Search

Summary Background Therapeutic synergism between radiotherapy and immune checkpoint blockade has been observed in preclinical models of hepatocellular carcinoma. We aimed to study the safety and efficacy of sequential radioembolisation with yttrium-90-resin microspheres (Y90-radioembolisation) followed by nivolumab in patients with advanced hepatocellular carcinoma. Methods Patients with Child-Pugh A cirrhosis and advanced hepatocellular carcinoma not suitable for curative surgery were treated with Y90-radioembolisation followed by intravenous nivolumab 240 mg 21 days after Y90-radioembolisation and every 2 weeks thereafter. The primary endpoint, assessed in the per-protocol population, was the objective response rate, determined by RECIST version 1.1, defined as the proportion of patients with a confirmed complete or partial response observed for lesions both within and outside the Y90-radioembolisation field. This study is registered with ClinicalTrials.gov , NCT03033446 and has been completed. Findings 40 patients were enrolled, of whom 36 received Y90-radioembolisation followed by nivolumab. One (3%) patient had a complete response and ten (28%) had a partial response; the objective response rate was 30·6% (95% CI 16·4–48·1). The most common treatment-related adverse events of any grade were pruritus (18 [50%] of 36 patients) and maculopapular rash (13 [36%]). Two (6%) patients experienced grade 3–4 treatment-related adverse events: one patient had a grade 3 increase in alanine aminotransferase levels, grade 3 bilirubin increase, and grade 4 increase in aspartate aminotransferase levels, while the other had a grade 3 maculopapular rash. Five (14%) patients had a treatment-related serious adverse event (Steven-Johnson syndrome, hepatitis E infection, fever, liver abscesses, and ascites). Interpretation Y90-radioembolisation followed by nivolumab resulted in an encouraging objective response rate in patients with advanced hepatocellular carcinoma, although the activity observed was not as high as the study was powered for. This strategy should be further evaluated in patients with Barcelona Clinic Liver Clinic (BCLC) stage B hepatocellular carcinoma that is ineligible or refractory to transarterial chemoembolisation and patients with BCLC C disease without extrahepatic spread. Funding National Medical Research Council Singapore, Bristol-Myers Squibb, Sirtex.

To describe the rationale, design and methodology of a geographically-representative and population-based study investigating the epidemiology, impact, personal and economic burden of age-related eye diseases, declining visual and other sensory systems in Asians aged >60 years in Singapore.PIONEER (The PopulatION HEalth and Eye Disease PRofilE in Elderly Singaporeans Study) is currently a cross-sectional study targeting 3152 Chinese, Malay and Indian adults who are Singapore citizens or permanent residents aged 60 years and older living across Singapore. The study is intended to be longitudinal, with several waves of data planned to be collected in the future. The sampling frame consisted of 7000 names derived from age, gender and ethnicity-stratified random sampling of individuals >60 years. Selected individuals were invited via letters, home visits, and telephone calls for a clinical assessment at the Singapore Eye Research Institute. Individuals with limited mobility were examined in a custom-designed mobile eye clinic. Questionnaires were subsequently administered at participants' homes by trained interviewers in their preferred language. A total of 3,299 participants (from East, West, North and South Singapore) were approached from December 2017 to November 2019. Of these, 953 (28.5%) were deemed ineligible. Out of 2,346 eligible participants, 904 (38.5%) refused, and 1,442 (61.5%) attended our clinical testing protocol, giving an initial response rate of 61.5%. Of these, 1,170 (81%) were cognitively able to complete the questionnaire assessment. The mean age±SD of our participants was 73.8±8.6 years; n=798 (55.3%) were female; and 828 (57.4%) were of Chinese ethnicity. The findings from this study will allow a deeper understanding of the risk factors and impact of aging in Asian populations, particularly in relation to the visual function and other functional system.

Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD), which is mediated by lung infiltration with inflammatory cells, enhanced oxidative stress, and tissue destruction. Anti-malarial drug artesunate has been shown to possess anti-inflammatory and anti-oxidative actions in mouse asthma models. We hypothesized that artesunate can protect against cigarette smoke-induced acute lung injury via its anti-inflammatory and anti-oxidative properties. Artesunate was given by oral gavage to BALB/c mice daily 2 h before 4% cigarette smoke exposure for 1 h over five consecutive days. Bronchoalveolar lavage (BAL) fluid and lungs were collected for analyses of cytokines, oxidative damage and antioxidant activities. Bronchial epithelial cell BEAS-2B was exposed to cigarette smoke extract (CSE) and used to study the mechanisms of action of artesunate. Artesunate suppressed cigarette smoke-induced increases in BAL fluid total and differential cell counts; levels of IL-1β, MCP-1, IP-10 and KC; and levels of oxidative biomarkers 8-isoprostane, 8-OHdG and 3-nitrotyrosine in a dose-dependent manner. Artesunate promoted anti-oxidant catalase activity and reduced NADPH oxidase 2 (NOX2) protein level in the lungs from cigarette smoke-exposed mice. In BEAS-2B cells, artesunate suppressed pro-inflammatory PI3 K/Akt and p44/42 MAPK signaling pathways, and increased nuclear Nrf2 accumulation in response to CSE. Artesunate possesses anti-inflammatory and anti-oxidative properties against cigarette smoke-induced lung injury, probably via inhibition of PI3K and p42/22 MAPK signaling pathways, augmentation of Nrf2 and catalase activities, and reduction of NOX2 level. Our data suggest that artesunate may have therapeutic potential for treating COPD.

Allergy is an acquired hypersensitivity reaction of the immune system mediated by cross-linking of the allergen-specific IgE-bound high-affinity IgE receptors, leading to immediate mast cell degranulation. Rottlerin is an active molecule isolated from Mallotus philippinensis, a medicinal plant used in Ayurvedic Medicine System for anti-allergic and anti-helminthic treatments. The present study investigated potential anti-allergic effects of rottlerin in animal models of IgE-dependent anaphylaxis and the anti-allergic mechanisms of action of rottlerin in mast cells. Anti-allergic actions of rottlerin were evaluated in passive cutaneous anaphylaxis and passive systemic anaphylaxis mouse models, and in anaphylactic contraction of bronchial rings isolated from sensitized guinea pigs. Direct mast cell-stabilizing effect of rottlerin was examined in RBL-2H3 mast cell line. Anti-allergic signaling mechanisms of action of rottlerin in mast cells were also examined. Rottlerin prevented IgE-mediated cutaneous vascular extravasation, hypothermia, elevation in plasma histamine level and tracheal tissue mast cell degranulation in mice in a dose-dependent manner. In addition, rottlerin suppressed ovalbumin-induced guinea pig bronchial smooth muscle contraction. Furthermore, rottlerin concentration-dependently blocked IgE-mediated immediate release of β-hexosaminidase from RBL-2H3 mast cells. Rottlerin was found to inhibit IgE-induced PLCγ1 and Akt phosphorylation, production of IP3 and rise in cytosolic Ca2+ level in mast cells. We report here for the first time that rottlerin possesses anti-allergic activity by blocking IgE-induced mast cell degranulation, providing a foundation for developing rottlerin for the treatment of allergic asthma and other mast cell-mediated allergic disorders.

Hydrogen sulfide (H2S) has complex effects in inflammation with both pro- and anti-inflammatory actions of this gas reported. Recent work suggests that a deficiency of H2S occurs in, and may contribute to, the chronic inflammation which underpins ongoing atherosclerotic disease. However, whether a high fat diet, predisposing to atherosclerosis, affects H2S metabolism is not known. In this study we assessed H2S metabolism in different tissues of mice fed a high fat diet for up to 16 weeks. Ex vivo biosynthesis of H2S was reduced in liver, kidney and lung of high fat fed mice. Western blotting revealed deficiency of cystathionine γ lyase (CSE) in liver and lung with increased expression of cystathionine β synthetase (CBS) in liver and kidney. Expression of 3-mercaptopyruvate sulfurtransferase (3-MST) was reduced in liver but not other tissues. Aortic endothelial cell CSE was also reduced in high fat fed animals as determined immunohistochemically. Plasma H2S concentration was not changed in these animals. No evidence of lipid deposition was apparent in aortae from high fat fed animals and plasma serum amyloid A (SAA) and C-reactive protein (CRP) were also unchanged suggesting lack of frank atherosclerotic disease. Plasma IL-6, IL12p40 and G-CSF levels were increased by high fat feeding whilst other cytokines including IL-1α, IL-1b and TNF-α were not altered. These results suggest that deficiency of tissue CSE and H2S occurs in mice fed a high fat diet and that this change takes place prior to development of frank atherosclerotic disease. ASTAR (Agency for Sci., Tech. and Research, S’pore) Accepted version

The endogenous gas, hydrogen sulfide (H2S), plays an important role in regulating cardiovascular function and inflammation. It also contributes to the pathogenesis of insulin resistance and glucose metabolism [1] , [2] , [3] . Recently, it has been reported that large amounts of H2S are formed from L-cysteine by cystathionine gamma lyase (CSE) in fat tissues and that H2S reduces glucose uptake in rat adipose tissue [3] . However, the biological roles of H2S in adipocytes and its contribution to adipogenesis are not yet clear. Here, we investigate whether H2S-synthesizing enzymes; cystathionine beta-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), are endogenously expressed in adipocytes and play a role in adipogenesis. In the present experiments, we stimulated the mouse fibroblast-like cell line, 3T3L1, to differentiate into adipocytes for up to one week as a model of adipogenesis in vitro. Over 7 days differentiation, whole cell lysate at day 0, 1, 3, 5 and 7 were collected for Western blotting of CBS, CSE and 3-MST. Endogenous CBS and 3-MST in adipocytes peaked at day 5 of differentiation whereas CSE protein expression was up-regulated in a time-dependent manner and maximally expressed at day 7 (n = 3, P [4] , [5] , [6] , but that these enzymes are also present in large amounts in adipose tissue and that their up-regulation during adipogenesis promotes adipocyte maturation and prevent lipid degradation. Thus, it is possible that modulation of the H2S system may be useful for obesity treatment.

Atherosclerosis is a progressive disease characterized by endothelial dysfunction and accumulation of lipids, cellular debris and fibrous elements within the intima of the vessel wall. Eventually, atherosclerosis triggers plaque formation, vascular remodeling and luminal obstruction [1] . It is becoming increasingly clear that atherosclerosis is an inflammatory disease, dependent on the interaction between inflammatory cells recruited in response to endothelial lipid accumulation, and to the local, wound-healing response of surrounding vascular smooth muscle cells. H2S is a gasomediator synthesized from cysteine predominantly by cystathionine-γ-lyase (CSE) [2] and been reported to exhibit both pro- and anti-inflammatory effects [3] . Administration of high fat diet over an extended period results in the development of a low grade inflammatory response which can predispose to atherosclerosis. In the present work we therefore investigated the role of H2S in high fat (HF) fed mice. Mice (C57/Black, male, 25 g) were fed either normally or given a HF diet (16% fat, 12.5% cholesterol and 5% sodium cholic acid) for up to 16 weeks. At the end of 8, 12 or 16 weeks, groups of mice were anaesthetised, blood obtained by cardiac puncture and centrifuged for plasma and heart, liver, kidney and lungs removed for biochemical and histological analyses. H2S synthesizing enzyme activity was measured in lung, kidney and liver homogenates incubated with l -cysteine(10 mM) and pyridoxal 5’-phosphate (2 mM) by the zinc trapping spectrophotometric assay. Plasma H2S levels were determined by high-performance liquid chromatography (HPLC) [4] . Lung, kidney and liver homogenates were used for western blotting of cystathionine-β-synthetase (CBS), CSE and 3-mercaptopyruvate synthase (3-MST). A range of cytokines and chemokines were also assayed in mouse plasma using a Bio-Plex Pro™ Assay. Plasma serum amyloid A (SAA) and C-reactive protein (CRP) levels were measured using ELISA kits. Oil red O staining was performed on aorta samples. Compared to normal diet mice, HF mice showed similar H2S synthesizing activity for lung (normal mice 0.28 ± 0.04 nmol/mg, n = 7; HF mice 0.22 ± 0.03 nmol/mg, n = 7), and decreased H2S synthesizing activity for kidney (normal mice 2.49 ± 0.79 nmol/mg, n = 7; HF mice 1.16 ± 0.32 nmol/mg, n = 7) and liver (normal mice 9.21 ± 2.44 nmol/mg, n = 7; HF mice 1.65 ± 0.34 nmol/mg, n = 7) (p

Atherosclerosis is a progressive disease characterized by endothelial dysfunction and accumulation of lipids, cellular debris and fibrous elements within the intima of the vessel wall. Eventually, atherosclerosis triggers plaque formation, vascular remodeling and luminal obstruction [1] . It is becoming increasingly clear that atherosclerosis is an inflammatory disease, dependent on the interaction between inflammatory cells recruited in response to endothelial lipid accumulation, and to the local, wound-healing response of surrounding vascular smooth muscle cells. H 2 S is a gasomediator synthesized from cysteine predominantly by cystathionine- γ -lyase (CSE) [2] and been reported to exhibit both pro- and anti-inflammatory effects [3] . Administration of high fat diet over an extended period results in the development of a low grade inflammatory response which can predispose to atherosclerosis. In the present work we therefore investigated the role of H 2 S in high fat (HF) fed mice. Mice (C57/Black, male, 25 g) were fed either normally or given a HF diet (16% fat, 12.5% cholesterol and 5% sodium cholic acid) for up to 16 weeks. At the end of 8, 12 or 16 weeks, groups of mice were anaesthetised, blood obtained by cardiac puncture and centrifuged for plasma and heart, liver, kidney and lungs removed for biochemical and histological analyses. H 2 S synthesizing enzyme activity was measured in lung, kidney and liver homogenates incubated with L-cysteine (10 mM) and pyridoxal 5′-phosphate (2 mM) by the zinc trapping spectrophotometric assay. Plasma H 2 S levels were determined by high-performance liquid chromatography (HPLC) [4] . Lung, kidney and liver homogenates were used for western blotting of cystathionine- β -synthetase (CBS), CSE and 3-mercaptopyruvate synthase (3-MST). A range of cytokines and chemokines were also assayed in mouse plasma using a Bio-Plex Pro ™ Assay. Plasma serum amyloid A (SAA) and C-reactive protein (CRP) levels were measured using ELISA kits. Oil red O staining was performed on aorta samples. Compared to normal diet mice, HF mice showed similar H 2 S synthesizing activity for lung (normal mice 0.28 ± 0.04 nmol/mg, n = 7; HF mice 0.22 ± 0.03 nmol/mg, n = 7), and decreased H 2 S synthesizing activity for kidney (normal mice 2.49 ± 0.79 nmol/mg, n = 7; HF mice 1.16 ± 0.32 nmol/mg, n = 7) and liver (normal mice 9.21 ± 2.44 nmol/mg, n = 7; HF mice 1.65 ± 0.34 nmol/mg, n = 7) (p 2 S levels (normal mice 256.7 ± 18.4 nM; HF mice 274.6 ± 17.1 nM, n = 5). For western blots, HF fed mice showed increased CBS in liver and kidney, decreased CSE in liver and lung, decreased 3-MST in liver, but similar expression of 3-MST in kidney and lung. HF fed mice had elevated plasma levels of IL-12 (p40), G-CSF, IL-6 and MCP-1. There was no difference between groups in terms of plasma SAA and CRP. There was little difference in aortic lipid content between groups as assessed histologically by oil red O staining. Taken the data together, we propose that changes in H 2 S metabolism are apparent in these animals prior to the development of frank atherosclerosis. Thus, it is possible that monitoring H 2 S may be useful as a predictive biomarker for atherosclerosis.