predict the severity of CAD and to have a treatment decision for revascularization by percutaneous coronary intervention (PCI) or bypass surgery, is still unknown. Which biology marker can be a predictor for higher Syntax score remains unknown as well.METHODS: Severity of 105 patients who underwent coronary angiography, were evaluated with Syntax score. Laboratory tests were conducted for routine blood test, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), high-sensitivity C-reactive protein (hs-CRP), total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, kidney function, creatinine and hemoglobin A1c (HbA1c).RESULTS: Ureum had significant correlation with Syntax score, p=0.016 and 95% confidence interval (CI)=0.05-0.55. Result of logistic regression analysis showed that HDL and haematocrit had significant correlation with Syntax score, with cut off point 34 (p=0.02, p=0.0033, respectively). Based on multi-variate analysis, Syntax score 34 = 16 (HDL ≤40 + Ureum ≥23) + 10 (Neutrophil/Lymphocyte ≥3.789) +10 (HbA1c ≥7 ) + 13 ( monocyte ≥9%).CONCLUSION: HDL cholesterol, ureum, neutrophil-lymphocyte ratio, HbA1c and monocyte altogether can predict the higher Syntax score.KEYWORDS: syntax, neutrophil, lymphocyte, HDL, cholesterol, ureum, HbA1c, monocyt
Comparison between Carotid Intima-Media Thickness and Coronary Artery Calcification in the Prediction of Atherosclerosis in Diabetic Patients
BACKGROUND: Cardiovascular disease is one of the atherosclerosis etiologies that can lead to death. Diabetes mellitus increases the risk of atherosclerosis. Screening tool is very beneficial for detecting atherosclerotic plaque, especially in subclinical atherosclerotic cases. Carotid intima-media thickness (CIMT) and coronary artery calcification score (CACS) are two kinds of tools that are widely used, and each of these tools has its own superiority. This study was aimed to investigate the sensitivity and specificity of both of these tools as screening tools.METHODS: The study was conducted with a cross sectional design involving 43 diabetic and 68 non-diabetic male subjects aged above 45 years old. All subjects fulfilled inclusion criteria. Carotid artery ultrasonography and CACS measurement were performed.RESULTS: Fischer exact test was used to show a significant correlation between CIMT and CACS (p<0.05). Diagnostic test was used to assess the sensitivity of CIMT toward CACS in above 75 percentile. The left common carotid artery (LCCA) showed the highest sensitivity either in diabetic (76.4%) or non-diabetic male subjects (90%).CONCLUSION: CIMT has the same sensitivity with CACS. CIMT can be used as the preferred screening tool for high risk patients and as a substitution tool to CACS for low risk patients in subclinical atherosclerosis detection.KEYWORDS: atherosclerosis, diabetes mellitus, carotid intima-media thickness, coronary artery calciication scor
MicroRNAs in Lipid Metabolism and Atherosclerosis
BACKGROUND: MicroRNAs (miRNA) are mediators of post-transcriptional gene expression that likely regulate most biological pathways and networks. The study of miRNAs is a rapidly emerging field; recent findings have revealed a significant role for miRNAs in atherosclerosis and lipoprotein metabolism.CONTENT: Results from recent studies demonstrated a role for miRNAs in endothelial integrity, macrophage inflammatory response to oxidized low-density lipoprotein, vascular smooth muscle cell proliferation and cholesterol synthesis. These mechanisms are all vital to the initiation and proliferation of atherosclerosis and cardiovascular disease. The importance of miRNAs has recently been recognized in cardiovascular sciences and miRNAs will likely become an integral part of our fundamental comprehension of atherosclerosis and lipoprotein metabolism. The extensive impact of miRNA mediated gene regulation and the relative ease of in vivo applicable modifications highlight the enormous potential of miRNA-based therapeutics in cardiovascular diseases.SUMMARY: miRNA studies in the field of lipid metabolism and atherosclerosis are in their infancy, and thus there is tremendous opportunity for discovery in this understudied area. The ability to target miRNAs in vivo through delivery of miRNA-mimics to enhance miRNA function, or antimiRNAs which inhibit miRNAs, has opened new avenues for the development of therapeutics for dyslipidemias and atherosclerosis, offers a unique approach to treating disease by modulating entire biological pathways. These exciting findings support the development of miRNA antagonists as potential therapeutics for the treatment of dyslipidaemia, atherosclerosis and related metabolic diseases.KEYWORDS: atherosclerosis, lipoprotein, HDL, miRN
Ser81 Survivin Induced Protein Kinase A (PKA)-dependent Phosphatidylinositol 3-kinase (PI3K) Activity
BACKGROUND: Our previous report showed that phosphorylated-survivin at Ser81 induces survivin back loop to activate protein kinase A (PKA) in the cytoprotection mechanism. Activated PKA could possibly induce the cytoprotection via Phosphatydilinositol 3-kinase (PI3K). Therefore our current study was conducted to investigate the possibility of survivin-PKA-PI3K signaling pathway.METHODS: Viral productions by BOSC23 cells of Survivin, Antisense Survivin (Survivin-AS) and Ser81Ala mutant (Survivin-S81A) in pMSCV-IRES-GFP vector with cytomegalovirus (CMV) promoter were conducted. L929 cells were pretreated with/without PKI 6-22 amide and infected with viral particle of Survivin, Survivin-AS, Survivin-S81A or vector only. Cells were harvested, lysed and immunoprecipitated with anti-PI3K (p85) antibody and immunoblotted to detect PI3K (p85) and phospho-(Tyr) p85 PI3K. To confirm PI3K activation, PI3K Activity Assay was conducted by using phosphoinositide fraction containing PtdIns(4,5)P2 and [32P]ATP.RESULTS: Immunoblot and PI3K activity results showed similar results. Upon infection of virus with survivin, a markedly increased level of tyrosine phosphorylation of p85 PI3K or PI3K activity in L929 cells was seen. Low levels of tyrosine phosphorylation of p85 PI3K or PI3K activity were observed for Survivin-AS and Survivin-S81A-viral-infected L929 cells. With higher concentrations of Survivin-viral-infection, levels of tyrosine phosphorylation of p85 PI3K or PI3K activity in L929 cells were gradually increased. However, when L929 cells were pretreated with PKI 6-22 amide, prior to Survivin-viral-infection, level of tyrosine phosphorylation level of p85 PI3K or PI3K activity was detected much lower.CONCLUSION: Our result suggest that Ser81 Survivin play role in inducing PI3K activation and the Survivin-PI3K signaling pathway was PKA-dependent.KEYWORDS: Ser81, Survivin, PKA, PI3K, L92
Proliferation of Peripheral Blood-derived Endothelial Progenitor Cells from Stable Angina Subjects
BACKGROUND: A population of circulating Endothelial Progenitor Cells (EPCs) has been reported to play important role in maintaining endothelial function and integrity. Since EPCs culture is crucial and an optimized medium is currently available. Therefore we conducted a study to investigate whether stable angina subjects peripheral blood-derived EPCs could be cultured in this medium. Here, we performed study to detect EPCs characteristics and extracellular signalregulated kinase (Erk)1/2 Mitogen-Activated Protein Kinase (MAPK) pathway as possible underlying pathway for EPCs proliferation.METHODS: Peripheral blood EPCs from 8 stable angina subjects were cultured in an optimized medium with/without addition of supplement for 1 or 3 days. Then, the membrane of cultured EPCs were detected with immunofluorescence method for CD34, Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) and CD133. Colony forming unit (CFU) enumeration was performed. XTT Cell proliferation assay was performed to assess EPCs growth after 1 and 3-days culture. The western blot analysis was performed to detect possible activation of Erk1/2 MAPK.RESULTS: Number of EPCs and CFU cultured for 3 days were significantly higher than the ones cultured for 1 day (p=0.012). EPCs membrane markers from stable angina subjects were detected as well as CFUs were formed. There were significant increase of EPCs number, CFUs number and phosphorylated-Erk2 amount when the groups with and without supplement were compared (p<0.05). Meanwhile U0126, a MAPK Erk1/2 (MEK1/2) inhibitor, significantly inhibited the supplement-induced EPCs number, CFUs number and phosphorylated-Erk2 amount (p<0.05).CONCLUSION: Our results showed that ERK2 MAPK signaling pathway might play an important role in supplement-induced peripheral blood EPCs proliferation in subjects with stable angina.KEYWORDS: endothelial progenitor cell, EPC, p42, Erk2, proliferatio
Comparison of IL-6, IL-8 Concentrations in H. pylori- and non-H. pylori-associated Gastritis
BACKGROUND: Helicobacter pylori is a non-invasive microorganism causing intense gastric mucosal inflammatory and immune reaction. The gastric mucosal levels of the proinflammatory cytokines Interleukin 6 (IL-6) and IL-8 have been reported to be increased in H. pylori infection, but the serum levels in H. pylori infection is still controversial. The purpose of this study was to investigate the serum levels of IL-6 and IL-8 in H. pylori infection.METHODS: A cross sectional study was done on eighty consecutive gastritis patients admitted to endoscopy units at Adam Malik General Hospital and Permata Bunda Hospital, Medan, Indonesia from May-October 2014. Histopathology was performed for the diagnosis of gastritis. Rapid urease test for diagnosis of H. pylori infection. Serum samples were obtained to determine circulating IL-6 and IL-8. Univariate and bivariate analysis (independent t test) were done.RESULTS: There were 41.25% patients infected with H. pylori. Circulatory IL-6 levels were significantly higher in H. pylori-infected patients compared to H. pylori negative, but there were no differences between serum levels of IL-8 in H. pylori positive and negative patients.CONCLUSION: The immune response to H. pylori promotes systemic inflammation, which was reflected in an increased level of serum IL-6. Serum levels of IL-8 were not significantly different between H. pylori positive and negative.KEYWORDS: Helicobacter pylori, gastritis, IL-6, IL-8, cytokin
Application of Umbilical Cord Blood Stem Cells in Regenerative Medicine
BACKGROUND: Since the first umbilical cord blood (UCB) transplant, performed 25 years ago, UCB banks have been established worldwide for the collection and cryopreservation of UCB for autologous and allogeneic transplants.CONTENT: Much has been learned in a relatively short time on the properties of UCB hematopoietic progenitors and their clinical application. More interestingly, non-hematopoietic stem cells have been isolated from UCB. These cells can be grown and differentiated into various tissues including bone, cartilage, liver, pancreas, nerve, muscle and so on. The non-hematopoietic stem cells have an advantage over other sources of stem cells, such as embryonic stem cells or induced pluripotent stem cells, because their supply is unlimited, they can be used in autologous or allogeneic situations, they need minimal manipulation and they raise no ethical concerns. Future studies will test the potential of UCB cells for the treatment of several diseases including, among other possibilities, diabetes, arthritis, burns, neurological disorder and myocardial infarction.SUMMARY: In addition to hematopoietic stem cells, UCB contain a large number of non-hematopoietic stem cells. In the absence of ethical concern, the unlimited supply of UCB cells explains the increasing interest of using UCB for developing regenerative medicine.KEYWORDS: UCB, transplantation, UCB bank, HSC, MSC, CD34, CD133, VSE
The Effect of the Transtheoretical Model Approach Towards the Body Mass Index of Overweight and Obese Children in Makassar
BACKGROUND: Overweight and obesity is now ranked fifth as a global risk for the cause of death. Urban lifestyle is fueled by excessive food intake and lack of activity in overweight and obese children. The assessment of the children’s Body Mass Index (BMI) is one of the indicators to assess the nutritional status of children. One strategy that can be used is the transtheoretical model (TTM) approach which is behavior change that helps children and parents in making more effective decisions to reduce health-risk behaviors among children so that the children’s BMI can decrease. This study aims to identify the differences between children's BMI before and after the TTM approach.METHODS: This research was conducted in the district area of Tamalanrea and Biringkanaya, Makassar. The design used is the Quasy Experiment namely pre- and post-test with control group design. Research subjects were overweight or obese children in the 4th, 5th, and 6th grade of elementary school. The samples in the treatment group consisted of 31 participants, while the control group consisted of 33 participants which were selected using purposive sampling. Intervention research was conducted during a six month period by providing guide books on healthy lifestyle, visiting the families every month using questionnaires, and measurements of children’s height, weight and BMI using WHO’s AntrhoPlus software year 2007.RESULTS: Post-pretest results showed the average value of the treatment group’s BMI was -1.48 and the control group was 1.35. This means that the treatment group experienced a decrease in BMI after the application of TTM. The Mann Whitney test results indicate that there is a difference in the BMI pre-post intervention with the value of p=0.00 (>0.05).CONCLUSION: The overweight and obese children’s BMI was influenced by TTM approach.KEYWORDS: transtheoretical model, children’s BMI, overweight, obesit