Effectiveness and safety of normoxic allogenic umbilical cord mesenchymal stem cells administered as adjunctive treatment in patients with severe COVID-19.

Inflammatory response in COVID-19 contributes greatly to disease severity. Mesenchymal Stem Cells (MSCs) have the potential to alleviate inflammation and reduce mortality and length of stay in COVID-19 patients. We investigated the safety and effectiveness of normoxic-allogenic umbilical cord (NA-UC)-MSCs as an adjunctive treatment in severe COVID-19 patients. A double-blind, multicentric, randomized, placebo-controlled trial involving severe COVID-19 patients was performed from January to June 2021 in three major hospitals across Java, Indonesia. Eligible participants (n = 42) were randomly assigned to two groups (1:1), namely the intervention (n = 21) and control (n = 21) groups. UC-MSCs dose was 1 × 106 /kg body weight on day D0, D3, and D6. The primary outcome was the duration of hospitalization. Meanwhile, the secondary outcomes were radiographical progression (Brixia score), respiratory and oxygenation parameters, and inflammatory markers, in addition to the safety profile of NA-UC-MSCs. NA-UC-MSCs administration did not affect the length of hospital stay of severe COVID-19 patients, nor did it improve the Brixia score or mMRC dyspnoea scale better than placebo. Nevertheless, NA-UC-MSCs led to a better recuperation in oxygenation index (120.80 ± 72.70 baseline vs. 309.63 ± 319.30 D + 22, p = 0.038) and oxygen saturation (97.24 ± 4.10% vs. 96.19 ± 3.75% in placebo, p = 0.028). Additionally, compared to the placebo group, the treatment group had a significantly smaller increase in PCT level at D + 22 (1.43 vs. 12.76, p = 0.011). No adverse effects, including serious ones, were recorded until D + 91. NA-UC-MSCs therapy is a very safe adjunct for COVID-19 patients. It improves the oxygenation profile and carries potential to suppress inflammation.

Difference between Microscopic and PCR Examination Result for Malaria Diagnosis and Treatment Evaluation in Sumba Barat Daya, Indonesia.

Microscopic examination is the backbone of malaria diagnosis and treatment evaluation in Indonesia. This test has limited ability to detect malaria at low parasite density. Inversely, nested polymerase chain reaction (PCR) can detect parasites at a density below the microscopic examination's detection limit. The objective of this study is to compare microscopic and PCR results when being used to identify malaria in suspected patients and patients who underwent dihydroartemisinin-piperaquine (DHP) therapy in the last 3-8 weeks with or without symptoms in Sumba Barat Daya, Nusa Tenggara Timur, Indonesia. Recruitment was conducted between April 2019 and February 2020. Blood samples were then taken for microscopic and PCR examinations. Participants (n = 409) were divided into three groups: suspected malaria (42.5%), post-DHP therapy with fever (4.9%), and post-DHP therapy without fever (52.6%). Microscopic examination found five cases of P. falciparum + P. vivax infection, while PCR found 346 cases. All microscopic examinations turned negative in the post-DHP-therapy group. Conversely, PCR result from the same group yielded 29 negative results. Overall, our study showed that microscopic examination and PCR generated different results in detecting Plasmodium species, especially in patients with mixed infection and in patients who recently underwent DHP therapy.

Prevalence and distribution of metabolic syndrome and its components among provinces and ethnic groups in Indonesia.

BACKGROUND: Global increase of metabolic syndrome (MetS) may have affected Indonesia, however, lack of data in this multiethnic group country warrants a nationwide study for MetS and its components. This study aims to determine the prevalence of metabolic syndrome and its components among Indonesian people based on the province and ethnic groups. METHODS: We obtained 8573 subjects from the Indonesian Family Life Survey Wave 4 (IFLS4), spread over 20 provinces in Indonesia and consisting of 27 ethnic groups. MetS was operationalized according to an adapted Harmonized MetS definition. Prevalence ratios with 95% confidence interval were estimated using log-binomial regression. RESULTS: The prevalence of MetS in Indonesia is 21.66% with provincial prevalence ranging from 0 to 50%, while the ethnic prevalence ranging from 0 to 45.45%. Significant higher MetS prevalence ratios were found in Jakarta (PR 1.826; 95CI 1.628-2.048), West Nusa Tenggara (PR 1.412; 95CI: 1.222-1.630), West Sumatra (PR 1.404; 95CI: 1.202-1.641), East Java province (PR 1.109; 95CI: 1.001-1.229) and in Sasak (PR 1.532; 95CI:1.304-1.800), Minangkabau (PR 1.469; 95CI:1.251-1.726), Betawi (PR 1.597; 95CI:1.346-1.895), Acehnese ethnic group (PR 2.101; 95CI:1.099-4.020) while significant lower prevalence ratios were observed in Central Java (PR 0.668; 95CI: 0.580-0.770), Yogyakarta (PR 0.695; 95CI: 0.575-0.840), Banten (PR 0.718; 95CI: 0.533-0.968), Bali province (PR 0.724; 95CI: 0.590-0.889) and in Javanese (PR 0.855; 95CI:0.788-0.928), also Balinese ethnic groups (PR 0.669; 95CI:0.535-0.836). The highest prevalence of MetS components among Indonesians was low HDL cholesterol (66.41%), followed by hypertension (64.45%), and central obesity (43.21%). CONCLUSIONS: The prevalence of MetS in Indonesia is moderate with provincial and ethnic prevalence varied. Provincial and ethnic group differences in MetS prevalence ratios were observed. The top two most prevalent MetS components in Indonesian were low HDL cholesterol and hypertension.

HPLC-based Measurement of Glycated Hemoglobin using Dried Blood Spots Collected under Adverse Field Conditions.

Glycated hemoglobin (HbA1c) measured using high-performance liquid chromatography (HPLC) assays with venous blood and dried blood spots (DBS) are compared for 143 paired samples collected in Aceh, Indonesia. Relative to gold-standard venous-blood values, DBS-based values reported by the HPLC are systematically upward biased for HbA1c<8% and the fraction diabetic (HbA1c ≥ 6.5%) is overstated almost five-fold. Inspection of chromatograms from DBS assays indicates the % glycosylated calculated by the HPLC excludes part of the hemoglobin A which is misidentified as a hemoglobin variant. Taking this into account, unbiased DBS-based values are computed using data from the machine-generated chromatograms. When the DBS are collected in a clinic-like setting, under controlled humidity/temperature conditions, the recalculated values are almost identical to venous-based values. When DBS are collected under field conditions, the recalculated values are unbiased, but only about half the HbA1c values are measured reliably, calling into question the validity of the other half. The results suggest that collection conditions, particularly humidity, affect the quality of the DBS-based measures. Cross-validating DBS-based HbA1c values with venous samples collected under exactly the same environmental conditions is a prudent investment in population-based studies.

Reactive oxygen species play an essential role in IGF-I signaling and IGF-I-induced myocyte hypertrophy in C2C12 myocytes.

IGF-I induces skeletal muscle hypertrophy by stimulating protein synthesis and suppressing the protein degradation pathway; the downstream signaling pathways Akt-mammalian target of rapamycin (mTOR)-p70-kDA-S6-kinase (p70S6K), and Forkhead box O1 (FoxO1) play essential roles in this regulation. Reactive oxygen species (ROS) modulate the signaling of various growth factors via redox regulation. However, the role of ROS in IGF-I signaling is not fully understood. In this study, we investigated whether ROS regulate the signaling and biological action of IGF-I in C2C12 myocytes. We found that IGF-I induces ROS in C2C12 myocytes. While treatment with H(2)O(2) significantly enhanced IGF-I-induced phosphorylation of the IGF-I receptor (IGF-IR), IGF-IR phosphorylation was markedly attenuated when cells were treated with antioxidants. The downstream signaling pathway, Akt-mTOR-p70S6K was subsequently down-regulated. Furthermore, the phosphorylation of FoxO1 by IGF-I decreased concomitantly with the restoration of the expression of its target genes, Atrogin-1 and muscle RING finger 1, which are related to muscle atrophy. Nox4 knockdown, which is reportedly to produce ROS in insulin signaling, attenuated IGF-I-induced IGF-IR phosphorylation, indicating that Nox4 is involved in the regulation of IGF-I signaling. Importantly, antioxidant treatments inhibited IGF-I-induced myocyte hypertrophy, demonstrating that ROS are necessary for IGF-I-induced myocyte hypertrophy in vitro. These results indicate that ROS play an essential role in the signaling and biological action of IGF-I in C2C12 myocytes.

W194XProp1 and S156insTProp1, both of which have intact DNA-binding domain, show a different DNA-binding activity to the Prop1-binding element in human Pit-1 gene.

Prop1 activates POU1F1 (Pit-1) gene expression, which in turn stimulates GH, PRL, TSHbeta and GHRH receptor gene expressions. Therefore the patients with Prop1 mutation show GH, PRL, and TSH deficiency. The mutation of Prop1 is a major abnormality causing combined pituitary hormone deficiency (CPHD). However, DNA-binding and activating functions of mutant Prop1 have not been examined fully because Prop1-binding elements (PBEs) in human POU1F1 gene were not identified until 2008. The aim of this study is to test DNA-binding and transcriptional activities of two mutant Prop1s (W194XProp1 and S156insTProp1, both of them were found in the patients with CPHD) whose mutation is located in putative transactivating domain but not in DNA-binding domain. W194XProp1 showed a marked DNA-binding to PBE as well as a consensus element of paired-like transcription factors (PRDQ9). Activating function for POU1F1 reporter genes expression was lost or decreased in W194XProp1 but still preserved for PRDQ9 reporter gene. S156insTProp1 did not bind PBE but bound PRDQ9. Consistent with the result, S156insTProp1 did not stimulate POU1F1 reporter gene but stimulated PRDQ9 reporter gene. These results support the inference that W194XProp1 is unable to increase POU1F1 gene expression by the defect of transactivating domain and that S156insTProp1 is unable to increase due to the loss of DNA-binding activity. DNA-binding domain that has been assumed is not sufficient to provide full DNA-binding activity of Prop1 and transactivating domain of Prop1 is likely to affect DNA binding to PBE.

Branched-chain amino acids protect against dexamethasone-induced soleus muscle atrophy in rats.

We investigated the utility of branched-chain amino acids (BCAA) in dexamethasone-induced muscle atrophy. Dexamethasone (600 microg/kg, intraperitoneally) and/or BCAA (600 mg/kg, orally) were administered for 5 days in rats, and the effect of BCAA on dexamethasone-induced muscle atrophy was evaluated. Dexamethasone decreased total protein concentration of rat soleus muscles. Concomitant administration of BCAA reversed the decrease. Dexamethasone decreased mean cross-sectional area of soleus muscle fibers, which was reversed by BCAA. Dexamethasone increased atrogin-1 expression, which has been reported to play a pivotal role in muscle atrophy. The increased expression of atrogin-1 mRNA was significantly attenuated by BCAA. Furthermore, dexamethasone-induced conversion from microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II, which is an indicator of autophagy, was blocked by BCAA. These findings suggest that BCAA decreased protein breakdown to prevent muscle atrophy. BCAA administration appears to be useful for prevention of steroid myopathy.

Branched-chain amino acids and arginine suppress MaFbx/atrogin-1 mRNA expression via mTOR pathway in C2C12 cell line.

The effect of amino acid on muscle protein degradation remains unclear. Recent studies have elucidated that proteolysis in catabolic conditions occurs through ubiquitin-proteasome proteolysis pathway and that muscle-specific ubiquitin ligases (atrogin-1 and MuRF1) play an important role in protein degradation. In the present study, we examined the direct effect of 5 mM amino acids (leucine, isoleucine, valine, glutamine and arginine) on atrogin-1 and MuRF1 levels in C2C12 muscle cells and the involved intracellular signal transduction pathway. Leucine, isoleucine and valine suppressed atrogin-1 and MuRF1 mRNA levels (approximately equal to 50%) at 6 and 24 h stimulations. Arginine showed a similar effect except at 24 h-treatment for atrogin-1 mRNA. However, glutamine failed to reduce atrogin-1 and MuRF1 mRNA levels. The inhibitory effect of leucine, isoleucine or arginine on atrogin-1 mRNA level was reversed by rapamycin, although wortmannin did not reverse the effect. PD98059 and HA89 reduced basal atrogin-1 level without influencing the inhibitory effects of those amino acids. The inhibitory effect of leucine, isoleucine or arginine on MuRF1 mRNA levels was not reversed by rapamycin. Taken together, these findings indicated that leucine, isoleucine and arginine decreased atrogin-1 mRNA levels via mTOR and that different pathways were involved in the effect of those amino acids on MuRF1 mRNA levels.

GHRP-2, a GHS-R agonist, directly acts on myocytes to attenuate the dexamethasone-induced expressions of muscle-specific ubiquitin ligases, Atrogin-1 and MuRF1.

Recent reports suggest that Atrogin-1 and MuRF1, E3 ubiquitin ligases, play a pivotal role in muscle atrophy. In the present study, effect of Growth Hormone Releasing Peptide-2 (GHRP-2), a GH secretagogue receptor (GHS-R) agonist, on the expressions of Atrogin-1 and MuRF1 in vivo rat muscles was examined. Dexamethasone administration increased Atrogin-1 mRNA level in rat soleus muscle. The increased mRNA level of Atrogin-1 was significantly attenuated by GHRP-2. In addition, GHRP-2 decreased MuRF1 mRNA level irrespective of the presence of dexamethasone. Although IGF-I is a well-known protective factor for muscle atrophy, GHRP-2 did not influence plasma IGF-I levels and IGF-I mRNA levels in muscles. To clarify a direct effect of GHRP-2, differentiated C2C12 myocytes were used. Ten micrometer dexamethasone increased both Atrogin-1 and MuRF1 mRNA levels in C2C12 cells. GHRP-2 attenuated dexamethasone-induced expression of them dose-dependently and decreased the basal level of MuRF1 mRNA. The suppressive effect on the expressions of Atrogin-1 and MuRF1 by GHRP-2 was blocked by [D-Lys(3)]-GHRP-6, a GHS-R1a blocker, suggesting the effect of GHRP-2 was mediated through GHS-R1a. Taken together, GHRP-2 directly attenuates Atrogin-1 and MuRF1 mRNA levels through ghrelin receptors in myocytes.

Involvement of mPOU (Brn-5), a class VI POU protein, in the gene expression of Pit-1 as well as PRL.

PRL is mainly expressed in the pituitary and its gene expression is regulated by a variety of transcription factors including Pit-1. Brn-5 is a transcription factor that binds to Pit-1 binding elements and stimulates PRL reporter gene expression. In this study, the role of Brn-5 was examined. RNA interference (RNAi) against Brn-5 leaded to reduction in PRL content of GH3 cells, indicating endogenous Brn-5 may play a role in PRL gene expression. Furthermore Brn-5 RNAi decreased Pit-1 mRNA. Transfection of expression vectors for mPOU (human ortholog of Brn-5) modestly but significantly stimulated activities of PRL-Luc and Pit-1-Luc reporter genes in GH3 and HEK 293 cells. In addition, mPOU showed synergistic action with Pit-1 and CBP on PRL-Luc expression. mPOU-FL, a splicing variant of mPOU, showed weaker activity than mPOU. Chip assay suggested binding of mPOU to PRL and Pit-1 promoters of genomic DNA. Taken together, these results suggest that mPOU (Brn-5) enhances PRL gene expression directly in association with Pit-1 and CBP, and indirectly via the activation of Pit-1 gene expression.

Neither intravenous nor intracerebroventricular administration of obestatin affects the secretion of GH, PRL, TSH and ACTH in rats.

To examine the effect of obestatin, a recently identified peptide derived from preproghrelin, on pituitary hormone secretion, obestatin was administered in anesthetized male rats. Intravenous administration of obestatin did not show any effect on plasma GH, PRL, ACTH and TSH levels. Since obestatin has been reported to have opposite effects of ghrelin in regulating food intake, gastric emptying and intestinal contractility, GH suppressive effect, which is opposite effect of ghrelin, was tested. Intravenous administration of GHRH or GHRP-2, a ghrelin receptor ligand, resulted in a marked plasma GH elevation. However obestatin did not show any effect on GHRH- or GHRP-2-induced GH rise. Furthermore intracerebroventricular administration of obestatin also did not influence plasma GH, PRL, ACTH and TSH levels. These findings suggest that obestatin has no effect on pituitary hormone secretions despite the presence of GPR39, a receptor for obestatin, in the pituitary.