Selective mitochondrial antioxidant MitoTEMPO reduces renal dysfunction and systemic inflammation in experimental sepsis in rats.

BACKGROUND: Excess mitochondrial reactive oxygen species (mROS) in sepsis is associated with organ failure, in part by generating inflammation through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. We determined the impact of a mitochondrial-targeted antioxidant (MitoTEMPO) on mitochondrial dysfunction in renal proximal tubular epithelial cells, peritoneal immune cell function ex vivo, and organ dysfunction in a rat model of sepsis. METHODS: The effects of MitoTEMPO were assessed ex vivo using adenosine triphosphate and lipopolysaccharide-stimulated rat peritoneal immune cells and fresh rat kidney slices exposed to serum from septic rats. We assessed mROS production and phagocytotic capacity (flow cytometry), mitochondrial functionality (multiphoton imaging, respirometry), and NLRP3 inflammasome activation in cell culture. The effect of MitoTEMPO on organ dysfunction was evaluated in a rat model of faecal peritonitis. RESULTS: MitoTEMPO decreased septic serum-induced mROS (P<0.001) and maintained normal reduced nicotinamide adenine dinucleotide redox state (P=0.02) and mitochondrial membrane potential (P<0.001) in renal proximal tubular epithelial cells ex vivo. In lipopolysaccharide-stimulated peritoneal immune cells, MitoTEMPO abrogated the increase in mROS (P=0.006) and interleukin-1ß (IL-1ß) (P=0.03) without affecting non-mitochondrial oxygen consumption or the phagocytotic-induced respiratory burst (P>0.05). In vivo, compared with untreated septic animals, MitoTEMPO reduced systemic IL-1ß (P=0.01), reduced renal oxidative stress as determined by urine isoprostane levels (P=0.04), and ameliorated renal dysfunction (reduced serum urea (P<0.001) and creatinine (P=0.05). CONCLUSIONS: Reduction of mROS by a mitochondria-targeted antioxidant reduced IL-1ß, and protected mitochondrial, cellular, and organ functionality after septic insults.

The Pharmacology and Therapeutic Utility of Sodium Hydroselenide.

Metabolically active gasotransmitters (nitric oxide, carbon monoxide and hydrogen sulfide) are important signalling molecules that show therapeutic utility in oxidative pathologies. The reduced form of selenium, hydrogen selenide (HSe-/H2Se), shares some characteristics with these molecules. The simple selenide salt, sodium hydroselenide (NaHSe) showed significant metabolic activity, dose-dependently decreasing ex vivo O2 consumption (rat soleus muscle, liver) and transiently inhibiting mitochondrial cytochrome C oxidase (liver, heart). Pharmacological manipulation of selenoprotein expression in HepG2 human hepatocytes revealed that the oxidation status of selenium impacts on protein expression; reduced selenide (NaHSe) increased, whereas (oxidized) sodium selenite decreased the abundance of two ubiquitous selenoproteins. An inhibitor of endogenous sulfide production (DL-propargylglycine; PAG) also reduced selenoprotein expression; this was reversed by exogenous NaHSe, but not sodium hydrosulfide (NaHS). NaHSe also conferred cytoprotection against an oxidative challenge (H2O2), and this was associated with an increase in mitochondrial membrane potential. Anesthetized Wistar rats receiving intravenous NaHSe exhibited significant bradycardia, metabolic acidosis and hyperlactataemia. In summary, NaHSe modulates metabolism by inhibition of cytochrome C oxidase. Modification of selenoprotein expression revealed the importance of oxidation status of selenium therapies, with implications for current clinical practice. The utility of NaHSe as a research tool and putative therapeutic is discussed.

Working accuracy of pulse oximetry in COVID-19 patients stepping down from intensive care: a clinical evaluation.

INTRODUCTION: UK guidelines suggest that pulse oximetry, rather than blood gas sampling, is adequate for monitoring of patients with COVID-19 if CO2 retention is not suspected. However, pulse oximetry has impaired accuracy in certain patient groups, and data are lacking on its accuracy in patients with COVID-19 stepping down from intensive care unit (ICU) to non-ICU settings or being transferred to another ICU. METHODS: We assessed the bias, precision and limits of agreement using 90 paired SpO2 and SaO2 from 30 patients (3 paired samples per patient). To assess the agreement between pulse oximetry (SpO2) and arterial blood gas analysis (SaO2) in patients with COVID-19, deemed clinically stable to step down from an ICU to a non-ICU ward, or be transferred to another ICU. This was done to evaluate whether the guidelines were appropriate for our setting. RESULTS: Mean difference between SaO2 and SpO2 (bias) was 0.4%, with an SD of 2.4 (precision). The limits of agreement between SpO2 and SaO2 were as follows: upper limit of 5.2% (95% CI 6.5% to 4.2%) and lower limit of -4.3% (95% CI -3.4% to -5.7%). CONCLUSIONS: In our setting, pulse oximetry showed a level of agreement with SaO2 measurement that was slightly suboptimal, although within acceptable levels for Food and Drug Authority approval, in people with COVID-19 judged clinically ready to step down from ICU to a non-ICU ward, or who were being transferred to another hospital's ICU. In such patients, SpO2 should be interpreted with caution. Arterial blood gas assessment of SaO2 may still be clinically indicated.

Sepsis - thoughtful management for the non-expert.

Sepsis is a common and often devastating medical emergency with a high mortality rate and, in many survivors, long-term morbidity. It is defined as the dysregulated host response to infection resulting in organ dysfunction, and its incidence is increasing as the population ages. However, it is a treatable and potentially reversible condition, especially if identified and treated promptly. A sound understanding of sepsis is crucial for optimal care. Although general guidelines are available for management, here we provide a foundation of understanding to encourage thoughtful, personalised management of sepsis during the acute phase. We provide an overview of the epidemiology, the new Sepsis-3 definitions, pathophysiology, clinical presentations, and investigation and management of sepsis for the non-expert.

CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.

OBJECTIVE: Immune paresis in patients with acute-on-chronic liver failure (ACLF) accounts for infection susceptibility and increased mortality. Immunosuppressive mononuclear CD14+HLA-DR- myeloid-derived suppressor cells (M-MDSCs) have recently been identified to quell antimicrobial responses in immune-mediated diseases. We sought to delineate the function and derivation of M-MDSC in patients with ACLF, and explore potential targets to augment antimicrobial responses. DESIGN: Patients with ACLF (n=41) were compared with healthy subjects (n=25) and patients with cirrhosis (n=22) or acute liver failure (n=30). CD14+CD15-CD11b+HLA-DR- cells were identified as per definition of M-MDSC and detailed immunophenotypic analyses were performed. Suppression of T cell activation was assessed by mixed lymphocyte reaction. Assessment of innate immune function included cytokine expression in response to Toll-like receptor (TLR-2, TLR-4 and TLR-9) stimulation and phagocytosis assays using flow cytometry and live cell imaging-based techniques. RESULTS: Circulating CD14+CD15-CD11b+HLA-DR- M-MDSCs were markedly expanded in patients with ACLF (55% of CD14+ cells). M-MDSC displayed immunosuppressive properties, significantly decreasing T cell proliferation (p=0.01), producing less tumour necrosis factor-alpha/interleukin-6 in response to TLR stimulation (all p<0.01), and reduced bacterial uptake of Escherichia coli (p<0.001). Persistently low expression of HLA-DR during disease evolution was linked to secondary infection and 28-day mortality. Recurrent TLR-2 and TLR-4 stimulation expanded M-MDSC in vitro. By contrast, TLR-3 agonism reconstituted HLA-DR expression and innate immune function ex vivo. CONCLUSION: Immunosuppressive CD14+HLA-DR- M-MDSCs are expanded in patients with ACLF. They were depicted by suppressing T cell function, attenuated antimicrobial innate immune responses, linked to secondary infection, disease severity and prognosis. TLR-3 agonism reversed M-MDSC expansion and innate immune function and merits further evaluation as potential immunotherapeutic agent.

Outcomes of endoscopic human thrombin injection in the management of gastric varices.

OBJECTIVE: Endoscopic human thrombin injection appears to be a technically simple and efficacious alternative to tissue adhesives with fewer complications; however, data remain limited. We analysed our experience using endoscopic human thrombin injection for gastric varices in a tertiary referral liver unit. METHODS: Thirty patients received thrombin injection for gastric varices between December 2008 and January 2013. Twenty patients (67%) had active bleeding or signs of recent bleeding at endoscopy. Ten patients (33%) received thrombin for prophylaxis of rebleeding: secondary (eight patients) and primary (two patients). RESULTS: The mean thrombin dose/injection was 1100 IU (range 400-2500); the mean number of sessions was two (range 1-9), with no reported complications. Haemostasis was achieved in 18 out of 20 (90%) patients treated acutely. Failure to control bleeding (bleeding before day 5) was seen in seven patients: three died and four were managed successfully [two with further thrombin and two using a salvage transjugular intrahepatic portosystemic shunt (TIPSS)]. Rebleeding occurred in a further four patients, all managed successfully with salvage TIPSS. In the prophylaxis group, rebleeding occurred in two out of 10 patients.The median follow-up period was 672 days (interquartile range 92-1331). One patient underwent liver transplantation. Ten deaths occurred in total: four due to gastric variceal bleeding. Six-week survival was 83%. In cases in which TIPSS was precluded, 91% of patients (10 out of 11 patients) were managed successfully with thrombin. CONCLUSION: Endoscopic thrombin therapy for gastric variceal bleeding may have most utility as a safe and easily applied bridge to more definitive therapy, in secondary prophylaxis of rebleeding and in cases in which TIPSS is precluded.

Character and temporal evolution of apoptosis in acetaminophen-induced acute liver failure*.

OBJECTIVE: To evaluate the role of hepatocellular and extrahepatic apoptosis during the evolution of acetaminophen-induced acute liver failure. DESIGN AND SETTING: A prospective observational study in two tertiary liver transplant units. PATIENTS: Eighty-eight patients with acetaminophen-induced acute liver failure were recruited. Control groups included patients with nonacetaminophen-induced acute liver failure (n = 13), nonhepatic multiple organ failure (n = 28), chronic liver disease (n = 19), and healthy controls (n = 11). MEASUREMENTS: Total and caspase-cleaved cytokeratin-18 (M65 and M30) measured at admission and sequentially on days 3, 7, and 10 following admission. Levels were also determined from hepatic vein, portal vein, and systemic arterial blood in seven patients undergoing transplantation. Protein arrays of liver homogenates from patients with acetaminophen-induced acute liver failure were assessed for apoptosis-associated proteins, and histological assessment of liver tissue was performed. MAIN RESULTS: Admission M30 levels were significantly elevated in acetaminophen-induced acute liver failure and non-acetaminophen induced acute liver failure patients compared with multiple organ failure, chronic liver disease, and healthy controls. Admission M30 levels correlated with outcome with area under receiver operating characteristic of 0.755 (0.639-0.885, p < 0.001). Peak levels in patients with acute liver failure were seen at admission then fell significantly but did not normalize over 10 days. A negative gradient of M30 from the portal to hepatic vein was demonstrated in patients with acetaminophen-induced acute liver failure (p = 0.042) at the time of liver transplant. Analysis of protein array data demonstrated lower apoptosis-associated protein and higher catalase concentrations in acetaminophen-induced acute liver failure compared with controls (p < 0.05). Explant histological analysis revealed evidence of cellular proliferation with an absence of histological evidence of apoptosis. CONCLUSIONS: Hepatocellular apoptosis occurs in the early phases of human acetaminophen-induced acute liver failure, peaking on day 1 of hospital admission, and correlates strongly with poor outcome. Hepatic regenerative/tissue repair responses prevail during the later stages of acute liver failure where elevated levels of M30 are likely to reflect epithelial cell death in extrahepatic organs.

Whole genome RNA expression profiling of endoscopic biliary brushings provides data suitable for biomarker discovery in cholangiocarcinoma.

BACKGROUND & AIMS: Molecular analyses of biliary brushings using microarray and qPCR have the potential to provide valuable information on the biology of biliary diseases. Microarray analysis of biliary strictures has rarely been applied to endoscopic biliary brushings. METHODS: Biliary brushings were obtained from patients with benign and malignant biliary disease at the time of ERCP. Microarray analysis of mRNA isolated using brushings from 10 patients was validated for a selection of genes by qPCR using the same source mRNA and a second fresh set of nine biliary brushings as well as surgical resection tissue. Cultured cholangiocytes were used to assess the impact of bile or X-ray contrast solution on RNA quality. RESULTS: RNA was of variable quantity (100-1500 ng) and poor quality (Agilent RNA Integrity Number (RIN)<5, estimated to be fragments 100 to 600 base pairs long). Reliable qPCR results required primer pairs designed to produce amplicons <130 bp. Differential gene expression by microarray analysis identified 1140 up-regulated genes and 1001 down-regulated genes between benign and malignant biliary strictures. The trends in a selection of 45 up-regulated genes, including various HOX genes, collagens, PVT1, MUC4, MUC5AC, and LEF1, were validated by qPCR using RNA from biliary strictures with a moderate to strong correlation coefficient between microarray and qPCR (r=0.41 to r=0.57). Immunohistochemistry of surgical resection tissues (n=23) showed elevated CD9, SERPINA3, and PNMA2 protein expression in cancer samples. CONCLUSIONS: RNA isolated from biliary brushings is suitable for molecular analysis of biliary diseases using qPCR and microarray.