Statistical primer: using prognostic models to predict the future: what cardiothoracic surgery can learn from Strictly Come Dancing.

OBJECTIVES: Prognostic models are widely used across medicine and within cardiothoracic surgery, where predictive tools such as EuroSCORE are commonplace. Such models are a useful component of clinical assessment but may be misapplied. In this article, we demonstrate some of the major issues with risk scores by using the popular BBC television programme Strictly Come Dancing (known as Dancing with the Stars in many other countries) as an example. METHODS: We generated a multivariable prognostic model using data from the then-completed 19 series of Strictly Come Dancing to predict prospectively the results of the 20th series. RESULTS: The initial model based solely on demographic data was limited in its predictive value (0.25, 0.22; R2 and Spearman's rank correlation, respectively) but was substantially improved following the introduction of early judges' scores deemed representative of whether contestants could actually dance (0.40, 0.30). We then utilize our model to discuss the difficulties and pitfalls in using and interpreting prognostic models in cardiothoracic surgery and beyond, particularly where these do not adequately capture potentially important prognostic information. CONCLUSION: Researchers and clinicians alike should use prognostic models cautiously and not extrapolate conclusions from demographic data alone.

Investigating potential confounding by indication when considering the association between proton pump inhibitor use, infection, hepatic encephalopathy and mortality in hospitalised decompensated cirrhosis: a post-hoc analysis of the ATTIRE trial.

Background: Proton pump inhibitors (PPIs) are commonly prescribed to prevent and treat upper gastrointestinal ulceration and bleeding. Studies have identified increased incidence of spontaneous bacterial peritonitis and hepatic encephalopathy (HE) in cirrhosis patients taking PPIs. However, results are conflicting, and as PPIs are prescribed for variceal bleeding, a major risk factor for infection and HE, it is challenging to discern whether these associations are causal. Methods: In this post-hoc analysis of the ATTIRE trial, we pooled all patient data to investigate the effects of PPI use on clinical outcomes. ATTIRE was a multicentre, open-label, randomised trial of targeted 20% human albumin solution (HAS) daily infusions versus standard care involving 777 adults with decompensated cirrhosis hospitalised with acute complications and albumin <30 g/L. Study recruitment was between Jan 25, 2016, and June 28, 2019, at 35 hospitals across England, Scotland, and Wales. Key exclusion criteria were advanced hepatocellular carcinoma with life expectancy <8 weeks and patients receiving palliative care. In ATTIRE, patients were grouped by PPI use at trial entry. We studied infection and HE at baseline and incidence of hospital acquired infection, new onset HE, renal dysfunction and mortality. We attempted with propensity score matching to account for differences in disease severity. Findings: Overall PPI use at baseline was not associated with increased incidence of infection, renal dysfunction or mortality, but was associated with significantly increased incidence of grade III/IV HE during hospital stay (P = 0.011). This was only significant for those taking intravenous PPIs and these patients had >10 times the incidence of variceal bleeding and near double the 28-day mortality compared to non-PPI patients. However, propensity score matching was not possible as there was such a strong selection of patients for PPI use, that we could not find sufficient non-PPI patients to match to. We found no impact of PPI use on plasma markers of bacterial translocation, infection or systemic inflammation. Interpretations: Our real-world data from a completed randomised trial show that PPIs are widely prescribed in the UK and judicious use appears safe in patients hospitalised with decompensated cirrhosis. However, patients prescribed PPIs had fundamentally different phenotypes to those not prescribed PPIs, a form of confounding by indication, which should be strongly considered when interpreting studies and making recommendations about their use. Funding: Wellcome Trust and Department of Health and Social Care.

Targeted Albumin Infusions Do Not Improve Systemic Inflammation or Cardiovascular Function in Decompensated Cirrhosis.

INTRODUCTION: Albumin is recommended in decompensated cirrhosis, and studies have shown potential immunomodulatory effects. However, 2 large trials of repeated albumin infusions demonstrated contrasting results between outpatients and hospitalized patients. We investigated markers of systemic inflammation, immune function, albumin binding, and cardiovascular function using samples from Albumin To prevenT Infection in chronic liveR failurE (ATTIRE) taken at baseline, day 5, and day 10 of the trial to identify why targeted albumin infusions had no effect in hospitalized patients. METHODS: Plasma samples were analyzed from 143 patients (n = 71 targeted albumin; n = 72 standard care at baseline) for cytokines, cardiovascular markers, prostaglandin E2, the effect of plasma on macrophage function, and albumin radioligand binding and oxidation status. The sample size was based on our feasibility study, and samples were selected by a trial statistician stratified by the serum albumin level and the presence of infection at randomization and analyses performed blinded to the study arm. Data were linked to 3-month mortality and treatment groups compared. RESULTS: Increased baseline model for end-stage liver disease score, white cell count, calprotectin, CD163, tumor necrosis factor, renin, atrial natriuretic peptide, and syndecan-1 were associated with 3-month mortality. Despite infusing substantially differing volumes of albumin, there were no significant differences in inflammatory markers, albumin-prostaglandin E2 binding, or cardiovascular markers between treatment arms. DISCUSSION: Contrary to many preclinical studies, targeted intravenous albumin therapy in hospitalized decompensated cirrhosis had no effect across a broad range of systemic inflammation, albumin function, and cardiovascular mediators and biomarkers compared with standard care, consistent with the null clinical findings.

Monocyte dysfunction in decompensated cirrhosis is mediated by the prostaglandin E2-EP4 pathway.

BACKGROUND & AIMS: Infection is a major problem in advanced liver disease secondary to monocyte dysfunction. Elevated prostaglandin (PG)E2 is a mediator of monocyte dysfunction in cirrhosis; thus, we examined PGE2 signalling in outpatients with ascites and in patients hospitalised with acute decompensation to identify potential therapeutic targets aimed at improving monocyte dysfunction. METHODS: Using samples from 11 outpatients with ascites and 28 patients hospitalised with decompensated cirrhosis, we assayed plasma levels of PGE2 and lipopolysaccharide (LPS); performed quantitative real-time PCR on monocytes; and examined peripheral blood monocyte function. We performed western blotting and immunohistochemistry for PG biosynthetic machinery expression in liver tissue. Finally, we investigated the effect of PGE2 antagonists in whole blood using polychromatic flow cytometry and cytokine production. RESULTS: We show that hepatic production of PGE2 via the cyclo-oxygenase 1-microsomal PGE synthase 1 pathway, and circulating monocytes contributes to increased plasma PGE2 in decompensated cirrhosis. Transjugular intrahepatic sampling did not reveal whether hepatic or monocytic production was larger. Blood monocyte numbers increased, whereas individual monocyte function decreased as patients progressed from outpatients with ascites to patients hospitalised with acute decompensation, as assessed by Human Leukocyte Antigen (HLA)-DR isotype expression and tumour necrosis factor alpha and IL6 production. PGE2 mediated this dysfunction via its EP4 receptor. CONCLUSIONS: PGE2 mediates monocyte dysfunction in decompensated cirrhosis via its EP4 receptor and dysfunction was worse in hospitalised patients compared with outpatients with ascites. Our study identifies a potential drug target and therapeutic opportunity in these outpatients with ascites to reverse this process to prevent infection and hospital admission. LAY SUMMARY: Patients with decompensated cirrhosis (jaundice, fluid build-up, confusion, and vomiting blood) have high infection rates that lead to high mortality rates. A white blood cell subset, monocytes, function poorly in these patients, which is a key factor underlying their sensitivity to infection. We show that monocyte dysfunction in decompensated cirrhosis is mediated by a lipid hormone in the blood, prostaglandin E2, which is present at elevated levels, via its EP4 pathway. This dysfunction worsens when patients are hospitalised with complications of cirrhosis compared with those in the outpatients setting, which supports the EP4 pathway as a potential therapeutic target for patients to prevent infection and hospitalisation.

Immune Regulatory Mediators in Plasma from Patients With Acute Decompensation Are Associated With 3-Month Mortality.

BACKGROUND & AIMS: Infection is a common cause of death in patients with cirrhosis. We investigated the association between the innate immune response and death within 3 months of hospitalization. METHODS: Plasma samples were collected on days 1, 5, 10, and 15 from participants recruited into the albumin to prevent infection in chronic liver failure feasibility study. Patients with acute decompensated cirrhosis were given albumin infusions at 10 hospitals in the United Kingdom. Data were obtained from 45 survivors and 27 non-survivors. We incubated monocyte-derived macrophages from healthy individuals with patients' plasma samples and measured activation following lipopolysaccharide administration, determined by secretion of tumor necrosis factor and soluble mediators of inflammation. Each analysis included samples from 4 to 14 patients. RESULTS: Plasma samples from survivors vs non-survivors had different inflammatory profiles. Levels of prostaglandin E2 were high at times of patient hospitalization and decreased with albumin infusions. Increased levels of interleukin 4 (IL4) in plasma collected at day 5 of treatment were associated with survival at 3 months. Incubation of monocyte-derived macrophages with day 5 plasma from survivors, pre-incubated with a neutralizing antibody against IL4, caused a significant increase in tumor necrosis factor production to the level of non-survivor plasma. Although baseline characteristics were similar, non-survivors had higher white cell counts and levels of C-reactive protein and renal dysfunction. CONCLUSIONS: We identified profiles of inflammatory markers in plasma that are associated with 3-month mortality in patients with acute decompensated cirrhosis given albumin. Increases in prostaglandin E2 might promote inflammation within the first few days after hospitalization, and increased levels of plasma IL4 at day 5 are associated with increased survival. Clinicaltrialsregister.eu: EudraCT 2014-002300-24.

Albumin Counteracts Immune-Suppressive Effects of Lipid Mediators in Patients With Advanced Liver Disease.

BACKGROUND & AIMS: Patients with acute decompensation and acute-on-chronic liver failure (AD/ACLF) have immune dysfunction, which increases their risk for infections; however, there are no effective treatments to restore their immune function. We investigated whether the potentially immune-restorative effects of albumin are mediated by its effects on prostaglandin E2 (PGE2) and other lipids. METHODS: We analyzed bloods samples from 45 of 79 patients with AD/ACLF and serum levels of albumin less than 30 g/L for whom infusion of 20% human albumin solution (HAS) increased serum levels of albumin 30 g/L or more in a feasibility study of effects of 20% HAS. Immune function was determined by comparison of macrophage function following addition of plasma samples. We also used samples from 12 healthy individuals. We measured binding of plasma proteins to PGE2 and serum levels of endotoxin (lipopolysaccharide) and cytokines; using 10 patients' samples, we investigated the effects of PGE2 inhibitors. We performed a comprehensive lipid metabolomic analysis using samples from 10 different patients, before and after HAS administration. RESULTS: At baseline, AD/ACLF patient plasma induced significantly lower production of tumor necrosis factor by healthy macrophages than plasma from healthy individuals (P < .0001). Plasma from patients after HAS infusion induced significantly higher levels of tumor necrosis factor production by macrophages (19.5 ± 4.8 ng/mL) compared with plasma collected before treatment (17.7 ± 4.5 ng/mL; P = .0013). There was a significantly lower proportion of plasma protein (albumin) binding to PGE2 from patients with AD/ACLF plasma (mean, 61.9%) compared with plasma from control subjects (77.1%; P = .0012). AD/ACLF plasma protein binding to PGE2 increased following HAS treatment compared with baseline (mean increase, 8.7%; P < .0001). Circulating levels of PGE2, lipopolysaccharide, and inflammatory or anti-inflammatory cytokines were higher in patients with AD/ACLF than healthy volunteers. Unexpectedly, HAS infusion had no effect on mediator levels. Principal component analysis of baseline levels of lipids that induce or resolve inflammation identified 2 distinct groups of patients that differed according to baseline plasma level of lipopolysaccharide. Sample analyses after HAS treatment indicated that albumin regulates circulating levels of lipid mediators, but this effect was distinct in each group. CONCLUSIONS: Analysis of blood samples from patients with AD/ACLF participating in a feasibility study of 20% HAS infusions has shown that infusions to raise serum albumin above 30 g/L reversed plasma-mediated immune dysfunction by binding and inactivating PGE2. We also describe a method to classify the inflammatory response in AD/ACLF, based on lipid profile, which could improve identification of patients most likely to respond to HAS treatment. A randomized controlled trial is needed to determine whether these effects of HAS reduce infections in AD/ACLF. Trial registered with European Medicines Agency (EudraCT 2014-002300-24) and adopted by NIHR (ISRCTN14174793).

Defective monocyte oxidative burst predicts infection in alcoholic hepatitis and is associated with reduced expression of NADPH oxidase.

OBJECTIVE: In order to explain the increased susceptibility to serious infection in alcoholic hepatitis, we evaluated monocyte phagocytosis, aberrations of associated signalling pathways and their reversibility, and whether phagocytic defects could predict subsequent infection. DESIGN: Monocytes were identified from blood samples of 42 patients with severe alcoholic hepatitis using monoclonal antibody to CD14. Phagocytosis and monocyte oxidative burst (MOB) were measured ex vivo using flow cytometry, luminometry and bacterial killing assays. Defects were related to the subsequent development of infection. Intracellular signalling pathways were investigated using western blotting and PCR. Interferon-γ (IFN-γ) was evaluated for its therapeutic potential in reversing phagocytic defects. Paired longitudinal samples were used to evaluate the effect of in vivo prednisolone therapy. RESULTS: MOB, production of superoxide and bacterial killing in response to Escherichia coli were markedly impaired in patients with alcoholic hepatitis. Pretreatment MOB predicted development of infection within two weeks with sensitivity and specificity that were superior to available clinical markers. Accordingly, defective MOB was associated with death at 28 and 90 days. Expression of the gp91 phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was reduced in patients with alcoholic hepatitis demonstrating defective MOB. Monocytes were refractory to IFN-γ stimulation and showed high levels of a negative regulator of cytokine signalling, suppressor of cytokine signalling-1. MOB was unaffected by 7 days in vivo prednisolone therapy. CONCLUSIONS: Monocyte oxidative burst and bacterial killing is impaired in alcoholic hepatitis while bacterial uptake by phagocytosis is preserved. Defective MOB is associated with reduced expression of NADPH oxidase in these patients and predicts the development of infection and death.

ATTIRE: Albumin To prevenT Infection in chronic liveR failurE: study protocol for a single-arm feasibility trial.

INTRODUCTION: Circulating prostaglandin E2 levels are elevated in acutely decompensated cirrhosis and have been shown to contribute to immune suppression. Albumin binds and inactivates this hormone. Human albumin solution could thus be repurposed as an immune restorative drug in these patients.This feasibility study aims to determine whether it is possible and safe to restore serum albumin to >30 g/L and maintain it at this level in patients admitted with acute decompensated cirrhosis using repeated 20% human albumin infusions according to daily serum albumin levels. METHODS AND ANALYSIS: Albumin To prevenT Infection in chronic liveR failurE (ATTIRE) stage 1 is a multicentre, open label dose feasibility trial. Patients with acutely decompensated cirrhosis admitted to hospital with a serum albumin of <30 g/L are eligible, subject to exclusion criteria. Daily intravenous human albumin solution will be infused, according to serum albumin levels, for up to 14 days or discharge in all patients. The primary end point is daily serum albumin levels for the duration of the treatment period and the secondary end point is plasma-induced macrophage dysfunction. The trial will recruit 80 patients. Outcomes will be used to assist with study design for an 866 patient randomised controlled trial at more than 30 sites across the UK. ETHICS AND DISSEMINATION: Research ethics approval was given by the London-Brent research ethics committee (ref: 15/LO/0104). The clinical trials authorisation was issued by the medicines and healthcare products regulatory agency (ref: 20363/0350/001-0001). RESULTS: Will be disseminated through peer reviewed journals and international conferences. Recruitment of the first participant occurred on 26/05/2015. TRIAL REGISTRATION NUMBER: The trial is registered with the European Medicines Agency (EudraCT 2014-002300-24) and has been adopted by the NIHR (ISRCTN 14174793). This manuscript refers to V.4.0 of the protocol; Pre-results.

Bile duct-ligated mice exhibit multiple phenotypic similarities to acute decompensation patients despite histological differences.

BACKGROUND & AIMS: Patients with decompensated cirrhosis are susceptible to infection. Innate immune dysfunction and development of organ failure are considered to underlie this. A rodent model of liver disease sharing these phenotypic features would assist in vivo study of underlying mechanisms and testing of therapeutics. We evaluated three models to identify which demonstrated the greatest clinical and immunological phenotypic similarity to patients with acutely decompensated (AD) cirrhosis. METHODS: We selected Bile Duct Ligation (BDL) rats at 4 weeks, BDL mice at 14 days and Carbon tetrachloride (CCl4 ) mice at 10 weeks (with studies performed 7 days after final CCl4 infection). We examined organ dysfunction, inflammatory response to carrageenan-in-paw, plasma eicosanoid concentrations, macrophage cytokine production and responses to peritoneal infection. RESULTS: Bile duct ligation caused sarcopenia, liver, cardiovascular and renal dysfunction whereas CCl4 mice demonstrated no clinical abnormalities. BDL rodents exhibited depressed response to carrageenan-in-paw unlike CCl4 mice. BDL rats have slightly elevated plasma eicosanoid levels and plasma showed partial PGE2 -mediated immune suppression whereas CCl4 mice did not. Plasma NOx was elevated in patients with acute or chronic liver failure (AoCLF) compared to healthy volunteers and BDL rodents but not CCl4 mice. Elevated nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates defective leucocyte trafficking in BDL rodent models. CONCLUSIONS: We conclude that BDL mice and rats are not simply models of cholestatic liver injury but may be used to study mechanisms underlying poor outcome from infection in AD and have identified elevated NO as a potential mediator of depressed leucocyte trafficking.

Systemic Inflammatory Response Syndrome After Major Abdominal Surgery Predicted by Early Upregulation of TLR4 and TLR5.

OBJECTIVES: To study innate immune pathways in patients undergoing hepatopancreaticobiliary surgery to understand mechanisms leading to enhanced inflammatory responses and identifying biomarkers of adverse clinical consequences. BACKGROUND: Patients undergoing major abdominal surgery are at risk of life-threatening systemic inflammatory response syndrome (SIRS) and sepsis. Early identification of at-risk patients would allow tailored postoperative care and improve survival. METHODS: Two separate cohorts of patients undergoing major hepatopancreaticobiliary surgery were studied (combined n = 69). Bloods were taken preoperatively, on day 1 and day 2 postoperatively. Peripheral blood mononuclear cells and serum were separated and immune phenotype and function assessed ex vivo. RESULTS: Early innate immune dysfunction was evident in 12 patients who subsequently developed SIRS (postoperative day 6) compared with 27 who did not, when no clinical evidence of SIRS was apparent (preoperatively or days 1 and 2). Serum interleukin (IL)-6 concentration and monocyte Toll-like receptor (TLR)/NF-κB/IL-6 functional pathways were significantly upregulated and overactive in patients who developed SIRS (P < 0.0001). Interferon α-mediated STAT1 phosphorylation was higher preoperatively in patients who developed SIRS. Increased TLR4 and TLR5 gene expression in whole blood was demonstrated in a separate validation cohort of 30 patients undergoing similar surgery. Expression of TLR4/5 on monocytes, particularly intermediate CD14CD16 monocytes, on day 1 or 2 predicted SIRS with accuracy 0.89 to 1.0 (areas under receiver operator curves). CONCLUSIONS: These data demonstrate the mechanism for IL-6 overproduction in patients who develop postoperative SIRS and identify markers that predict patients at risk of SIRS 5 days before the onset of clinical signs.

Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2.

Liver disease is one of the leading causes of death worldwide. Patients with cirrhosis display an increased predisposition to and mortality from infection due to multimodal defects in the innate immune system; however, the causative mechanism has remained elusive. We present evidence that the cyclooxygenase (COX)-derived eicosanoid prostaglandin E2 (PGE2) drives cirrhosis-associated immunosuppression. We observed elevated circulating concentrations (more than seven times as high as in healthy volunteers) of PGE2 in patients with acute decompensation of cirrhosis. Plasma from these and patients with end-stage liver disease (ESLD) suppressed macrophage proinflammatory cytokine secretion and bacterial killing in vitro in a PGE2-dependent manner via the prostanoid type E receptor-2 (EP2), effects not seen with plasma from patients with stable cirrhosis (Child-Pugh score grade A). Albumin, which reduces PGE2 bioavailability, was decreased in the serum of patients with acute decompensation or ESLD (<30 mg/dl) and appears to have a role in modulating PGE2-mediated immune dysfunction. In vivo administration of human albumin solution to these patients significantly improved the plasma-induced impairment of macrophage proinflammatory cytokine production in vitro. Two mouse models of liver injury (bile duct ligation and carbon tetrachloride) also exhibited elevated PGE2, reduced circulating albumin concentrations and EP2-mediated immunosuppression. Treatment with COX inhibitors or albumin restored immune competence and survival following infection with group B Streptococcus. Taken together, human albumin solution infusions may be used to reduce circulating PGE2 levels, attenuating immune suppression and reducing the risk of infection in patients with acutely decompensated cirrhosis or ESLD.

Pathways mediating resolution of inflammation: when enough is too much.

Patients with critical illness, and in particular sepsis, are now recognized to undergo unifying, pathogenic disturbances of immune function. Whilst scientific and therapeutic focus has traditionally been on understanding and modulating the initial pro-inflammatory limb, recent years have witnessed a refocusing on the development and importance of immunosuppressive 'anti-inflammatory' pathways. Several mechanisms are known to drive this phenomenon; however, no overriding conceptual framework justifies them. In this article we review the contribution of pro-resolution pathways to this phenotype, describing the observed immune alterations in terms of either a failure of resolution of inflammation or the persistence of pro-resolution processes causing inappropriate 'injurious resolution'-a novel hypothesis. The dysregulation of key processes in critical illness, including apoptosis of infiltrating neutrophils and their efferocytosis by macrophages, are discussed, along with the emerging role of specialized cell subtypes Gr1(+) CD11b(+) myeloid-derived suppressor cells and CD4(+) CD25(+) FoxP3(+) T-regulatory cells.

Reversal of life-threatening, drug-related potassium-channel syndrome by glibenclamide.

We describe three critically ill patients who received drugs with K(ATP) channel-opening properties and subsequently developed severe life-threatening complications, including hyperkalaemia and cardiovascular disturbances. Administration of the sulfonylurea-receptor inhibitor glibenclamide promptly reversed these abnormalities. Over the past 3 years, we have seen this syndrome and response in five patients taking nicorandil, ciclosporin, or isoflurane, which suggests that this disorder arises more frequently than is currently realised.