Immunogenicity and safety of SARS-CoV-2 recombinant protein subunit vaccine (IndoVac) adjuvanted with alum and CpG 1018 in Indonesian adults: A phase 3, randomized, active-controlled, multicenter trial.

BACKGROUND: Bio Farma has developed a recombinant protein subunit vaccine (IndoVac) that is indicated for active immunization in population of all ages. This article reported the results of the phase 3 immunogenicity and safety study in Indonesian adults aged 18 years and above. METHODS: We conducted a randomized, active-controlled, multicenter, prospective intervention study to evaluate the immunogenicity and safety of IndoVac in adults aged 18 years and above. Participants who were SARS-CoV-2 vaccine-naïve received two doses of either IndoVac or control (Covovax) with 28 days interval between doses and were followed up until 12 months after complete vaccination. RESULTS: A total of 4050 participants were enrolled from June to August 2022 and received at least one dose of vaccine. The geometric mean ratio (GMR) of neutralizing antibody at 14 days after the second dose was 1.01 (95 % confidence interval (CI) 0.89-1.16), which met the WHO non-inferiority criteria for immunobridging (95 % CI lower bound > 0.67). The antibody levels were maintained through 12 months after the second dose. The incidence rate of adverse events (AEs) were 27.95 % in IndoVac group and 32.15 % in Covovax group with mostly mild intensity (27.70 %). The most reported solicited AEs were pain (14.69 %) followed by myalgia (7.48 %) and fatigue (6.77 %). Unsolicited AEs varied, with each of the incidence rate under 5 %. There were no serious AEs assessed as possibly, probably, or likely related to vaccine. CONCLUSIONS: IndoVac in adults showed favourable safety profile and elicited non-inferior immune response to Covovax. (ClinicalTrials.gov: NCT05433285, Indonesian Clinical Research Registry: INA-R5752S9).

Characteristics, attitudes, and the odds for positive attitude toward clinical trial: A study on Indonesian COVID-19 vaccine trial participants.

Aims: This study was performed to understand the Indonesian population's characteristics and the factors that contribute to a more positive attitude toward participation in a clinical trial. Methods: A cross-sectional survey was conducted involving 402 COVID-19 vaccine trial participants in Semarang, Indonesia, utilizing self-reporting questionnaires consisting of questions related to socio-demographic characteristics and statements in a 5-scaled Likert Scale to assess the attitude toward vaccine trial. The odds for positive attitude were analyzed using Ordinal Logistic Regression to obtain the odd-ratio and 95% confidence interval. The P < 0.05 was considered statistically significant. Results: Most of the respondents were adults aged 22-64-year-old (89.30%), males (63.68%), married (77.61%), worked as an employee (59.70%), obtained information about the clinical trial from the Public Health Service (41.29%), had a low education level (40.80%), a low monthly income level (68.41%), with no previous participation in a clinical trial (90.80%). All respondents showed a good attitude toward the trial, with low education level, nonemployment status, fewer or no previous participation in clinical trials, and getting the information from the public health centers were the main predictors for better attitude toward vaccine trials. Conclusion: There was a positive attitude toward vaccine trials in the Indonesian population. The positive attitude could be driven by having a low education level, nonemployment status, fewer or no previous participation in the clinical trial, and getting information from public health centers.

The Effects of Hepatogomax Enteral Formula on Systemic Inflammation, Caecum Short-Chain Fatty Acid Levels, and Liver Histopathology in Thioacetamide-Induced Rats.

Liver damage characterized by fibrosis and necrosis can worsen the condition of liver disease. Liver disease is associated with impaired immune response and may affect short-chain fatty acid (SCFA) gut metabolites. Hepatogomax enteral formula was developed, which contains brain-chain amino acids (BCAAs) and middle-chain triglycerides (MCTs), which could repair liver tissue damage, improve the inflammatory status, and modulate SCFA in liver damage. The study aimed to determine the effect of hepatogomax on liver tissue repair, inflammation (TNF-α and IL-6), and SCFA levels in thioacetamide (TAA)-induced rats. The induction of TAA causes liver steatosis, increasing TNF-α and IL-6, and decreasing SCFA levels. Hepatogomax at a dose of 14.6 g/200 gBW significantly reduces TNF-α and IL-6 levels and increases SCFA levels (p < 0.05). The number of steatosis between groups P2 and P3 was lower as compared to a group of negative control [K2] (p < 0.05). Hepatogomax, in a dose-dependent manner, may repair liver tissue and improve inflammatory response and SCFA levels in TAA-induced rats.

The Effect of Curcumin Nanoparticles on Paracetamol-induced Liver Injury in Male Wistar Rats.

INTRODUCTION: Curcumin is a naturally occurring compound that has antioxidant properties, acts as a hepatoprotective, and lowers lipid peroxidation. However, curcumin's low solubility and bioavailability are its primary drawbacks and prevent its use as a therapeutic agent. In this study, curcumin nanoparticles will be created using the ultrasonic-assisted extraction method, and their effectiveness against paracetamol-induced changes in ALT, AST, SOD, MDA, and TNF-α will be compared to that of pure curcumin. PURPOSE: This study aimed to determine the hepatoprotective effect of curcumin nanoparticles in paracetamol- induced rats as a model for liver injury. METHODS: Thirty-six male Wistar rats, aged 6 to 8 weeks, with a minimum weight of 120 grams, were used in an experimental laboratory investigation with a post-test-only group design. Rats in each group received 100 mg/kgBW pure curcumin, 100 mg/kgBW curcumin nanoparticles, and 50 mg/kgBW curcumin nanoparticles for 7 days before paracetamol induction. On day 8, 300 mg/kgBW of paracetamol was intraperitoneally injected to cause liver damage. One of the groups received NAC as an antidote 10 hours after paracetamol induction. Detection of ALT and AST using a Chemistry Analyzer. ELISA approach for the detection of SOD, MDA, and TNF-α. The Roenigk score was calculated by two examiners after the liver histopathology preparations were stained using the Hematoxylin-Eosin method. Post hoc analyses were performed after the One Way Annova and Kruskal Wallis tests to examine the data. RESULTS: According to PSA results, the smallest formula that formed curcumin nanoparticles (10.2 nm) was 8 g of curcumin formula mixed with a mixture of Tween 20 4.5 ml, Kolliphor EL 1.5 ml, Propylene Glycol 1.5 ml, and Capryol 90 1 ml for 21 minutes using an ultrasonic process. MDA and TNF-α levels, as well as the liver's histological Roenigk score, were significantly lower in the 100 mg/kgBB pure curcumin group (C100) when compared to the model group (model). The levels of AST, MDA, TNF-α, and the liver histopathology score were significantly lower in the 100 mg/kgBB (NC100) and 50 mg/kgBB (NC50) curcumin nanoparticle groups compared to the model group (model) and pure curcumin group (C100) (p< 0.05). CONCLUSION: Curcumin nanoparticles showed better hepatoprotective ability than pure curcumin.

The efficiency of selective pooling strategy in a COVID-19 diagnostic laboratory.

INTRODUCTION: Mass testing is essential in the surveillance strategy for fighting the COVID-19 pandemic. It allows early detection of suspected cases and subsequently early isolation to mitigate spread. However, the high cost and limited consumables and reagents hinder the mass testing strategy in developing countries such as Indonesia. The specimen pooling strategy is an option to perform mass screening with limited resources. This study aims to determine the positivity rate cut-off and to evaluate the efficiency of pooling strategy for the laboratory diagnosis of COVID-19. METHODOLOGY: Between August 4th, 2020, and November 11th, 2020, a four-sample pooling strategy testing to detect SARS-CoV-2 was carried out at the Microbiology Diagnostic Laboratory of Diponegoro National Hospital, Semarang, Indonesia. Pools with positive results were subjected to individual specimen retesting. Spearman's correlation and linear regression analysis were used to determine the best positivity rate cut-off to apply pooling strategy. RESULTS: A total of 15,216 individual specimens were pooled into 3,804 four-sample pools. Among these pools, 1,007 (26.47%) were positive. Five hundred and ten (50.64%) were 1/4 positive. A maximum positivity rate of 22% is needed to save at least 50% extraction and qRT-PCR reactions in a four-sample pooling strategy. CT values between individual specimens and pools showed a good interval agreement. CONCLUSIONS: Pooling strategy could reduce personnel workload and reagent cost, and increase laboratory capacity by up to 50% when the positivity rate is less than 22%.

The effect of glutathione as adjuvant therapy on levels of TNF-α and IL-10 in wistar rat peritonitis model.

BACKGROUND: Peritonitis is the second most common cause of severe sepsis that associated with a significant mortality rate. Due to a large gap of newer antibiotics innovation and antibiotic resistance emergence, the use of antioxidant has a possible alternative as adjuvant therapy in peritonitis management. It has been studied that glutathione as an alternative in the development of new anti-inflammatory effect. Thus, the aim of this study was to evaluate the levels of TNF-α and IL-10 after glutathione administration as adjuvant therapy in rat peritonitis model. MATERIALS AND METHODS: Male wistar rats were divided into four groups (n = 6 per group), Group 1: control group (C), Group 2: peritonitis group (P), Group 3: peritonitis + Ceftriaxone group (P + Cef), Group 4: peritonitis + Ceftriaxone + Glutathione group (P + Cef + Glu). Twenty-four hours after peritonitis induction, the blood samples were taken to evaluate TNF-α and IL-10 levels. RESULTS: There was a significantly increase of mean TNF-α level in group 2 (P) 473,86 ± 388,99 pg/ml (p value 0,00) and significantly decrease of mean TNF-α level after glutathione injection in group 4 (P + Cef + Glu) (p value 0,02). No significant changes in IL-10 levels in rats peritonitis model. CONCLUSIONS: Glutathione supplementation is significantly decrease the mean level of TNF-α in rats induced peritonitis, however there is no difference compare to antibiotic only. Moreover, there no significant changes level of IL-10 in rats induced peritonitis after glutathione injection.

Superiority of Purple Okra (Abelmoschus esculentus) to Green Okra in Insulin Resistance and Pancreatic ß Cell Improvement in Diabetic Rats.

INTRODUCTION: Antidiabetic medicinal plants are increasingly used in the treatment of diabetes as they are generally assumed to pro-duce minimal side effects. Okra is a quercetin-containing plant which can induce pancreas regeneration and has antidiabetic effect. There has been a lot of research that demonstrate that purple okra contains more quercetin than green okra. AIM: To demonstrate the advantages of purple okra over green okra on the diabetic markers improvement in diabetic rats. MATERIALS AND METHODS: Fifteen male 2-month-old Wistar rats were injected intraperitoneally with 65 mg streptozotocin and 110 mg niacinamide. Their blood glucose levels were measured three days after the injection. The induction of diabetes was deemed  successful if the glucose level of the rats got higher than 250 mg/dL, and then such rats were considered diabetic. The diabetic rats were divided into three groups: an acarbose group, a purple okra powder group, and a green okra powder group. The latter two were given, respectively, purple and green okra powder for 28 days. Blood serum was taken to examine the fasting blood glucose, insulin, HOMA-B and GLUT-4 levels. Pancreas was examined histologically for damage using hematoxylin eosin staining. RESULTS: Fasting blood glucose, insulin, HOMA-B, and GLUT-4 levels of diabetic rats that received purple okra powder (p<0.05) were better than those of the rats that received green okra powder. The least damage (p<0.05) to pancreatic beta cells was found in the purple okra powder group. CONCLUSIONS: Purple okra is superior to green okra in terms of improving the diabetic markers of rats.

Uric Acid as a Risk Factor for Chronic Kidney Disease and Cardiovascular Disease　- Japanese Guideline on the Management of Asymptomatic Hyperuricemia.

Serum uric acid (UA) is taken up by endothelial cells and reduces the level of nitric oxide (NO) by inhibiting its production and accelerating its degradation. Cytosolic and plasma xanthine oxidase (XO) generates superoxide and also decreases the NO level. Thus, hyperuricemia is associated with impaired endothelial function. Hyperuricemia is often associated with vascular diseases such as chronic kidney disease (CKD) and cardiovascular disease (CVD). It has long been debated whether hyperuricemia is causally related to the development of these diseases. The 2020 American College of Rheumatology Guideline for the Management of Gout (ACR2020) does not recommend pharmacological treatment of hyperuricemia in patients with CKD/CVD. In contrast, the Japanese Guideline on Management of Hyperuricemia and Gout (JGMHG), 3rdedition, recommends pharmacological treatment of hyperuricemia in patients with CKD. In a FREED study on Japanese hyperuricemic patients with CVD, an XO inhibitor, febuxostat, improved the primary composite endpoint of cerebro-cardio-renovascular events, providing a rationale for the use of urate-lowering agents (ULAs). Since a CARES study on American gout patients with CVD treated with febuxostat revealed increased mortality, ACR2020 recommends switching to different ULAs. However, there was no difference in the mortality of Japanese patients between the febuxostat-treated group and the placebo or allopurinol-treated groups in either the FEATHER or FREED studies.

M3 Muscarinic Receptor Signaling Stabilizes a Novel Mutant Human Ether-a-Go-Go-Related Gene Channel Protein via Phosphorylation of Heat Shock Factor 1 in Transfected Cells.

BACKGROUND: Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.Methods and Results:A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG. CONCLUSIONS: M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1.

BACKGROUND: The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). METHODS: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. RESULTS: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. CONCLUSIONS: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.

E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells.

Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70.

Apoptosis induced by an uromodulin mutant C112Y and its suppression by topiroxostat.

BACKGROUND: Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder caused by mutations in UMOD that encodes uromodulin. Topiroxostat, a novel non-purine analog, selectively inhibits xanthine oxidase and reduces the serum uric acid levels and the urinary albuminuria. METHODS: Genomic DNA of a patient was extracted from peripheral white blood. Exons and flanking sequences of UMOD were amplified by PCR with primers. Mutation analysis was performed by direct sequencing of the PCR products. The wild-type and mutant uromodulin were expressed in HEK293 cells and analyzed by western blotting, immunoprecipitation, immunofluorescence, and flow cytometry. RESULTS: We identified an FJHN patient who carried a novel UMOD mutation G335A (C112Y). The levels of both cytosolic and secreted C112Y protein were significantly decreased compared with the wild-type, whereas the level of ubiquitination was higher in C112Y than that in the wild type. The half-life of C112Y was shortened and it was restored by a proteasome inhibitor MG132. Immunofluorescence revealed decreased levels of C112Y in the Golgi apparatus and on the plasma membrane. Expression of C112Y induced cellular apoptosis as revealed by flow cytometry. Apoptosis induced by C112Y was suppressed by topiroxostat. CONCLUSION: C112Y causes its protein instability resulting cellular apoptosis which could be suppressed with topiroxostat.