The Indonesian Biomedical Journal

11-28

Authors: Anna Meiliana, Nurrani Mustika Dewi, Andi Wijaya

BACKGROUND: Aging is an unavoidable part of life, defined by a gradual loss in tissue and organ function and an increasing chance of death. Current studies of aging connected the genetic and epigenetic changes to cause this process. CONTENT: When the aging-related epigenetic alterations is accumulated, it may result in irregulated gene expression, metabolic instability, stem cell senescence and exhaustion, and imbalance of tissue homeostasis, which all accelerate the aging process. Altered epigenetic gene regulatory mechanisms such as DNA methylation, histone modification and chromatin remodeling, and non-coding RNAs can induce aging process, thus manipulating these processes give a chance for the success of age-delaying interventions. SUMMARY: Given updated tools and technologies to investigate the epigenetic regulation affecting aging processes, new therapeutic strategies to delay this process can be developed to increase longevity and improve quality of life.

36-44

Authors: Mykyta Valilshchykov, Volodymyr Babalyan, Тetiana Markina, Marina Kumetchko, Liudmyla Boiko, Sergey Romaev

BACKGROUND: In patients with a fracture of the proximal femur and concomitant arterial hypertension, there is a disturbance of the reparative processes of bone tissue. This research aimed to study the regulation of the reparative processes of fractures of the proximal femur with intramedullary osteosynthesis during the correction of concomitant hypertension, which was examined based on some markers using the rat model. METHODS: The study involved healthy Wistar rats and spontaneously hypertensive rats (SHR). The subjects were then grouped into healthy rats without exposure (1.1) SHR without exposure (2.1), healthy rats with modeled fractures of the proximal femur (1.2), SHR with modeled fractures of the proximal femur (2.2), SHR underwent hypertension correction with enalapril in subgroups without fracture (2.3) and SHR underwent hypertension correction with enalapril in subgroups with fracture (2.4). The levels of interleukin (IL)-6, tumor necrosis factor alpha (TNF-a), IL-10, amino-terminal propeptide procollagen type III (PIIINP), glucose, uric acid, creatinine, urea, cholesterol, and albumin were determined in the blood serum of the animals. Femur preparations were examined after the removal of intramedullary fixation. RESULTS: Serum IL-6 level of animal in group 2.4 (2.297±0.361 pg/mL) were reduced compared to the corresponding indicators of rats in group 2.3 (4.054±0.491 pg/mL, p<0.05). Serum glucose and urea levels of animal in group 2.4 (3.951±0.156 mmol/L, 6.552±0.426 mmol/L, respectively) were significantly reduced in comparison with the group 2.3 (6.384±0.890 mmol/L, 10.369±0.888 mmol/L, respectively). The histological results indicated a positive effect of the drug enalapril on the healing of fractures of the proximal femur in animals with hypertension. CONCLUSION: Correction of arterial hypertension with enalapril in fractures of the proximal femur improves the reparative processes of bone tissue.

52-8

Authors: Dewi Wulandari, Alida Roswita Harahap, Suhendro Suhendro, R. Tedjo Sasmono, Aryati Aryati, Herdiman Theodorus Pohan, Iris Rengganis, Saptawati Bardosono

BACKGROUND: Dengue infection remains a major public health problem in Indonesia. Severe dengue associated with plasma leakage require hospitalization and potentially life threatening. However, the mechanism remains unclear, and the occurrence is unpredictable. The role of anti-endothelial antibody is predicted play an important role in the pathogenesis of plasma leakage, as severe dengue is more prevalent in secondary infection or post vaccinated individuals. METHODS: Serum samples from 127 single Dengue Virus (DENV) serotype infected subjects were obtained in day 2 of fever onset. Subjects were divided into plasma leakage and non-plasma leakage based on World Health Organization (WHO) criteria. Anti-endothelial antibody in patient sera were detected using western blot of Human Umbilical Vein Endothelial Cells (HUVEC). To confirm cross-reactivity, the sera was preabsorb with mix-DENV lysate. RESULTS: Three prominent bands were identified on western blot strips that inhibited by pre-absorption with DENV lysate. Plasma leakage patient expressed significantly more antibodies, with 51.7% of plasma leakage patients expressed at least two bands out of those three, compared to 18.5% of non-plasma leakage. CONCLUSION: Antibodies found in sera of dengue patients with plasma leakage cross-reacted with DENV proteins and endothelial proteins 37 kDa, 75 kDa, 120 kDa, and therefore may be involved in the pathogenesis of plasma leakage. Proteomic identification of those protein targets is needed and may be useful for vaccine studies and further development of predictor marker for plasma leakage in dengue.

66-73

Authors: Todung Silalahi, Idrus Alwi, Frans Suyatna, Katarina Dewi Sartika, Christopher Surya Suwita

BACKGROUND: Percutaneous coronary intervention (PCI) is the most common intervention for coronary artery disease (CAD) with very low complications. High oxidative stress post-PCI is associated with further atherosclerosis progression. Curcumin, extracted from a specific type of herbs, exhibits anti-oxidant properties by acting as hydrogen and electron donor for superoxide radicals. The aim of this study is to determine the effect of curcumin’s antioxidant properties in reducing oxidative stress of post-PCI in stable CAD. METHODS: This study was a double-blind parallel randomized controlled trial among 50 stable CAD patients undergoing PCI in Cipto Mangunkusumo General Hospital and Jakarta Heart Center. The subjects received either 45 mg/day curcumin or placebo 7 days pre-PCI until 48 hours post-PCI. Reduced oxidative stress markers (decreased MDA or increased GSH) were measured in 3 phases (7 days pre-PCI, 24 hours post-PCI, 48 hours post-PCI). RESULTS: Curcumin group showed increased MDA from baseline to 24 hours (Δ1=0.01 vs. 0.03; p=0.3) and decreased MDA from baseline to 48 hours (Δ2=-0.06 vs. 0.03; p=0.9). While, curcumin group showed decreased GSH from baseline to 24 hours (Δ1=-49.7% vs. 12.2%; p=0.4) and from baseline to 48 hours (Δ2=-19.09% vs. 11.4%; p=0.6). However, no significant changes were found in malondialdehide (MDA) and glutathione (GSH) level after the intervention. CONCLUSION: The 45 mg/day curcumin supplementation from 7 days pre-PCI until 48 hours post-PCI had no significant antioxidant effect in stable CAD post-PCI.

84-90

Authors: Kusmardi Kusmardi, Bagas Ariffandi, Nadar Sukri Lubis, Tri Wahyuni Lestari, Putri Reno Intan, Alfred Pakpahan

BACKGROUND: Malaria remains a global health concern and an endemic disease in Indonesia. Sambiloto (Andrographis paniculata) and spirulina (Spirulina platensis) are two potential antimalarial agents which act as antioxidants and antiinflammatories that can suppress morbidities during chronic inflammation in malaria, such as proliferation in the colon. This study aims to investigate the effects of A. paniculata extract and S. platensis administration on Ki-67 expression in medial colon epithelial cells of Plasmodium berghei-infected mice measured by H-score. METHODS: Thirty P. berghei-infected male Swiss-Webster mice were divided into five groups: negative controls (carboxymethyl cellulose/CMC); positive controls (dihydroartemisinin-piperaquine/DHP); A. paniculata extract alone (AP); A. paniculata extract in combination with S. platensis extract (AP+ES); and with S. platensis powder (AP+PS). All mice were infected with P. berghei on day 0. The treatment for each group were given 3 days before infection (D-3) until the day of infection (D0) for 28 days after infection. Colon tissues were processed with immunohistochemistry to detect Ki-67. RESULTS: A difference in Ki-67 expression was observed among the groups (p<0.01). The mean H-score for the CMC control group is 135.503±6.723. The lowest level of Ki-67 expression was observed in the AP+PS group (H-score= 110.941±7.079). AP group did not show a significant difference from the CMC group (p=0.514) and neither did the AP+ES group (p=0.234). CONCLUSION: In conclusion, administration of A. paniculata extract and S. platensis powder lowers Ki-67 expression in medial colon epithelial cells of P. berghei-infected mice.

98-103

Authors: Mabruratussania Maherdika, Indranila Kustarini Samsuria, Meita Hendrianingtyas, Nyoman Suci Widyastiti, Muji Rahayu

BACKGROUND: Inflammation occurs in chronic diseases such as type 2 diabetes mellitus (DM). Insulin resistance and inflammation in type 2 DM with obesity can increase interleukin (IL)-6, causing an increase in hepcidin synthesis in the liver. Increased inflammation can exacerbate the course of type 2 DM. This study aims to prove that there are differences in the levels of hepcidin and IL-6 between obese and non-obese type 2 DM. METHODS: This cross-sectional study was conducted on 61 patients with type 2 DM, consist of 22 male and 39 female with an age of more than 40 years. Type 2 DM subjects were obtained from a doctor's diagnosis and were divided into obese and non-obese groups based on body mass index (BMI). Hepcidin and IL-6 levels were examined using the Enzyme-Linked Immunosorbent Assay (ELISA) principle. The data were analyzed using an independent t-test and Mann-Whitney test. RESULTS: The mean level of hepcidin in the obese with type 2 DM group was 25.32±11.54 ng/mL, and non-obese was 11.94±5.31 ng/mL. The median level of IL-6 in the obese with type 2 DM group was 11.9 (5-61) pg/mL, and non-obese 4.8 (1.5-9.8) pg/mL. There was a significant difference in hepcidin and IL-6 levels between the obese and non-obese groups (p=0.000). CONCLUSION: Hepcidin and IL-6 levels in the obese group with type 2 DM were higher than non-obese group.

350-4

Authors: Pudji Rahaju, Ayunita Tri Wirattami, Ferry Sandra, Steffi Kurniawan, Khairun Nisa, Soehartono Soehartono, Edi Handoko, Ahmad Dian Wahyudiono, Hendradi Surjotomo, Hendy Setyo Yudhanto, Nanik Setjowati

BACKGROUND: Progression of laryngeal carcinoma can be classified with the clinical staging, however there are different patterns of progressions observed in the patient with the same clinical stage which also affects their prognoses. Therefore biomarkers should be used. Nuclear factor (NF)-ĸB, Cyclin-D1, vascular endothelial growth factor (VEGF), cyclooxygenase (Cox)-2 have been reported for laryngeal carcinoma. However, it is still unclear how these markers are expressed and correlated in advanced stage laryngeal carcinoma. Therefore current study was conducted to investigate the expressions of NF-ĸB, Cyclin-D1, VEGF and Cox-2 and their correlations in advanced stage laryngeal carcinoma. METHODS: Subjects were recruited and laryngeal biopsies were collected, fixed in formalin and prepared for immunohistochemistry. The immunohistochemistry was performed using mouse monoclonal anti-NF-kB p65, anti-Cyclin-D12 anti-VEGF, and anti-Cox-2 antibodies. The immunohistochemistry results were documented and measured using ImmunoRatio. Pearson or Spearman correlation test was used based on the results of Shapiro-Wilk test of normality. A p-value of less than 0.05 is considered statistically significant. RESULTS: Twelve male subjects were included in this study. Expressions of NF-ĸB, Cyclin-D1, VEGF dan Cox-2 were clearly observed. Mean of NF-ĸB, Cyclin-D1, VEGF dan Cox-2 IHC expression levels measured with ImmunoRatio were 57.50±20.06%, 45.00±24.31%, 43.33±17.23% and 40.42±16.98%, respectively. There was significant correlation between the expressions of VEGF dan Cox-2 (p=0.031, r=0.622). CONCLUSION: Since correlation between the VEGF and Cox-2 expressions was statistically significant, VEGF and Cox-2 might have important roles in the growth, invasion and metastasis of laryngeal carcinoma.

364-74

Authors: Nur Ainina Abdollah, Nabil Izzatie Mohamad Safiai, Muhammad Khairi Ahmad, Kumitaa Theva Das, Siti Razila Abdul Razak

BACKGROUND: Molecular alterations of microRNA130a (miR130a) are observed in many types of cancers, including non-small cell lung cancer (NSCLC). However, the role of miR130a in NSCLC has been poorly studied. METHODS: In this study, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 was utilised to knockdown miR130a. The gRNA was designed to target the stem loop, 3’ and 5’ sites of miR130a and stably expressed in A549 cells. Post-treatment, mature levels of miR130a-3p and 5p were quantified, and proliferation and migration assays were conducted. RESULTS: Result showed significant suppression of miR130a-3p and -5p by two and three-fold respectively, when the CRISPR/Cas9 targeted at the 3’ site and stem loop of the miR130a gene. Suppression of miR130a-3p significantly increased the growth and migration of A549 cells, but no significant changes were observed in cells with suppressed expression of miR130a-5p. CONCLUSION: Our encouraging results highlight that the suppression of the miR130a is achievable using CRISPR/Cas9, and suppression of the miR-130a-3p could play an important role in the regulation of NSCLC.

383-95

Authors: Nurul Syuhada Nordin, Yong Yoke Keong, Siti Farah Md Tohid, Zetty Nadia Md Zain, Muhammad Nazrul Hakim

BACKGROUND: Eventhough 6-Mercaptopurine act as a major drug for rheumatoid arthritis (RA) treatment, however, its toxicity become a limitation. Therefore, this current study investigated whether 6-hydroxy-2-mercaptopurine (6H2MP) and 6-thioguanine (6TG) compounds are purine nucleoside analogues as a potential treatment of RA. The objective was to evaluate the therapeutics effects, especially the anti-inflammatory potential of 6H2MP and 6TG in the lipopolysaccharides(LPS)-induced RAW264.7 cells and phorbol myristate acetate (PMA)-induced HIG-82 cells. METHODS: Macrophage cells (RAW264.7) and rabbit synoviocytes (HIG-82) cells were induced using LPS and PMA to evaluate the anti-inflammatory potential of 6H2MP and 6TG. The cytotoxicity assessment was done by using MTT assay, while enzyme-linked immunosorbent assay (ELISA) was used to determine the anti‑inflammatory potential, including tumour necrosis factor (TNF-α), interleukins (IL-1β, and IL-6). RESULTS: Upon LPS-induced, RAW 264.7 macrophages demonstrated that 6H2MP and 6TG could suppress the production of nitric oxide (NO) in vitro. The half-maximal inhibitory concentration (IC50) value of 6TG and 6H2MP were 10.73 and 13.31, respectively. Further studied in PMA-induced HIG-82 synovial fibroblast cells showed that 6H2MP and 6TG also suppressed the release of NO, Prostaglandin E2 (PGE2), and inflammatory cytokines such as TNF-α, IL-1β and IL-6. The 6TG is more effective to reduce inflammatory reactions compared to 6H2MP, by the lower dose needed compared to 6H2MP in all experiments except in PGE2. CONCLUSION: The inhibition of inflammatory mediators is an important mechanism by which 6TG and 6H2MP may alleviate pain and articular inflammation. These results indicated that 6H2MP and 6TG are effective candidates for ameliorating inflammatory-associated complications.

403-8

Authors: Hamsu Kadriyan, Eka Sunarwidhi Prasedya, Nova Audrey Luetta Pieter, Masyita Gaffar, Amsyar Akil, Agusalim Bukhari, Budu Budu, Andi Alfian Zainuddin, Rina Masadah, Achmad Chusnu Rhomdoni, Abdul Qadar Punagi

BACKGROUND: Nasopharyngeal cancer (NPC) is known to release a specific exosome. NPC-derived exosome (NPC-Exo) could carry p53. However, information regarding the type of p53 carrier on NPC-Exo remains limited. This study aims to introduce our important findings regarding the type of p53 NPC-Exo cargo. METHODS: Serum from patients with NPC were prepared for exosome isolation with Seramir Exoquick by following the manual instructions. RT-PCR was conducted to determine the expression levels of latent membrane protein 1 (LMP-1) and p53 in the exosome isolate. Partial sequencing of p53 amplicon was conducted to determine mutation type of p53. RESULTS: There were 8 patients enrolled in this study. According to RT-PCR results, the expression levels of LMP-1 and p53 varied in the NPC-Exo isolate. Based on sequencing analysis, 1 case of p53 mutation was noticeable. CONCLUSION: According to current results, the NPC-derived exosome potentially carries not only wild type but also mutant type p53. Further research is needed to explore deeper the effect of the mutant type p53 as an exosome carrier in the clinical application.

418-25

Authors: Dina Keumala Sari, Ridha Dharmajaya, Mutiara Indah Sari, Dewi Masyithah Darlan

BACKGROUND: The presence of vitamin D receptor (VDR) polymorphism and high levels of inflammatory markers are predisposing factors indicating disease progression and malnutrition. To meet nutritional needs in maintaining nutritional status in tuberculosis patients with VDR gene polymorphisms (TaqI or FokI), food that is easily absorbed and high in vitamin D, calcium, and protein is needed. This study was conducted to determine whether high vitamin D and calcium porridge called soy–catfish–anchovy–rice (SCAR) porridge would increase 25(OH)D serum levels and other parameters, including calcium, albumin, high-sensivity C-reactive protein (hs-CRP), and blood glucose serum levels. METHODS: The study was a parallel, open, clinical trial. There were 22 subjects in the intervention (I) group who received 50 g of SCAR porridge once per day along with dietary counseling, and 21 subjects in the control (C) group who only received dietary counseling. All subjects with TC and CC (TaqI) or TC and CC (FokI) genotype were included in this study. The intervention lasted for 14 days, and the parameters such as 25(OH)D, calcium, albumin, hs-CRP, and blood glucose serum levels were assessed before and after intervention between groups. RESULTS: All subjects in both groups completed the study. After 14 days of intervention, there was a significant increase in 25(OH)D (p=0.01) and decrease in hs-CRP (p=0.02) serum levels in the I group, and no change was observed in the C group. There was no significant difference in albumin and blood glucose serum levels. CONCLUSION: The results show that 50 g of SCAR porridge per day for 14 days is an effective supplementation that can increase 25(OH)D and decrease hs-CRP serum levels in tuberculosis patients with VDR gene polymorphism.

433-7

Authors: Ferry Sandra, Jennifer Putri, Hilary Limen, Blanca Sarizta

BACKGROUND: The potential of the caffeic acid in other important Receptor Activator Nuclear Factor kB Ligand (RANKL)-Tumor Necrosis Factor (TNF)a-induced osteoclastogenic signaling pathways has not been known. Therefore, the current study was conducted to explore as well as to understand the inhibition potential of caffeic acid. METHODS: RAW264.7 cells were cultured, treated with caffeic acid, RANKL and TNFa. Tartrate Resistant Acid Phosphatase (TRAP) staining was performed to detect TRAP+ osteoclast-like polynuclear cells. To detect the activity of p44/42 Mitogen Activated Protein Kinase (MAPK), Akt, and Transforming Growth Factor-β-activated Kinase (TAK)1, the phosphorylated forms of the proteins were investigated with the immunoblotting assay. RESULTS: Pre-treatment of caffeic acid inhibited the RANKL and TNFa-induced differentiation of RAW264.7 cells into TRAP+ osteoclast-like polynuclear cells. RANKL and TNFa induced phosphorylation of p44/42 MAPK at Thr202/Tyr204, phosphorylation of Akt at both Ser473 and Thr308 and phosphorylation of TAK1 at Ser412. Pretreatment with caffeic acid prior to the RANKL and TNFa induction, inhibited the phosphorylation of MAPK, and TAK1, but not Akt. CONCLUSION: Caffeic acid might regulate the RANKL-TNFa-induced osteoclastogenic pathway in RAW264.7 by targeting TAK1, which later activation of p44/42 MAPK was abolished.

242-55

Authors: Chandra Agung Purnama, Anna Meiliana, Melisa Intan Barliana, Keri Lestari Dandan, Andi Wijaya

BACKGROUND: Millions of cells in multicellular organisms regenerate every day to replace aged and died cells. Effective cell clearance (efferocytosis) is critical for tissue homeostasis, as the human body recycles its cellular components. We summarize what is known about the mechanisms of efferocytosis and how it impacts the physiology of the organism, effects on inflammation and the adaptive immune response, as well as the consequences of defects in this critical homeostatic mechanism in this review. CONTENT: Cell death is the process by which the human body replaces aged or damaged cells with new ones. It can be triggered by genetically encoded machinery or regulated cell death, or by specific pharmacologic or genetic interventions, resulting in accidental cell death. Dying cells release signals that entice phagocytes to engulf them in a process known as efferocytosis. Efferocytosis is a multistep process involving the release of “find me” and “eat me” signals and destruction of death cells by phagocytes. Different types of cell death including apoptosis and necroptosis can express pro- or anti-inflammatory signals via macrophage activity modulation. SUMMARY: Failed or ineffective efferocytosis can result in disruption of tissue homeostasis, which can contribute to the development of chronic inflammatory diseases such as atherosclerosis, obesity, diabetes, and heart failure. Therefore, any therapeutic strategy that enhances efferocytosis will have a beneficial effect on the treatment of these metabolic disorders.

261-70

Authors: Aigerim Turgimbayeva, Sailau Abeldenov, Nurgul Sarina, Bekbolat Khassenov, Saule Eskendirova

BACKGROUND: Active proliferation is specific property of a tumor cells. However, the cost of the analysis is high due to commercial anti-Ki-67 mAbs used as the main immunoreagent for reliable identification of proliferating cells. In this study, recombinant protein was used to obtain specific mAbs for Ki-67 biomarker immunodetection. METHODS: Codon optimized fragment of ki-67 gene was cloned into the pET28c(+)vector. The recombinant protein was purified by immobilized metal affinity chromatography (IMAC) and confirmed by liquid chromatography–mass spectrometry (LC-MS)/MS. Hybridoma cells were obtained by fusing myeloma cells with mouse spleen cells immunized with recombinant antigen. The specificity and activity of mAbs was determined by enzyme-linked immunosorbent assay (ELISA), Western blot and immunocytochemistry. RESULTS: The pET-28c(+)/ki-67 plasmid, which encodes 355 amino acid protein, was obtained. Analysis by LC-MS/MS of the recombinant antigen showed that 77.5% of the amino-acid sequence belonged to Ki-67 protein. Recombinant fragment of Ki-67 protein was used to obtain specific hybridoma strains. ELISA and Western blot demonstrated high affinity and the specificity of obtained mAbs against Ki-67 protein. Newly generated anti-Ki67 mAbs detected target protein in proliferating cells of MCF-7 cell line by immunocytochemistry. CONCLUSION: Newly developed mAbs are potentially useful as an immunodiagnostic tool for assessing the proliferative activity of breast tumor cells using immunocytochemistry.

281-8

Authors: Dewinda Candrarukmi, Annang Giri Moelyo, Muhammad Riza

BACKGROUND: Hyperglycemia is one of the most common endocrine complications in children with β thalassemia major. Though the current diagnostic marker either requires fasting, has low reproducibility, or it is not an accurate for thalassemia patients. Glycated albumin (GA) is a quick and easy alternative marker for hyperglycemia detection and monitoring of glycemic control. However to date, no studies have analyzed the role of GA value in detection of hyperglycemia in children with thalassemia major. This study analyzed the value of GA as an alternative screening marker for hyperglycemia detection in children with β thalassemia major. METHODS: A single-blind prospective diagnostic test was conducted in 9 to 18 years old children with β thalassemia major and who were treated at the Dr. Moewardi Regional General Hospital between October 1, 2018 and December 31, 2019. In a single, fasted study visit, height, weight, fasting blood glucose (FPG), GA, oral glucose tolerance test (OGTT) were measured. The area under a receiver operating characteristic curve (AUC) was used to determine the cut-off value at which hyperglycemia prediction (OGTT ≥140 mg/dL) display optimal sensitivity and specificity. RESULTS: Among the 53 children with β thalassemia major, 1 (1.9%) had diabetes mellitus and 4 (7.5%) had prediabetes based on their OGTT values. The median GA value in this study was 10.9% (range: 7.6–12.4%). GA had a low AUC (0.646, p=0.287) for hyperglycemia detection in pediatric patients with β thalassemia major. At a cut-off of 11.45%, GA demonstrated 60% sensitivity and 60.4% specificity. CONCLUSION: GA cannot be used as an alternative marker for hyperglycemia detection in children with β thalassemia major.

295-302

Authors: Dwita Permatasari, Hendrian Dwikoloso Soebagjo, Ismi Zuhria, Nila Kurniasari, Hari Basuki Notobroto, Cinintha Nandini

BACKGROUND: Usually, surgical intervention is needed to eradicate the fungal microorganism that cause fungal corneal ulcers. However, since surgical intervention is invasive, the latest technology uses cryotherapy in treating it. Cryotherapy plays a vital role in the wound healing process. We aimed to evaluate is to study the decreased expression of matrix metalloproteinase 9 (MMP-9) and transforming growth factor β1 (TGF-β1) in fungal corneal ulcers after the administration of cryotherapy. METHODS: Aspergillus flavus fungus was injected to the intrastromal corneas of all Sprague Dawley rats. The rats were divided into four groups, the first group was not given any therapy, the second group was given topical natamycin therapy, the third group was given cryotherapy, and the fourth group was given a combination between cryotherapy and topical natamycin therapy. Therapy was given after five days of follow up on the formation of a corneal ulcer. After four days of therapy, the eyes were enucleated to determine MMP-9 and TGF-β1 expression. RESULTS: The result in the third group showed lower MMP-9 expression (20.0±10.0% cells per field of view) compared to the second group (40.0±20.0% cells per field of view) and the fourth group (30.0±25.0% cells per field of view), but had the same MMP-9 expression value as the first group. There was no significant difference in MMP-9 expression between the four groups (p=0.356). The third group reduced more TGF-β1 expression (10.0±12.50% cells per field of view) compared to the fourth group (30±27.5% cells per field of view) and the first group (30±32.5% cells per field of view). There was also no significant difference in TGF-β1 between the four groups (p=0.315). CONCLUSION: There is no significant difference in the expression of TGF-β1 and MMP-9 after the cryotherapy treatment.

310-5

Authors: Fatmawati Fatmawati, Ellyza Nasrul, Nasrul Zubir, Ferry Sandra

BACKGROUND: T lymphocyte activation depends on the balance of co-stimulatory and co-inhibitory signals determined by Cluster of Diffrentiation (CD)28 and Programmed Cell Death Protein 1 (PD-1) expression. Alteration in CD28 and PD-1 expression might affect the progression of chronic hepatitis B (CHB). Current study was conducted to evaluate the correlations of the CD28 and PD-1 expressions of T lymphocytes and CHB progression. METHODS: Subjects were recruited, selected and divided into 3 groups, inactive CHB, active CHB and CHB with End-Stage Liver Disease (ESLD). HBeAg was determined by using Enzyme-Linked Fluorescence Assay while HBV-DNA was carried out by the RT-PCR method. Numbers of T lymphocytes expressing CD3, CD4, CD8, CD45, CD28 and PD-1 molecules were determined by flowcytometry. RESULTS: There was no significant difference in the expression of CD28 by CD4+ and CD8+ T lymphocytes of inactive CHB, active CHB and CHB with ESLD subjects. There was also no significant difference in the expression of PD-1 in CD4+ lymphocytes of inactive CHB, active CHB and ESLD subjects. In contrast there was a significant increase in the expression of PD-1 in CD8+ T lymphocytes of ESLD subjects. CONCLUSION: CD28 expression among CHB subjects was within normal range and not related to disease progression, but PD-1 expression of CD8+ T lymphocyte was increased along with disease progression, especially in CHB subjects with ESLD. This suggests that PD-1-overexpressed CD8+ T lymphocyte play a role in increasing CHB disease progression.

324-31

Authors: Thianti Sylviningrum, Ismiralda Oke Putranti, Octavia Permata Sari, Fitranto Arjadi, Triasari Oktavriana

BACKGROUND: Psoriasis is an autoimmune disease involving genetic-environmental factors. Human Leukocyte Antigen (HLA)-Cw6 allele is the main genetic risk factor of psoriasis, but its prevalence varies widely. HLA-Cw6 allele correlates with psoriasis clinical type and treatment responses. Narrowband ultraviolet B (NB-UVB) and methotrexate (MTX) are effective psoriasis treatments but their association with HLA-Cw6 allele has not been identified. The study aims to determine the association between HLA-Cw6 allele expression, NB-UVB, and MTX treatment responses in Javanese-Indonesian psoriasis subjects. METHODS: Ninety Javanese-Indonesian psoriasis subjects were recruited in this study, 45 subjects were treated using NB UVB, while the other 45 subjects were treated using MTX, respectively, for 12 weeks. The psoriasis diagnosis and treatment responses were evaluated by dermatologists using Psoriasis Area Severity Index (PASI) score. The HLA-Cw6 allele was examined using the single-specific-primer polymerase chain reaction method. Fisher's Exact and Chi-square tests were employed, where p-value<0.05 was considered of significant association. RESULTS: The HLA-Cw6 allele positivity was identified in 23 psoriasis subjects (25.56%), while the other 67 subjects expressed HLA-Cw6 allele negative (74.44%). Female with HLA-Cw6 allele positivity who did not have comorbid disease show good response to NB-UVB than MTX. Meanwhile, subjects who were treated with MTX showed no association between therapeutic response and HLA-Cw6. HLA-Cw6 status was not correlated with the onset of psoriasis, family history, and comorbid diseases in all subjects. CONCLUSION: HLA-Cw6 allele expression is more associated with good NB-UVB treatment response than with MTX treatment response in female Javanese-Indonesian psoriasis subjects without comorbid disease.

114-39

Authors: Anna Meiliana, Nurrani Mustika Dewi, Andi Wijaya

BACKGROUND: A lot of contemporary cancer research has concentrated on genetic influence. However, cancer also involves biochemical changes, such as metabolic adaptation to support the aberrant cell proliferation. CONTENT: The fast cell proliferation in cancer cells enforce a metabolic re-arrangement to promote their long-term survival. The increased glucose uptake and fermentation of glucose to lactate are common features of this altered metabolism known as “the Warburg effect”. These metabolic pathways regulation enable cancer cells to produce adenosine triphosphate (ATP) in an efficient way. Epigenetic and metabolic changes also both affect molecular rewiring in cancer cells and promote cancer development and progression. SUMMARY: Metabolic rewiring and epigenetic remodeling establishing a direct link between metabolism and nuclear transcription to promote the survival of tumor cells. A further understanding of how metabolic remodeling can result in epigenetic changes in tumors, affecting cancer cell differentiation, proliferation, and/or apoptosis, will lead to a new strategy for cancer therapy.

147-54

Authors: Erna Susanti, Endang Susilowati

BACKGROUND: Signaling pathways contributing to cholesterol efflux and inhibitory inflammation in atherosclerosis that has not been explored is the liver X receptor (LXR). Catechin as LXR agonist influences the expression of Niemann-pick C1 like 1 (NPC1L1) protein transporter that triggers the inhibition of cholesterol absorption. This study aimed to examine the effect of Catechins on the expression of intestinal transporters: NPC1L1, ATP-binding cassete-proteins G5 (ABCG5) and G8 (ABCG8). METHODS: Twenty-five experimental animals were divided into five treatment groups, with 5 rats in each group. The groups were normal diet rats (group 1), high fat diets-induced rats (group 2), high fat diets-induced rats treated with 30 mg/kg body weight (BW) of Catechins (group 3), high fat diets-induced rats treated with 60 mg/kg BW of Catechins (group 4), and high fat diets-induced rats treated with 120 mg/kg BW of Catechins (group 5). After one-month, all rats were sacrificed, blood and intestine were collected. Lipid profile were determined enzymatically, mRNA levels were determined by reversetranscriptase polymerase chain reaction (RT-PCR), while the expression of protein transporter were determined by immunohistochemistry. RESULTS: Catechins treatment decreased the expression of NPC1L1, but increased the expression of ABCG5 and ABG8. CONCLUSION: Catechins can be developed as a candidate for NPC1L1 inhibitor to mediate the inhibiting absorption of intestinal cholesterol, therefore increasing the inhibitory effect of atherogenesis.

163-9

Authors: Astuti Giantini, Ina Susianti Timan, Erlin Listiyaningsih, Rahajuningsih Dharma, Rianto Setiabudy, Idrus Alwi, Lia Gardenia Partakusuma, Nuri Dyah Indrasari, Alida Roswita Harahap, Siskawati Suparmin, Windy Sahar, Renan Sukmawan

BACKGROUND: Light transmission aggregometry (LTA) and VerifyNow is commonly used to measure platelet responsiveness to clopidogrel. This study aimed to compare the results of LTA and VerifyNow P2Y12 assay for assessing the clopidogrel resistance in patients undergoing percutaneous coronary intervention and determine factors affecting clopidogrel resistance. METHODS: The subjects were 119 patients who underwent percutaneous coronary intervention (PCI) and had given loading dose of 600 mg clopidogrel. Blood samples were taken at 6 hour after clopidogrel loading dose. Platelet aggregation was measured by LTA and VerifyNow. RESULTS: LTA and VerifyNow assay showed fair agreement with Kappa=0.270, p=0.001. The proportion of resistance to clopidogrel using VerifyNow was 21.8% and LTA was 47.1%. Patients with diabetes melitus were more likely to develop clopidogrel resistance than patients without diabetes (OR of 7.67; 95% CI: 1.87-31.50; p=0.005). CONCLUSION: The ability of LTA and VerifyNow in detecting clopidogrel resistance were not comparable. Multivariate analysis results for VerifyNow shows diabetes mellitus as the greatest predictors of clopidogrel resistance.

178-85

Authors: Mahmood Thamer Altemimi, Alaa Khattar Musa, Abbas Ali Mansour

BACKGROUND: Obese women with polycystic ovary syndrome (PCOS) are at high risk for developing type 2 diabetes mellitus (T2DM). A baseline oral glucose tolerance test (2-h OGTT) is important to screen for dysglycemia in PCOS particularly those high risk women. Due to its advantages by fasting is not required and less day-to-day variability, glycated hemoglobin (HbA1c) might be a convenient screening tool. This study aimed to evaluate the performance of HbA1c vs. 2-h OGTT in the diagnosis of glycemic disorders in PCOS and to evaluate the correlation between glycemic disorders, insulin resistance (IR), and anthropometric measures. METHODS: One hundred and twenty nine women diagnosed with PCOS according to the Rotterdam 2003 criteria in Basrah were included in the study. All subjects were examined for weight, body mass index (BMI) and waist circumference (WC). Then they were tested for fasting glucose, 2-h OGTT, HbA1c, and fasting insulin to assess IR. RESULTS: The result of 2-h OGTT test showed that there were 21 subjects (16.1%) showed to have IGT. The result of HbA1c test showed that 25 subjects (19.4%) were diagonised with prediabetes. Meanwhile FPG test result showed that 34 subjects (26.4%) were having IFG. The HbA1c examination showed an underdetected the diagnosis of T2DM (0.8%) and an overdeteced diagnosis of prediabetes (19.4%) (p=0.021) and at HbA1c 5.55% (37.2 mmol/mol), the specificity was (74.3%) and sensitivity (56.5%) to discriminate normal from abnormal glucose status. Ninety nine women (76.7%) were either overweight or obese and most of them had IR (76.8%). CONCLUSION: Screening of glycemic disorders is crucial for PCOS by using 2-h OGTT regardless of risk factor and HbA1c seems to be an unsatisfactory screening tool to predict glycemic disorders in women with PCOS.

192-200

Authors: Anggi Laksmita Dewi, Dewi Kartikawati Paramita, Jajah Fachiroh

BACKGROUND: Single nucleotide variations (SNV) have been mapped to be associated with several human conditions and diseases. To validate the association between SNV to certain human traits or diseases, a large number of subjects must be included. Thus, in need of a fast, relatively economic, and reliable genotyping method. This can be achieved through the use of tetra-primer amplification refractory mutation system polymerase chain reaction (Tetra-primer ARMS PCR). This study reports strategy to develop Tetra-primer ARMS PCR-based genotyping of CHRNA3 rs8040868. METHODS: The optimization of Tetra-primer ARMS PCR was done through these steps: identification of gene sequence and position of single mutation; designing outer and inner PCR primers; amplification of target gene fragments through PCR by using outer primer; confirming genotype of the PCR product by using sequencing; determining an optimum ratio of outer and inner primer; and determining optimum annealing temperature and cycles for the PCR program. The PCR products were run in 2% gel agarose electrophoresis and visualized under UV illumination. RESULTS: Outer and inner primer ratio of 1:3 with annealing temperature of 64.4°C and 40x cycles was found to be the most optimum condition. Tetra-primer ARMS PCR was able to confirm the results of the DNA sequence of 2 samples, confirming wild-type variants (TT allele) and the heterozygous mutant (CT allele). CONCLUSION: Tetra-primer ARMS PCR was able to genotype rs8040868 of the CHRNA3 gene.

208-15

Authors: Dwi Wahyuni, Joko Waluyo, Jekti Prihatin, Fersty Isna Kusumawardani, Aditya Kurniawan

BACKGROUND: Traditional medicine that might be useful to treat typhoid fever is earthworms (Pheretima javanica K.). It is important to know whether any substances contained in Pheretima javanica K. extract are toxic and have pharmacological effects, which can be observed from the eosinophil and immunoglobulin E (IgE) levels in the blood. METHODS: Thirty Rattus norvegicus B. were included in this study and were separated into 5 groups, with 6 rats in each group. T1, T2, T3, and T4 were groups of rats treated with 50, 300, 2000, and 5000 mg/kg BW of Pheretima javanica K. ethanol extract granules (PJEEG), respectively, while the control group was a group of rats treated with 0.36 mg Ciprofloxacin. The eosinophil number and the IgE concentration were detected. Physical observations were also conducted to detect any allergic symptoms, toxicity symptoms, or histopathological changes. RESULTS: Administration of PJEEG significantly increased the number of eosinophil in every group, from 49.82 cells/μL to 259.26 cells/μL on average (p<0.001). PJEEG did not significantly affect IgE levels (p=0.624). The results showed that the administration did not cause any allergic and toxicity symptoms. Histopathological analysis of rats’ kidneys and liver also showed that the administration had no significant effect on histopathological changes. CONCLUSION: The administration of PJEEG does not cause allergic symptoms, toxicity symptoms, and histopathological changes in rats. The administrations also significantly increase eosinophil number in rats’ blood. However, the concentration of IgE in rats’ blood was not significantly changed after administration of PJEEG.

19-26

Authors: Miftakh Nur Rahman, Ajeng Diantini, Miswar Fattah, Melisa Intan Barliana

BACKGROUND: There is a continuous rise in the prevalence of central obesity and become a pressing health problem in the world. Central obesity followed by many metabolic disorders especially Type 2 Diabetes Mellitus (T2DM). The pathogenesis started from overnutrition signal that force pancreatic beta cells to produce a large number of insulin. Low-grade chronic inflammation that occurred also affects the organs sensitivity against insulin and caused beta cells to compensated this situation and at the end become exhausted and loss its function. CONTENT: Along compensation mechanism, certain nutrients were support the beta cells to maintain their mass and function to produce insulin. Short chain fatty acids (SCFAs) are gut microbiota fermentation product that act as nutrient and give an advantage to the proliferation and survivability of the beta cells. Zinc (Zn) also plays an important role in every step of insulin production. Moreover, these nutrients protecting pancreas against inflammation and oxidative stress through certain mechanism. Most of patients with central obesity are unaware of the presence of this disturbance at early stage. Whereas, at molecular level there is a magnitude of SCFAs and Zn level in the blood that would become an early signal and predict the damage of beta cells. SUMMARY: Quantification of these two nutrients in the blood expected to provide an early warning tool to maintain insulin adequacy and predict the possibility of beta cell failure in central obesity with promising performance.

34-9

Authors: Aileen Clarissa Dauhan, Aridamuriany Dwiputri Lubis, Munar Lubis

BACKGROUND: Early detection and treatment of sepsis can prevent septic shock and reduce mortality rate. Troponin can become a prognostic factor in sepsis. However, not all health facilities are equipped to assess troponin levels. Vasoactive-inotropic score (VIS) is a simpler and more accessible method to describe hemodynamic status. The aim of this study was to assess the suitability of VIS score as early prognosis and mortality predictor of sepsis METHODS: A retrospective study was conducted to determine the correlation between VIS and troponin levels for sepsis cases in Pediatric Intensive Care Unity (PICU) Haji Adam Malik Hospital, Medan from January 2018 to December 2019. VIS score at 48 hours, maximum VIS score, pediatric logistic organ dysfunction-2 (PELOD-2) score, cardiac troponin levels at 48 hours were taken from medical records. RESULTS: There were 54 samples analyzed. VIS scores were positively correlated (p<0.001) to troponin T and troponin I levels at 48 hours (r=0.670 and r=0.606, respectively). VIS at 48 hours and maximum VIS were related to mortality (p=0.001 and p<0.001, respectively). VIS score at 48 hours could be used as a predictive factor for mortality (area under the curve (AUC): 79.7%, p<0.001) with a cut-off point at 11 (74.4% sensitivity and 80% specificity). High VIS at 48 hours indicated poor outcomes of sepsis in children with odd ratio (OR) value: 1.99 (95% confidence interval (CI): 1.25-3.19). CONCLUSION: Vasoactive-inotropic score was suitable as an alternative to cardiac troponin T and troponin I levels at 48 hours to early detect myocardial dysfunctions and mortality in children.

48-54

Authors: Mardiah Suci Hardianti, Syahru Agung Setiawan, Nungki Anggorowati, Wiwiek Probowati

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with dysregulation of somatic hypermutation (SHM) and class switch recombination (CSR) have been known to contribute for its lymphomagenesis. Activation-induced cytidine deaminase (AID) enzyme plays a vital role for both processes. Multiple myeloma oncogene 1 (MUM1) is known to upregulate the AID expression in normal and pathological conditions. However, both AID and MUM1 expression association in DLBCL is still unexplored using immunohistochemistry method. We examined DLBCL samples and then retrospectively tested its correlation with clinical findings. METHODS: A retrospective cohort study with 20 cases of DLBCL biopsy tissue with AID and MUM1 antibody was conducted. The samples were then classified into concordant (AID+/MUM1+ or AID-/MUM1-) and discordant group (AID-/MUM1+). The clinicopathological comparison was performed to observe any association between immunohistochemistry expression and clinical findings. RESULTS: Among 20 samples of DLBCL, concordant expression rate of AID and MUM1 was 80% with kappa Cohen’s of 0.578 (p=0.004). A significant association was observed between AID and MUM1 expression with a prevalence ratio of 2.25 (95% CI: 1.08-4.67; p=0.008). Clinical characteristics were not significantly different between each group. Restricted mean survival time was shorter in the concordant group compared with the discordant group but statistically insignificant (21.16 vs. 22.5 months; p=0.531). CONCLUSION: The result of this study showed the association between AID and MUM1 expression in DLBCL. However, whether the association may add further molecular heterogeneity of DLBCL is still to be confirmed by expanding the study.

61-7

Authors: Novalia Chumaladewi Guntarno, Anny Setijo Rahaju, Nila Kurniasari

BACKGROUND: Bladder cancer is the tenth-most common cancer in worldwide and the majority are urothelial carcinomas. The depth of invasion plays important role in the prognostic and therapeutic factor in urothelial carcinomas. The expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) may associated with the progression of bladder carcinoma, such as depth of invasion. In this study, the correlation between these two markers in urothelial bladder carcinoma invasion will be analyzed. METHODS: An analytical observational research with cross-sectional were conducted on 54 formalin fixed paraffin-embedded tissue from radical cystectomy (RC) which were diagnosed as bladder urothelial carcinoma at the Anatomical Pathology Laboratory of Dr. Soetomo Academic Hospital, Surabaya and divided based on the T stage were immunostained using VEGF and MMP-9 monoclonal antibodies. The difference of VEGF and MMP-9 expression in T stage of bladder urothelial carcinoma were analyzed using Kruskal-Wallis and Anova test, the correlation between VEGF and MMP-9 expression in various T stage of bladder urothelial carcinoma were analyzed using Spearman test. RESULTS: This study showed no significant difference of VEGF expression among T stage of bladder urothelial carcinoma (p>0.05) but there was significant difference of MMP-9 expression in T stage of bladder urothelial carcinoma (p=0.043). There was a correlation between VEGF and MMP-9 in various T stage of bladder urothelial carcinoma (rs= 0.50, p=0.001). CONCLUSION: The significant correlation of VEGF and MMP-9 expression in bladder urothelial carcinoma may prove the synergistically role of both proteins in tumor invasion by MMP-9 degradation extracellular matrix.

75-83

Authors: Erwin Astha Triyono, Sarah Firdausa, Heru Prasetyo, Joni Susanto, James Hutagalung, Lilis Masyfufah, Budiono Budiono, Ivan Hoesada

BACKGROUND: Human Immunodeficiency Virus (HIV) is an infectious disease that targets the human immune system by attacking cluster of differentiation (CD)4 cells. The use of propolis in HIV patients is expected to be safe and beneficial in terms of increasing endurance and immunity by its role in increasing CD4 level. This study aimed to analyze the influence of propolis supplementation in increasing the CD4 level in anti-retroviral (ARV)-treated HIV patients. METHODS: Double-blind randomized controlled clinical trial was conducted in 50 HIV patients who took regular ARV therapy. The subjects were divided into two groups, one group was treated with ARV and propolis, while another one was given ARV and placebo. The CD4 cell count was measured during pre-treatment, in the 3rd month, in the 6th month after treatment. The level of hemoglobin, leukocyte, and platelets were also measured. The SF-12 questionnaire was used to evaluate quality of life of the subject. RESULTS: Out of 50 subjects, 43 subjects completed the study, which were 19 subjects from the propolis group and 24 subjects from the placebo group. After 3-month of treatment, there was a statistically significant difference in the increase of CD 44 level in propolis group, while the increment was not significant in the placebo group. After 6-month treatment, the increase of CD4 level was occurred in both groups, propolis and placebo, however the increment was not statistically significant. The levels of hemoglobin, leukocyte, and platelets were not altered by the treatment and remained normal throughout the study. The quality of life was improved during the study; however, it was also not statistically significant. Mild adverse events occurred in 3 subjects which were relieved after the treatment stopped. CONCLUSION: Based on the result of this study, the administration of propolis on HIV patients receiving ARV bring significant difference in the increase of CD4 in propolis group from baseline to 3 month after the treatment. While in placebo group, this increment was not significant. At the end of study, CD4 count continued to rise up, however the increase was not statistically significant. There are no hemoglobin, leukocyte, platelets, and quality of life abnormalities. Therefore, it is necesary to do further research with a spesific CD4 count. However, it may be beneficial in relieving the clinical symptoms and quality of life of patient living with HIV.

91-6

Authors: Diana Nur Afifah, Fauzia Purnamasari, Luthfiatul Khusna, Noviasti Rahma Utami, Aida Fitri Nazillah, Syafira Noor Pratiwi, Fillah Fithra Dieny, Aryu Candra, Ayu Rahadiyanti, Rachma Purwanti, Enny Probosari, Martha Ardiaria, Nyoman Suci Widiastiti, Ferry Sandra

BACKGROUND: Administration of resistant starch (RS) influences the diversity and the composition of microbiota as well as inhibits the growth of cancer cell. Banana as a potential source of RS has been reported. Although Musa paradisiaca has been reported to induce apoptosis in colon cancer cells, Musa balbisiana, which has low glycemic index and suitable for particular patients, has not been investigated yet. METHODS: Starches of M. balbisiana and M. paradisiaca were prepared and mixed with other components to make 3 types of mouse pellets. Mouse model for colon cancer was prepared and fed with different types of mouse pellets. Blood was collected and processed for measuring β-glucuronidase and malondialdehyde (MDA) with Enzyme-linked Immunosorbent Assay (ELISA) method. Resected ceca were incised to collect the inner part for short-chain fatty acid (SCFA) measurement with gas chromatography analysis. Resected colas were fixed and processed for immunohistochemistry to detect Caspase-3. RESULTS: Colon-cancer-mice fed with the M. balbisiana and M. paradisiaca starches-contained pellets had significant higher concentrations of total SCFA (p=0.003), acetic acid (p=0.000), propionic acid (p=0.000) and butyric acid (p=0.000); lower concentration of β-glucuronidase (p<0.001); higher Caspase-3 score (p=0.040); and lower MDA concentration (p<0.001) than colon-cancer-mice fed with standard pellet (control). CONCLUSION: M. balbisiana and M. paradisiaca starches could be suggested as potential anti-colon cancer RS. Further research should be carried out to disclose the starches mechanisms in colon cancer cell.