LP-110 development of GSTA1, CYP2C19, and CYP2B6 gene polymorphism detection methods on the response of cyclophosphamide therapy for lupus nephritis patients

Background Cyclophosphamide, an immunosuppressant in patients with lupus nephritis, aims to prevent recurrence and is a steroid-sparing agent. The clinical response (efficacy and toxicity) of lupus nephritis patients receiving cyclophosphamide varied.1 Cyclophosphamide is a pro-drug metabolized by hydroxylation via the CYP2C19 and CYP2B6 enzymes and detoxification via the glutathione-S-transferase (GST) enzyme.2 The most common type of mutation that occurs is single nucleotide polymorphism (SNP). Several SNPs from previous studies associated with cyclophosphamide response, metabolism, and toxicity in patients with lupus nephritis, namely rs3957356 in the GSTA1, rs4244285 in the CYP2C19, rs7254579 and rs4802101 in the CYP2B6.3 Therefore, detecting and screening SNP genotypes in lupus nephritis patients can help analyze cyclophosphamide response variation. This study aims to develop a method for detecting the genotypes of the four target SNPs and several surrounding SNPs using PCR-Sanger sequencing. Methods The gene polymorphism analysis method was optimized before taking patient samples at the hospital. PCR and Sanger sequencing methods are used for gene polymorphism analysis because they produce chromatograms with target amplicon base lengths and several SNPs that can be analyzed simultaneously. Results The analysis results of the four target SNPs of the GSTA1, CYP2C19, and CYP2B6 (rs3957356, rs4244285, rs7254579, and rs4802101) showed one synonymous SNP and three SNPs (regulatory and intergenic). We have developed an SNP detection assay using four fragments from the GSTA1, CYP2C19, and CYP2B6 that can detect 15 SNPs simultaneously (figure 1). In addition, this method succeeded in distinguishing wild-type, heterozygous and homozygous genotypes. Furthermore, this method can be used to analyze GSTA1, CYP2C19, and CYP2B6 gene polymorphisms in lupus nephritis patients receiving cyclophosphamide therapy, especially in a population in West Java, Indonesia. Conclusions PCR-sanger sequencing is a reliable, accurate, and simple method for determining SNP genotypes from blood samples

LP-110 development of GSTA1, CYP2C19, and CYP2B6 gene polymorphism detection methods on the response of cyclophosphamide therapy for lupus nephritis patients

Background Cyclophosphamide, an immunosuppressant in patients with lupus nephritis, aims to prevent recurrence and is a steroid-sparing agent. The clinical response (efficacy and toxicity) of lupus nephritis patients receiving cyclophosphamide varied.1 Cyclophosphamide is a pro-drug metabolized by hydroxylation via the CYP2C19 and CYP2B6 enzymes and detoxification via the glutathione-S-transferase (GST) enzyme.2 The most common type of mutation that occurs is single nucleotide polymorphism (SNP). Several SNPs from previous studies associated with cyclophosphamide response, metabolism, and toxicity in patients with lupus nephritis, namely rs3957356 in the GSTA1, rs4244285 in the CYP2C19, rs7254579 and rs4802101 in the CYP2B6.3 Therefore, detecting and screening SNP genotypes in lupus nephritis patients can help analyze cyclophosphamide response variation. This study aims to develop a method for detecting the genotypes of the four target SNPs and several surrounding SNPs using PCR-Sanger sequencing. Methods The gene polymorphism analysis method was optimized before taking patient samples at the hospital. PCR and Sanger sequencing methods are used for gene polymorphism analysis because they produce chromatograms with target amplicon base lengths and several SNPs that can be analyzed simultaneously. Results The analysis results of the four target SNPs of the GSTA1, CYP2C19, and CYP2B6 (rs3957356, rs4244285, rs7254579, and rs4802101) showed one synonymous SNP and three SNPs (regulatory and intergenic). We have developed an SNP detection assay using four fragments from the GSTA1, CYP2C19, and CYP2B6 that can detect 15 SNPs simultaneously (figure 1). In addition, this method succeeded in distinguishing wild-type, heterozygous and homozygous genotypes. Furthermore, this method can be used to analyze GSTA1, CYP2C19, and CYP2B6 gene polymorphisms in lupus nephritis patients receiving cyclophosphamide therapy, especially in a population in West Java, Indonesia. Conclusions PCR-sanger sequencing is a reliable, accurate, and simple method for determining SNP genotypes from blood samples

Genetic polymorphisms associated with cyclophosphamide outcome and risk of toxicity in patients with lupus nephritis

A 6-month cyclophosphamide induction therapy followed by maintenance therapy every three months is the first-line treatment for Class III, IV, and V lupus nephritis. Among the 139 single nucleotide polymorphisms (SNPs) associated with cyclophosphamide, four SNPs, namely rs4244285, rs4802101, rs7254579 and rs3957356, are related to the response and risk of toxicity in patients with lupus nephritis. Although pharmacogenetic studies in patients with lupus nephritis (LN) have not been conducted previously in Indonesia, data on rs4244285 are available for several ethnic groups, including Papuans, Bataks, Balinese, Dayaks, Javanese, Bugis, Chinese, Timorese and Malays, even though direct evidence in LN patients is less detectable. However, this can be followed up prior to cyclophosphamide therapy based on the identification of genetic markers. Therefore, clinical studies in patients with lupus nephritis are deemed necessary to evaluate the potential of these markers

Genetic polymorphisms associated with cyclophosphamide outcome and risk of toxicity in patients with lupus nephritis

A 6-month cyclophosphamide induction therapy followed by maintenance therapy every three months is the first-line treatment for Class III, IV, and V lupus nephritis. Among the 139 single nucleotide polymorphisms (SNPs) associated with cyclophosphamide, four SNPs, namely rs4244285, rs4802101, rs7254579 and rs3957356, are related to the response and risk of toxicity in patients with lupus nephritis. Although pharmacogenetic studies in patients with lupus nephritis (LN) have not been conducted previously in Indonesia, data on rs4244285 are available for several ethnic groups, including Papuans, Bataks, Balinese, Dayaks, Javanese, Bugis, Chinese, Timorese and Malays, even though direct evidence in LN patients is less detectable. However, this can be followed up prior to cyclophosphamide therapy based on the identification of genetic markers. Therefore, clinical studies in patients with lupus nephritis are deemed necessary to evaluate the potential of these markers

Aktivitas Makrofag Meningkat Pada Aorta Tikus Hiperkolesterolemia

Aterosklerosis merupakan, kondisi inflamasi kronik, faktor resiko penyakit kardiovaskular disebabkan oleh tingginya kadar kolesterol. Tujuan penelitian ini mengevaluasi peran mieloperoksidase (MPO) dan makrofag di aorta dan jantung tikus yang diinduksi hiperkolesterolemia. Tikus dikelompokkan menjadi kelompok normal dan hiperkolesterolemia. Induksi hiperkolesterolemia dilakukan dengan pemberian pakan tinggi kolesterol, kolesterol murni, asam kolat dan propiltiourasil (KKT) selama 5 bulan. Kolesterol total diukur sebelum induksi, pertengahan, dan setelah induksi. HDL, trigliserida (TG), LDL, indeks aterogenik (IA), jumlah sel darah merah dan sel darah putih diukur setelah induksi. Deteksi ekspresi mieloperoksidase (MPO) dan CD68 pada aorta dan jantung dilakukan dengan metode dot blot dan ELISA. Induksi hiperkolesterolemia selama 5 bulan menghasilkan kadar kolesterol total (364,10 ± 148,46 mg/dL), HDL (7,90 ± 1,29 mg/dL), LDL (307,47 ± 116,91 mg/dL), dan Indeks Aterogenik (1,04 ± 0,23). Kadar kolesterol yang tinggi meningkatkan jumlah sel darah putih yang bersirkulasi namun tidak mempengaruhi jumlah sel darah merah. Jumlah makrofag yang berada di jaringan aorta dan jantung kelompok hiperkolesterolemia meningkat secara signifikan dibanding dengan kelompok normal. Namun, peningkatan aktivitas makrofag yang diukur dari ekspresi MPO hanya teramati pada aorta hewan hiperkolesterolemia, tidak pada jantung. Simpulan, aktivitas makrofag meningkat hanya pada aorta hewan hiperkolesterolemia diduga berperan dalam pembentukan plak ateroma di aorta. Kata kunci: Aorta, CD68, hiperkolesterolemia, makrofag, mieloperoksidase   Macrophage Activity Increases in Hypercholesterolemia Rat Aorta   Abstract   Atherosclerosis, which is an inflammatory chronic condition, is one of the major risk factors of cardiovascular disease caused by hypercholesterolemia. This study aimed to evaluate role of myeloperoxidase (MPO) and macrophage in aorta and heart of rat hypercholesterolemia. Rats were divided into normal and hypercholesterolemia groups. Hypercholesterolemia was induced by cholesterol feeding and CCT (cholesterol, cholic acid and propiltiourasil) oral administration for 5 months. Total cholesterol was measured before induction (T0), in the middle (T2.5), and after induction (T5). HDL, triglyceride (TG), LDL, aterogenic index (IA), red blood, and white blood cell count was measured after induction (T5). Success of induction was proven by the elevation of cholesterol total value of hypercholesterolemia group compared to normal group. Myeloperoxidase (MPO) and CD68 in aorta and heart hypercholesterolemia rat was detected by dot blot and ELISA method. Hypercholesterolemia group showed significant differences in total cholesterol value (364.10±148.46 mg/dL), HDL value (7.90±1.29 mg/dL ), LDL value (307.47±116.91) and Atherogenic Index (1.04±0.23). High level of cholesterol increases circulating white blood cells but have no effect on  circulating red blood cells. Macrophage in the  hypercholesterolemia group increased significantly compared to the normal group. However, the increase in macrophage activity identified throughMPO expression was only seen in hypercholesterolemic aorta but not  in the heart. It is concluded that macrophage activities increase in the aortic tissue, but  not in the heart tissue of the hypercholesterolemia group, which may contribute to the formation of atheroma plaque in aorta. Key words: Aorta, CD68, hypercholesterolemia, macrophage, myeloperoxidas

Design of Adenovirus 5 Vector with Adenovirus 26 Hexon Hypervariable Region Sequence using In Silico Approach

Adenovirus type 5 (Ad5) is one of the vaccine vectors, including the COVID-19 vaccine. Pre-existing immunity to Ad5 may suppress the immunogenicity and efficacy of adenovirus vectored vaccine. The neutralizing antibodies are directed specifically toward seven hypervariable regions (HVR) of hexon proteins located on the outer surface of the capsid. This study aims to design an Ad5 vector that may circumvent anti-Ad5 immunity by designing a chimera Ad5 vector with the sequence of Ad26 HVR (Ad5HVR26) using in silico approach. Substitution of the Ad5 HVR DNA sequence may affect the alternative splicing process of adenovirus mRNA, which then influence the protein product. The splice site prediction of Ad5HVR26 chimera vector was found at HVR5, 6, and 7. The codon change in the splice site was performed to decrease the possibility of incorrect splicing, while retaining the original amino acid sequence. The HVR substitution in chimera vector Ad5HVR26 may also affect the interaction of hexon in the capsid. The HVR2 and HVR4 hexon proteins individually interact with other hexon proteins and IX protein. Thus, two designs of the Ad5HVR26 chimera vector were created in this research. The first design was the Ad5 chimera vector with complete substitution of HVR hexon by Ad26 sequence, with codon modification on the splice site. The second design was Ad5HVR26 chimera vector without the HVR2 and HVR4 substitution to maintain the hexon protein interaction with the capsid proteins. Production of the designed vectors are needed to prove the reduction of vector neutralization by pre-existing immunity

The long and stumble way to find potential active compounds from plants for defeating hepatitis B and C: review

Hepatitis is a liver illness caused by virus such as hepatitis A virus, hepatitis B virus and hepatitis C virus. Hepatitis B and C are considerably more usual and induce more cirrhosis and dead worldwide than hepatitis A. Although drugs that are currently often used in the medication of hepatitis B and C, the finding of recent drug from various resources including herbal has been intensively developed. Therefore, the purpose of this review is to consider the possibility of plant’s compounds as anti-HBV and anti-HCV. From the results of a review of several articles, several plant’s compound have shown effectiveness againts HBV and HCV by in silico, in vitro and in vivo studies. In conclusion, several plant’s active compounds are possibility to be developed as anti-hepatitis B and C

Genetic Polymorphisms Associated with Cyclophosphamide Outcome and Risk of Toxicity in Patients with Lupus Nephritis

A 6-month cyclophosphamide induction therapy followed by maintenance therapy every three months is the first-line treatment for Class III, IV, and V lupus nephritis. Among the 139 single nucleotide polymorphisms (SNPs) associated with cyclophosphamide, four SNPs, namely rs4244285, rs4802101, rs7254579 and rs3957356, are related to the response and risk of toxicity in patients with lupus nephritis. Although pharmacogenetic studies in patients with lupus nephritis (LN) have not been conducted previously in Indonesia, data on rs4244285 are available for several ethnic groups, including Papuans, Bataks, Balinese, Dayaks, Javanese, Bugis, Chinese, Timorese and Malays, even though direct evidence in LN patients is less detectable. However, this can be followed up prior to cyclophosphamide therapy based on the identification of genetic markers. Therefore, clinical studies in patients with lupus nephritis are deemed necessary to evaluate the potential of these markers

Molecular Docking, Dynamics Simulation, and Scanning Electron Microscopy (SEM) Examination of Clinically Isolated <i>Mycobacterium tuberculosis</i> by Ursolic Acid: A Pentacyclic Triterpenes

The purpose of this study was to analyze the inhibitory action of ursolic acid (UA) as an antitubercular agent by computational docking studies and molecular dynamics simulations. The effect of UA on the cell wall of Mycobacterium tuberculosis (MTB) was evaluated by using Scanning Electron Microscopy (SEM). UA was used as a ligand for molecular interaction and investigate its binding activities to a group of proteins involved in the growth of MTB and the biosynthesis of the cell wall. Computational docking analysis was performed by using autodock 4.2.6 based on scoring functions. UA binding was confirmed by 30 ns molecular dynamics simulation using gromacs 5.1.1. H37Rv sensitive strain and isoniazid-resistant strain were used in the SEM study. UA showed to have the optimum binding affinity to inhA (Two-trans-enoyl-ACP reductase enzyme involved in elongation of fatty acid) with the binding energy of -9.2 kcal/mol. The dynamic simulation showed that the UA-inhA complex relatively stable and found to establish hydrogen bond with Thr196 and Ile194. SEM analysis confirms that UA treatment in both sensitive strain and resistant strain affected the morphology cell wall of MTB. This result indicated that UA could be one of the potential ligands for the development of new antituberculosis drugs