Triple therapy with artemether-lumefantrine plus amodiaquine versus artemether-lumefantrine alone for artemisinin-resistant, uncomplicated falciparum malaria: an open-label, randomised, multicentre trial.

BACKGROUND: Late treatment failures after artemisinin-based combination therapies (ACTs) for falciparum malaria have increased in the Greater Mekong subregion in southeast Asia. Addition of amodiaquine to artemether-lumefantrine could provide an efficacious treatment for multidrug-resistant infections. METHODS: We conducted an open-label, randomised trial at five hospitals or health centres in three locations (western Cambodia, eastern Cambodia, and Vietnam). Eligible participants were male and female patients aged 2-65 years with uncomplicated Plasmodium falciparum malaria. Patients were randomly allocated (1:1 in blocks of eight to 12) to either artemether-lumefantrine alone (dosed according to WHO guidelines) or artemether-lumefantrine plus amodiaquine (10 mg base per kg/day), both given orally as six doses over 3 days. All received a single dose of primaquine (0·25 mg/kg) 24 h after the start of study treatment to limit transmission of the parasite. Parasites were genotyped, identifying artemisinin resistance. The primary outcome was Kaplan-Meier 42-day PCR-corrected efficacy against recrudescence of the original parasite, assessed by intent-to-treat. Safety was a secondary outcome. This completed trial is registered at ClinicalTrials.gov (NCT03355664). FINDINGS: Between March 18, 2018, and Jan 30, 2020, 310 patients received randomly allocated treatment; 154 received artemether-lumefantrine alone and 156 received artemether-lumefantrine plus amodiaquine. Parasites from 305 of these patients were genotyped. 42-day PCR-corrected treatment efficacy was noted in 151 (97%, 95% CI 92-99) of 156 patients with artemether-lumefantrine plus amodiaquine versus 146 (95%, 89-97) of 154 patients with artemether-lumefantrine alone; hazard ratio (HR) for recrudescence 0·6 (95% CI 0·2-1·9, p=0·38). Of the 13 recrudescences, 12 were in 174 (57%) of 305 infections with pfkelch13 mutations indicating artemisinin resistance, for which 42-day efficacy was noted in 89 (96%) of 93 infections with artemether-lumefantrine plus amodiaquine versus 73 (90%) of 81 infections with artemether-lumefantrine alone; HR for recrudescence 0·44 (95% CI 0·14-1·40, p=0·17). Artemether-lumefantrine plus amodiaquine was generally well tolerated, but the number of mild (grade 1-2) adverse events, mainly gastrointestinal, was greater in this group compared with artemether-lumefantrine alone (vomiting, 12 [8%] with artemether-lumefantrine plus amodiaquine vs three [2%] with artemether-lumefantrine alone, p=0·03; and nausea, 11 [7%] with artemether-lumefantrine plus amodiaquine vs three [2%] with artemether-lumefantrine alone, p=0·05). Early vomiting within 1 h of treatment, requiring retreatment, occurred in no patients of 154 with artemether-lumefantrine alone versus five (3%) of 156 with artemether-lumefantrine plus amodiaquine, p=0·06. Bradycardia (≤54 beats/min) of any grade was noted in 59 (38%) of 154 patients with artemether-lumefantrine alone and 95 (61%) of 156 with artemether-lumefantrine plus amodiaquine, p=0·0001. INTERPRETATION: Artemether-lumefantrine plus amodiaquine provides an alternative to artemether-lumefantrine alone as first-line treatment for multidrug-resistant P falciparum malaria in the Greater Mekong subregion, and could prolong the therapeutic lifetime of artemether-lumefantrine in malaria-endemic populations. FUNDING: Bill & Melinda Gates Foundation, Wellcome Trust.

Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells.

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.

Evaluation of in vitro cytotoxicity and in vivo potential toxicity of the extract from in vitro cultivated Anoectochilus roxburghii Lindl.

Anoectochilus roxburghii Lind. (A. roxburghii) has promising anti-oxidant, hyperglycemic, hepatoprotective, and immunomodulatory activities as well as anti-tumor effects. However, the pharmacological actions of in vitro cultured plants remain to be determined. Therefore, the objective of the study was to assess in vitro cytotoxicity and in vivo potential toxicity of an extract derived from in vitro cultivated A. roxburghii, termed as iARE. The total flavonoid content and predominant flavonoid compounds of extract were identified and quantitatively analyzed. The in vitro cytotoxicity of iARE was examined using several cancer and normal cell lines. The apoptotic activity and expression of apoptosis-associated genes were also examined in MCF7 cells to determine the underlying mechanisms related to anti-proliferative effects. In vivo potential toxicity of iARE was assessed following acute and subchronic oral administration in Sprague Dawley rats. Quercetin, kaempferol, and isorhamnetin were three flavonoid components identified in iARE. The extract exerted cytotoxic effects on various cancer cells but not normal fibroblasts. Apoptosis in MCF7 cells was induced by iARE in a concentration-dependent manner associated with increased Bax/Bcl-2 ratio and reduced mitochondrial membrane potential ΔΨm, leading to release of cytochrome c, activation of caspase-3/7 and caspase-9, and cleavage of PARP. In the acute oral toxicity study, no mortality or toxicological signs were observed in rats at 1000 or 5000 mg/kg. In a subchronic oral toxicity study, iARE at a dosage of up to 1000 mg/kg produced no mortality or treatment-related adverse effects on general behavior, food intake, body weight, relative organ weights. No apparent marked changes in the histopathology of the liver and kidney were detected. Data demonstrated that iARE induced in vitro cytotoxic effects in cancer cells are associated with lackof invivo toxicity. Thus, iARE was suggested to be considered as apotential therapeutic candidate for cancer treatment.

Tetrahydrobiopterin enhances mitochondrial biogenesis and cardiac contractility via stimulation of PGC1α signaling.

Tetrahydrobiopterin (BH4) shows therapeutic potential as an endogenous target in cardiovascular diseases. Although it is involved in cardiovascular metabolism and mitochondrial biology, its mechanisms of action are unclear. We investigated how BH4 regulates cardiovascular metabolism using an unbiased multiple proteomics approach with a sepiapterin reductase knock-out (Spr-/-) mouse as a model of BH4 deficiency. Spr-/- mice exhibited a shortened life span, cardiac contractile dysfunction, and morphological changes. Multiple proteomics and systems-based data-integrative analyses showed that BH4 deficiency altered cardiac mitochondrial oxidative phosphorylation. Along with decreased transcription of major mitochondrial biogenesis regulatory genes, including Ppargc1a, Ppara, Esrra, and Tfam, Spr-/- mice exhibited lower mitochondrial mass and severe oxidative phosphorylation defects. Exogenous BH4 supplementation, but not nitric oxide supplementation or inhibition, rescued these cardiac and mitochondrial defects. BH4 supplementation also recovered mRNA and protein levels of PGC1α and its target proteins involved in mitochondrial biogenesis (mtTFA and ERRα), antioxidation (Prx3 and SOD2), and fatty acid utilization (CD36 and CPTI-M) in Spr-/- hearts. These results indicate that BH4-activated transcription of PGC1α regulates cardiac energy metabolism independently of nitric oxide and suggests that BH4 has therapeutic potential for cardiovascular diseases involving mitochondrial dysfunction.

Morphological changes during replicative senescence in bovine ovarian granulosa cells.

The objective of this study was to evaluate replicative senescence of bovine granulosa cells (bGCs) during in vitro long-term culture. WST-1 assay analysis showed that bGCs proliferation was reduced from primary culture to 14th passage. The several bGCs from the 3rd passage and 7th passage exposed the weak activity of beta-galactosidase, while a strongly positive staining of beta-galactosidase was observed in bGCs from 14th passage. Flow cytometry analysis showed that bGCs were induced to cell cycle arrest at G0/G1 phase through in vitro expansion. TERT transcript expression of bGCs was downregulated from primary culture to 14th passage. The cell and nuclear area of bGCs were dramatically increased from 14th passage to 25th passage. The nucleocytoplasmic ratio of bGCs was dramatically reduced in 22th passage (4.32%) and 25th passage (2.45%), comparing to previous passages: primary culture (10.67%), 7th passage (9.21%), or 14th passage (10.33%). The number of microfilament bundle of bGCs was increased in 22nd passage (67.42 ± 17.76) and 25th passage (56.31 ± 22.45). The diameter of microfilament bundle of bGCs in 25th passage was dramatically increased to 1.88 ± 0.32 µm comparing to the primary culture (1.15 ± 0.03 µm). In this study, we also assessed the nuclear form factor which illustrates the level of nuclear circular form. A reduction of nuclear form factor was observed in bGCs during long-term in vitro expansion. The changes of nuclear form factor were correlated to other senescent characteristics, especially the nucleocytoplasmic ratio.

NecroX-5 exerts anti-inflammatory and anti-fibrotic effects via modulation of the TNFα/Dcn/TGFß1/Smad2 pathway in hypoxia/reoxygenation-treated rat hearts.

Inflammatory and fibrotic responses are accelerated during the reperfusion period, and excessive fibrosis and inflammation contribute to cardiac malfunction. NecroX compounds have been shown to protect the liver and heart from ischemia-reperfusion injury. The aim of this study was to further define the role and mechanism of action of NecroX-5 in regulating infl ammation and fi brosis responses in a model of hypoxia/reoxygenation (HR). We utilized HR-treated rat hearts and lipopolysaccharide (LPS)-treated H9C2 culture cells in the presence or absence of NecroX-5 (10 µmol/L) treatment as experimental models. Addition of NecroX-5 signifi cantly increased decorin (Dcn) expression levels in HR-treated hearts. In contrast, expression of transforming growth factor beta 1 (TGFß1) and Smad2 phosphorylation (pSmad2) was strongly attenuated in NecroX-5-treated hearts. In addition, signifi cantly increased production of tumor necrosis factor alpha (TNFα), TGFß1, and pSmad2, and markedly decreased Dcn expression levels, were observed in LPS-stimulated H9C2 cells. Interestingly, NecroX-5 supplementation effectively attenuated the increased expression levels of TNFα, TGFß1, and pSmad2, as well as the decreased expression of Dcn. Thus, our data demonstrate potential antiinflammatory and anti-fibrotic effects of NecroX-5 against cardiac HR injuries via modulation of the TNFα/Dcn/TGFß1/Smad2 pathway.

NecroX-5 protects mitochondrial oxidative phosphorylation capacity and preserves PGC1α expression levels during hypoxia/reoxygenation injury.

Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment (10 µM) and non-treatment were employed on isolated rat hearts during hypoxia/reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC1α) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving PGC1α during cardiac HR injuries.

Effects of the novel angiotensin II receptor type I antagonist, fimasartan on myocardial ischemia/reperfusion injury.

BACKGROUND: The aim of this study was to investigate the cardioprotective effect of fimasartan, a newly developed angiotensin II receptor type I blocker (ARB), against myocardial ischemia/reperfusion (I/R) injury and to identify the mechanism by which it reduces mitochondrial damage. METHODS: Fimasartan was administered intravenously to Sprague-Dawley rats (3mg/kg), cardiomyocytes (50 µM), and H9c2 cells (50 µM) before ischemia or hypoxia. Myocardial infarction (MI), echocardiograms, DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling, immunoblotting, oxygen consumption, confocal microscopic appearance, and L-type Ca(2+) current (ICa,L) were then assessed. RESULTS: Fimasartan pretreatment remarkably reduced the rate of MI and improved cardiac performance well after I/R (n = 9/group). Fimasartan also reduced apoptotic cell death both in vivo and in hypoxia/reoxygenation (H/R)-treated H9c2 cells (n = 5~8/group). H/R-induced mitochondrial O2(-) production and collapse of membrane potential were markedly attenuated in fimasartan-treated cardiomyocytes (n = 4 ~ 6/group). Additionally, mitochondrial Ca(2+) overload during reoxygenation was suppressed by fimasartan (n = 4~6/group), and this was found to be possibly related to the inhibition of ICa,L and mitochondrial Ca(2+) uniporter. Furthermore, fimasartan pretreatment increased phosphorylations of Akt and glycogen synthase kinase-3ß (n = 5 ~ 7/group), decreased pro-apoptotic p53 levels, and increased anti-apoptotic Bcl-2 levels (n = 4) during reperfusion. CONCLUSIONS: Fimasartan preconditioning has the potential to modulate Bcl-2 and suppress I/R-induced Ca(2+) overload by inhibiting ICa,L and MCU. These beneficial effects could prevent the mitochondrial dysfunction and apoptosis accompanied by I/R.

The angiotensin receptor blocker and PPAR-γ agonist, telmisartan, delays inactivation of voltage-gated sodium channel in rat heart: novel mechanism of drug action.

Telmisartan is an angiotensin II receptor blocker and partial peroxisome proliferator-activated receptor gamma agonist that modulates the renin-angiotensin-aldosterone system. It is used primarily to manage hypertension, diabetic nephropathy, and congestive heart failure. Recent studies have reported that myocardial infarction (MI) has occurred in telmisartan-treated patients. The purpose of the study was to investigate the specific conditions and underlying mechanisms that may result in telmisartan-induced MI. We evaluated the effect of telmisartan on whole hearts, cardiomyocytes, and cardiac sarcolemmal ion channels. Hearts of 8-week-old male Sprague-Dawley rats were perfused with 3, 10, 30, or 100 µM telmisartan or losartan or with normal Tyrode's solution (control) for 3 h. We found that telmisartan induced myocardial infarction, with an infarct size of 21 % of the total at 30 µM (P < 0.0001) and 63 % of the total area at 100 µM (P < 0.001). Telmisartan also induced cardiac dysfunction (e.g., decreased heart rate, diminished coronary flow, hypercontracture, and arrhythmia). Confocal microscopy demonstrated that 30 µM telmisartan significantly elevated the intracellular Ca(2+) level, leading to hypercontracture and cell death. Patch clamp analysis of isolated cardiomyocytes revealed that telmisartan induced Na(+) overload by slowing the inactivation of voltage-gated Na(+) current (I (Na)), activating the reverse mode of Na(+)-Ca(2+) exchanger activity, and causing Ca(2+) overload. Telmisartan significantly delayed the inactivation of the voltage-gated Na(+) channel, causing cytosolic Na(+) overload, prolonged action potential duration, and subsequent Ca(2+) overload. Above 30 µM, telmisartan may potentially cause cardiac cell death and MI.

In vivo susceptibility of Plasmodium falciparum to artesunate in Binh Phuoc Province, Vietnam.

BACKGROUND: By 2009, there were worrying signs from western Cambodia that parasitological responses to artesunate-containing treatment regimens for uncomplicated Plasmodium falciparum malaria were slower than elsewhere which suggested the emergence of artemisinin resistance. Vietnam shares a long land border with Cambodia with a large number of migrants crossing it on a daily basis. Therefore, there is an urgent need to investigate whether there is any evidence of a change in the parasitological response to the artemisinin derivatives in Vietnam. METHODS: From August 2010 to May 2011, a randomized controlled clinical trial in uncomplicated falciparum malaria was conducted to compare two doses of artesunate (AS) (2mg/kg/day versus 4 mg/kg/day for three days) followed by dihydroartemisinin-piperaquine (DHA-PPQ) and a control arm of DHA-PPQ. The goal was characterization of the current efficacy of artesunate in southern Vietnam. The primary endpoint of this study was the parasite clearance half-life; secondary endpoints included the parasite reduction ratios at 24 and 48 hours and the parasite clearance time. RESULTS: 166 patients were recruited into the study. The median parasite clearance half-lives were 3.54 (AS 2mg/kg), 2.72 (AS 4mg/kg), and 2.98 hours (DHA-PPQ) (p=0.19). The median parasite-reduction ratio at 24 hours was 48 in the AS 2mg/kg group compared with 212 and 113 in the other two groups, respectively (p=0.02). The proportions of patients with a parasite clearance time of >72 hours for AS 2mg/kg, AS 4mg/kg and DHA-PPQ were 27%, 27%, and 22%, respectively. Early treatment failure occurred in two (4%) and late clinical failure occurred in one (2%) of the 55 patients in the AS 2mg/kg group, as compared with none in the other two study arms. The PCR-corrected adequate clinical and parasitological response (APCR) rates in the three groups were 94%, 100%, and 100% (p=0.04). CONCLUSIONS: This study demonstrated faster P. falciparum parasite clearance in southern Vietnam than in western Cambodia but slower clearance in comparison with historical data from Vietnam. Further studies to determine whether this represents the emergence of artemisinin resistance in this area are needed. Currently, the therapeutic response to DHA-PPQ remains satisfactory in southern Vietnam. TRIAL REGISTRATION: NTC01165372.

NecroX-5 prevents hypoxia/reoxygenation injury by inhibiting the mitochondrial calcium uniporter.

AIMS: Preservation of mitochondrial function is essential to limit myocardial damage in ischaemic heart disease. We examined the protective effects and mechanism of a new compound, NecroX-5, on rat heart mitochondria in a hypoxia/reoxygenation (HR) model. METHODS AND RESULTS: NecroX-5 reduced mitochondrial oxidative stress, prevented the collapse in mitochondrial membrane potential, improved mitochondrial oxygen consumption, and suppressed mitochondrial Ca(2+) overload during reoxygenation in an in vitro rat heart HR model. Furthermore, NecroX-5 reduced the ouabain- or histamine-induced increase in mitochondrial Ca(2+). CONCLUSION: These findings suggest that NecroX-5 may act as a mitochondrial Ca(2+) uniporter inhibitor to protect cardiac mitochondria against HR damage.