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Background Supine chest radiography for bedridden patients in intensive care units (ICUs) is one of the most frequently ordered imaging studies worldwide. Purpose To evaluate the diagnostic performance of a neural network-based model that is trained on structured semiquantitative radiologic reports of bedside chest radiographs. Materials and Methods For this retrospective single-center study, children and adults in the ICU of a university hospital who had been imaged using bedside chest radiography from January 2009 to December 2020 were reported by using a structured and itemized template. Ninety-eight radiologists rated the radiographs semiquantitatively for the severity of disease patterns. These data were used to train a neural network to identify cardiomegaly, pulmonary congestion, pleural effusion, pulmonary opacities, and atelectasis. A held-out internal test set (100 radiographs from 100 patients) that was assessed independently by an expert panel of six radiologists provided the ground truth. Individual assessments by each of these six radiologists, by two nonradiologist physicians in the ICU, and by the neural network were compared with the ground truth. Separately, the nonradiologist physicians assessed the images without and with preliminary readings provided by the neural network. The weighted Cohen κ coefficient was used to measure agreement between the readers and the ground truth. Results A total of 193 566 radiographs in 45 016 patients (mean age, 66 years ± 16 [SD]; 61% men) were included and divided into training (

Soluble receptor activator of nuclear factor κ B ligand (sRANKL) is a member of the tumor necrosis factor receptor superfamily, and therefore, involved in various inflammatory processes. The role of sRANKL in the course of bone remodeling via activation of osteoclasts as well as chronic disease progression has been described extensively. However, the potential functional importance of sRANKL in critically ill or septic patients remained unknown. Therefore, we measured sRANKL serum concentrations in 303 critically ill patients, including 203 patients with sepsis and 100 with non-sepsis critical illness. Results were compared to 99 healthy controls. Strikingly, in critically ill patients sRANKL serum levels were significantly decreased at intensive care unit (ICU) admission (p = 0.011) without differences between sepsis and non-sepsis patients. Inline, sRANKL was correlated with markers of metabolic dysregulation, such as pre-existing diabetes and various adipokines (e.g., adiponectin, leptin receptor). Importantly, overall mortality of critically ill patients in a three-year follow-up was significantly associated with decreased sRANKL serum concentrations at ICU admission (p = 0.038). Therefore, our study suggests sRANKL as a biomarker in critically ill patients which is associated with poor prognosis and overall survival beyond ICU stay.

BACKGROUND & AIMS: Homozygosity for the Pi∗Z variant of the gene that encodes the alpha-1 antitrypsin peptide (AAT), called the Pi∗ZZ genotype, causes a liver and lung disease called alpha-1 antitrypsin deficiency. Heterozygosity (the Pi∗MZ genotype) is a risk factor for cirrhosis in individuals with liver disease. Up to 4% of Europeans have the Pi∗MZ genotype; we compared features of adults with and without Pi∗MZ genotype among persons without preexisting liver disease. METHODS: We analyzed data from the European Alpha-1 Liver Cohort, from 419 adults with the Pi∗MZ genotype, 309 adults with the Pi∗ZZ genotype, and 284 individuals without the variant (noncarriers). All underwent a comprehensive evaluation; liver stiffness measurements (LSMs) were made by transient elastography. Liver biopsies were analyzed to define histologic and biochemical features associated with the Pi∗Z variant. Levels of serum transaminases were retrieved from 444,642 participants, available in the United Kingdom biobank. RESULTS: In the UK biobank database, levels of serum transaminases were increased in subjects with the Pi∗MZ genotype compared with noncarriers. In the Alpha-1 Liver Cohort, adults with Pi∗MZ had lower levels of gamma-glutamyl transferase in serum and lower LSMs than adults with the Pi∗ZZ variant, but these were higher than in noncarriers. Ten percent of subjects with the Pi∗MZ genotype vs 4% of noncarriers had LSMs of 7.1 kPa or more (adjusted odds ratio, 4.8; 95% confidence interval, 2.0-11.8). Obesity and diabetes were the most important factors associated with LSMs ≥7.1 kPa in subjects with the Pi∗MZ genotype. AAT inclusions were detected in liver biopsies of 63% of subjects with the Pi∗MZ genotype, vs 97% of subjects with the Pi∗ZZ genotype, and increased with liver fibrosis stages. Subjects with the Pi∗MZ genotype did not have increased hepatic levels of AAT, whereas levels of insoluble AAT varied among individuals. CONCLUSIONS: Adults with the Pi∗MZ genotype have lower levels of serum transaminases, fewer AAT inclusions in liver, and lower liver stiffness than adults with the Pi∗ZZ genotype, but higher than adults without the Pi∗Z variant. These findings should help determine risk of subjects with the Pi∗MZ genotype and aid in counseling. ispartof: GASTROENTEROLOGY vol:159 issue:2 pages:534-+ ispartof: location:United States status: published

ObjectiveAlpha-1 antitrypsin deficiency (AATD) is a common, potentially lethal inborn disorder caused by mutations in alpha-1 antitrypsin (AAT). Homozygosity for the ‘Pi*Z’ variant of AAT (Pi*ZZ genotype) causes lung and liver disease, whereas heterozygous ‘Pi*Z’ carriage (Pi*MZ genotype) predisposes to gallstones and liver fibrosis. The clinical significance of the more common ‘Pi*S’ variant remains largely undefined and no robust data exist on the prevalence of liver tumours in AATD.DesignBaseline phenotypes of AATD individuals and non-carriers were analysed in 482 380 participants in the UK Biobank. 1104 participants of a multinational cohort (586 Pi*ZZ, 239 Pi*SZ, 279 non-carriers) underwent a comprehensive clinical assessment. Associations were adjusted for age, sex, body mass index, diabetes and alcohol consumption.ResultsAmong UK Biobank participants, Pi*ZZ individuals displayed the highest liver enzyme values, the highest occurrence of liver fibrosis/cirrhosis (adjusted OR (aOR)=21.7 (8.8–53.7)) and primary liver cancer (aOR=44.5 (10.8–183.6)). Subjects with Pi*MZ genotype had slightly elevated liver enzymes and moderately increased odds for liver fibrosis/cirrhosis (aOR=1.7 (1.2–2.2)) and cholelithiasis (aOR=1.3 (1.2–1.4)). Individuals with homozygous Pi*S mutation (Pi*SS genotype) harboured minimally elevated alanine aminotransferase values, but no other hepatobiliary abnormalities. Pi*SZ participants displayed higher liver enzymes, more frequent liver fibrosis/cirrhosis (aOR=3.1 (1.1–8.2)) and primary liver cancer (aOR=6.6 (1.6–26.9)). The higher fibrosis burden was confirmed in a multinational cohort. Male sex, age ≥50 years, obesity and the presence of diabetes were associated with significant liver fibrosis.ConclusionOur study defines the hepatobiliary phenotype of individuals with the most relevant AATD genotypes including their predisposition to liver tumours, thereby allowing evidence-based advice and individualised hepatological surveillance.

Journal of Clinical Medicine 12(1), 253 (2022). doi:10.3390/jcm12010253 special issue: "Topical Collection "Clinical Research in Hepatology" / Yulia A.Nevzorova, Editor", Published by MDPI, Basel

Soluble receptor activator of nuclear factor κ B ligand (sRANKL) is a member of the tumor necrosis factor receptor superfamily, and therefore, involved in various inflammatory processes. The role of sRANKL in the course of bone remodeling via activation of osteoclasts as well as chronic disease progression has been described extensively. However, the potential functional importance of sRANKL in critically ill or septic patients remained unknown. Therefore, we measured sRANKL serum concentrations in 303 critically ill patients, including 203 patients with sepsis and 100 with non-sepsis critical illness. Results were compared to 99 healthy controls. Strikingly, in critically ill patients sRANKL serum levels were significantly decreased at intensive care unit (ICU) admission (p = 0.011) without differences between sepsis and non-sepsis patients. Inline, sRANKL was correlated with markers of metabolic dysregulation, such as pre-existing diabetes and various adipokines (e.g., adiponectin, leptin receptor). Importantly, overall mortality of critically ill patients in a three-year follow-up was significantly associated with decreased sRANKL serum concentrations at ICU admission (p = 0.038). Therefore, our study suggests sRANKL as a biomarker in critically ill patients which is associated with poor prognosis and overall survival beyond ICU stay.

Perilipin 2 (PLIN2) is a lipid droplet protein with various metabolic functions. However, studies investigating PLIN2 in the context of inflammation, especially in systemic and acute inflammation, are lacking. Hence, we assessed the relevance of serum PLIN2 in critically ill patients. We measured serum PLIN2 serum in 259 critically ill patients (166 with sepsis) upon admission to a medical intensive care unit (ICU) compared to 12 healthy controls. A subset of 36 patients underwent computed tomography to quantify body composition. Compared to controls, serum PLIN2 concentrations were elevated in critically ill patients at ICU admission. Interestingly, PLIN2 independently indicated multiple organ dysfunction (MOD), defined as a SOFA score &gt, 9 points, at ICU admission, and was also able to independently predict MOD after 48 h. Moreover, serum PLIN2 levels were associated with severe respiratory failure potentially reflecting a moribund state. However, PLIN2 was neither a predictor of ICU mortality nor did it reflect metabolic dysregulation. Conclusively, the first study assessing serum PLIN2 in critical illness proved that it may assist in risk stratification because it is capable of independently indicating MOD at admission and predicting MOD 48 h after PLIN2 measurement. Further evaluation regarding the underlying mechanisms is warranted.

Diagnostics 11(9), 1650 (2021). doi:10.3390/diagnostics11091650 special issue: "Special Issue "Novel Approaches in Oncologic Imaging" / Special Issue Editors: Dr. Thomas Geyer, Guest Editor; Dr. Saif Afat, Guest Editor", Published by MDPI, Basel

BMC medicine 19, 39 (2021). doi:10.1186/s12916-021-01917-6 special issue: "Editor���s Choice: World's Digestive Health Day 2021", Published by BioMed Central, London

Hypothermic oxygenated machine perfusion (HOPE) was recently tested in preclinical trials in kidney transplantation (KT). Here we investigate the effects of HOPE on extended-criteria-donation (ECD) kidney allografts (KA). Fifteen ECD-KA were submitted to 152 ± 92 min of end-ischemic HOPE and were compared to a matched group undergoing conventional-cold-storage (CCS) KT (n = 30). Primary (delayed graft function-DGF) and secondary (e.g. postoperative complications, perfusion parameters) endpoints were analyzed within 6-months follow-up. There was no difference in the development of DGF between the HOPE and CCS groups (53% vs. 33%, respectively; p = 0.197). Serum urea was lower following HOPE compared to CCS (p = 0.003), whereas the CCS group displayed lower serum creatinine and higher eGFR rates on postoperative days (POD) 7 and 14. The relative decrease of renal vascular resistance (RR) following HOPE showed a significant inverse association with serum creatinine on POD1 (r = − 0.682; p = 0.006) as well as with serum urea and eGFR. Besides, the relative RR decrease was more prominent in KA with primary function when compared to KA with DGF (p = 0.013). Here we provide clinical evidence on HOPE in ECD-KT after brain death donation. Relative RR may be a useful predictive marker for KA function. Further validation in randomized controlled trials is warranted.Trial registration: clinicaltrials.gov (NCT03378817, Date of first registration: 20/12/2017).

Alpha-1 antitrypsin deficiency (AATD) is a systemic disorder affecting mainly the lung and the liver and is caused by mutations in SERPINA1, the AAT gene. A homozygous "Pi*Z" mutation (Pi*ZZ genotype) may cause liver fibrosis on its own independently of pulmonary AATD manifestation, while heterozygous Pi*Z carriage (Pi*MZ genotype) is considered a strong risk factor for development of liver cirrhosis in patients with concomitant liver disease such as alcoholic and non-alcoholic liver disease. In Pi*ZZ homozygotes, liver disease constitutes the second leading cause of death and is highly heterogeneous. About 35% of Pi*ZZ individuals display significant liver fibrosis on biopsy (i.e., fibrosis stage ≥ 2 on scale 0-4). Among non-invasive methods for liver fibrosis assessment, liver stiffness measurement (LSM) via vibration-controlled transient elastography (VCTE) has been most widely evaluated. Based on these data, Pi*ZZ adults have 20x increased odds of developing advanced liver fibrosis (i.e., fibrosis stage ≥ 3) than adults without AAT mutation. Risk factors for accelerated fibrosis progression are male sex, age ≥ 50 years, alcohol misuse, obesity, diabetes mellitus, or metabolic syndrome. Unlike VCTE, other ultrasound- and magnetic resonance-based elastography methods have been assessed in small cohorts of Pi*ZZ individuals and remain to be comprehensively validated. Among blood-based fibrosis tests, AST-to-platelet ratio index (APRI) correlates moderately with histologic fibrosis stage and LSM. Given APRI's wide availability, it can be used for risk stratification as an adjunct to LSM or when LSM is not at hand. Despite recent efforts, AATD-related liver disease, especially for genotypes other than Pi*ZZ, remains greatly understudied. AATD individuals should be offered liver biochemistry, liver ultrasound, and non-invasive fibrosis assessment at the time of diagnosis to detect potential complications and for proper risk stratification. If signs of AATD-related liver disease occur (i.e., pathologic fibrosis test or repeatedly elevated liver enzymes), patients should be referred to a health care center specialized in AATD-related liver disease and be screened for potentially treatable comorbidities. To exclude the latter, they may need a liver biopsy. Moreover, every health care provider of an AATD individual should be aware of the potential liver manifestation, counsel their patient on modifiable hepatic risk factors, and offer them regular liver check-ups.

Objectives Severe alcoholic hepatitis (sAH) confers substantial mortality, but the disease course is difficult to predict. As iron parameters are attractive outcome predictors in other liver diseases, we tested their prognostic ability in sAH. Methods Serum ferritin, transferrin, iron, transferrin saturation, nontransferrin-bound iron, soluble transferrin receptor, and hepcidin were measured in 828 patients with sAH recruited prospectively through the STOPAH trial. The cohort was randomly divided into exploratory (n = 200) and validation sets (n = 628). Results Patients with sAH had diminished serum transferrin but increased transferrin saturation. Among iron parameters, baseline transferrin was the best predictor of 28-day (area under the receiver operated characteristic 0.72 [95% confidence interval 0.67-0.78]) and 90-day survival (area under the receiver operated characteristic 0.65 [0.61-0.70]). Transferrin's predictive ability was comparable with the composite scores, namely model of end-stage liver disease, Glasgow alcoholic hepatitis score, and discriminant function, and was independently associated with survival in multivariable analysis. These results were confirmed in a validation cohort. Transferrin did not correlate with markers of liver synthesis nor with non-transferrin-bound iron or soluble transferrin receptor (as markers of excess unbound iron and functional iron deficiency, respectively). Discussion In patients with sAH, serum transferrin predicts mortality with a performance comparable with commonly used composite scoring systems. Hence, this routinely available parameter might be a useful marker alone or as a component of prognostic models.

Background Keratins (Ks) represent tissue-specific proteins. K18 is produced in hepatocytes while K19, the most widely used ductular reaction (DR) marker, is found in cholangiocytes and hepatic progenitor cells. K18-based serum fragments are commonly used liver disease predictors, while K19-based serum fragments detected through CYFRA21-1 are established tumor but not liver disease markers yet. Since DR reflects the severity of the underlying liver disease, we systematically evaluated the usefulness of CYFRA21-1 in different liver disease severities and etiologies. Methods Hepatic expression of ductular keratins (K7/K19/K23) was analyzed in 57 patients with chronic liver disease (cohort i). Serum CYFRA21-1 levels were measured in 333 Austrians with advanced chronic liver disease (ACLD) of various etiologies undergoing hepatic venous pressure gradient (HVPG) measurement (cohort ii), 231 French patients with alcoholic cirrhosis (cohort iii), and 280 hospitalized Germans with decompensated cirrhosis of various etiologies (cohort iv). Results (i) Hepatic K19 levels were comparable among F0–F3 fibrosis stages, but increased in cirrhosis. Hepatic K19 mRNA strongly correlated with the levels of other DR-specific keratins. (ii) In ACLD, increased serum CYFRA21-1 associated with the presence of clinically significant portal hypertension (CSPH; HVPG ≥ 10 mmHg) (OR = 5.87 [2.95–11.68]) and mortality (HR = 3.02 [1.78–5.13]; median follow-up 22 months). (iii) In alcoholic cirrhosis, elevated serum CYFRA21-1 indicated increased risk of death/liver transplantation (HR = 2.59 [1.64–4.09]) and of HCC (HR = 1.74 [1.02–2.96]) over the long term (median follow-up 73 months). (iv) In decompensated cirrhosis, higher serum CYFRA21-1 predicted 90-day mortality (HR = 2.97 [1.92–4.60]) with a moderate accuracy (AUROC 0.64), independently from established prognostic scores. Conclusions Hepatic K19 mRNA and serum CYFRA21-1 levels rise in cirrhosis. Increased CYFRA21-1 levels associate with the presence of CSPH and reliably indicate mortality in the short and long term independently of conventional liver biochemistry markers or scoring systems. Hence, the widely available serum CYFRA21-1 constitutes a novel, DR-related marker with prognostic implications in patients with different settings of advanced liver disease.

Hepatocytes synthesise the majority of serum proteins. This production occurs in the endoplasmic reticulum (ER) and is adjusted by complex local and systemic regulatory mechanisms. Accordingly, serum levels of hepatocyte-made proteins constitute important biomarkers that reflect both systemic processes and the status of the liver. For example, C-reactive protein is an established marker of inflammatory reaction, whereas transferrin emerges as a liver stress marker and an attractive mortality predictor. The high protein flow through the ER poses a continuous challenge that is handled by a complex proteostatic network consisting of ER folding machinery, ER stress response, ER-associated degradation and autophagy. Various disorders disrupt this delicate balance and result in protein accumulation in the ER. These include chronic hepatitis B infection with overproduction of hepatitis B surface antigen or inherited alpha1-antitrypsin deficiency that give rise to ground glass hepatocytes and alpha1-antitrypsin aggregates, respectively. We review these ER storage disorders and their downstream consequences. The interaction between proteotoxic stress and other ER challenges such as lipotoxicity is also discussed. Collectively, this article aims to sharpen our view of liver hepatocytes as the central hubs of protein metabolism.

The presence of the homozygous &lsquo, Pi*Z&rsquo, variant of alpha-1 antitrypsin (AAT) (&lsquo, Pi*ZZ&rsquo, genotype) predisposes to liver fibrosis development, but the role of iron metabolism in this process remains unknown. Therefore, we assessed iron metabolism and variants in the Homeostatic Iron Regulator gene (HFE) as the major cause of hereditary iron overload in a large cohort of Pi*ZZ subjects without liver comorbidities. The human cohort comprised of 409 Pi*ZZ individuals and 254 subjects without evidence of an AAT mutation who were recruited from ten European countries. All underwent a comprehensive work-up and transient elastography to determine liver stiffness measurements (LSM). The corresponding mouse models (Pi*Z overexpressors, HFE knockouts, and double transgenic [DTg] mice) were used to evaluate the impact of mild iron overload on Pi*Z-induced liver injury. Compared to Pi*Z non-carriers, Pi*ZZ individuals had elevated serum iron, transferrin saturation, and ferritin levels, but relevant iron overload was rare. All these parameters were higher in individuals with signs of significant liver fibrosis (LSM &ge, 7.1 kPa) compared to those without signs of significant liver fibrosis. HFE knockout and DTg mice displayed similar extent of iron overload and of fibrosis. Loss of HFE did not alter the extent of AAT accumulation. In Pi*ZZ individuals, presence of HFE mutations was not associated with more severe liver fibrosis. Taken together, Pi*ZZ individuals display minor alterations in serum iron parameters. Neither mild iron overload seen in these individuals nor the presence of HFE mutations (C282Y and H63D) constitute a major contributor to liver fibrosis development.

ObjectiveHomozygous alpha1-antitrypsin (AAT) deficiency increases the risk for developing cirrhosis, whereas the relevance of heterozygous carriage remains unclear. Hence, we evaluated the impact of the two most relevant AAT variants (‘Pi*Z’ and ‘Pi*S’), present in up to 10% of Caucasians, on subjects with non-alcoholic fatty liver disease (NAFLD) or alcohol misuse.DesignWe analysed multicentric case–control cohorts consisting of 1184 people with biopsy-proven NAFLD and of 2462 people with chronic alcohol misuse, both cohorts comprising cases with cirrhosis and controls without cirrhosis. Genotyping for the Pi*Z and Pi*S variants was performed.ResultsThe Pi*Z variant presented in 13.8% of patients with cirrhotic NAFLD but only in 2.4% of counterparts without liver fibrosis (pConclusionThe Pi*Z variant is the hitherto strongest single nucleotide polymorphism-based risk factor for cirrhosis in NAFLD and alcohol misuse, whereas the Pi*S variant confers only a weak risk in alcohol misusers. As 2%–4% of Caucasians are Pi*Z carriers, this finding should be considered in genetic counselling of affected individuals.

In addition to carbon tetrachloride (CCl4), thioacetamide (TAA) represents a second widely used model for the induction of experimental liver fibrosis, but can also be employed for the development of acute liver failure and liver tumours. While TAA itself is not hepatotoxic, its reactive metabolites covalently bind to proteins and lipids thereby causing oxidative stress and centrilobular necrosis. Compared with CCl4, TAA leads to more periportal infiltrates and more pronounced ductal proliferation. While TAA has been shown to induce liver fibrosis development in several different mouse strains, wide variations in the administration routes, doses and treatment durations have been reported. Therefore, an adoption of a universal standard operating procedure for the administration of TAA is urgently needed. For that purpose, we are presenting here two TAA models (intraperitoneal administration of 150 mg/kg of TAA three times per week for 11 weeks in rats, and TAA administration in drinking water at 300 mg/L for 2–4 months in mice) with which we have had success in reliably and reproducibly developing chronic liver injury and fibrosis.

Background & Aims Alpha-1 antitrypsin deficiency (AATD) is among the most common genetic disorders. Severe AATD is caused by a homozygous mutation in the SERPINA1 gene that encodes the Glu342Lys substitution (called the Pi*Z mutation, Pi*ZZ genotype). Pi*ZZ carriers may develop lung and liver diseases. Mutation-associated lung disorders have been well studied, but less is known about the effects in liver. We assessed the liver disease burden and associated features in adults with this form of AATD. Methods We collected data from 554 Pi*ZZ adults (403 in an exploratory cohort, 151 in a confirmatory cohort), in 9 European countries, with AATD who were homozygous for the Pi*Z mutation, and 234 adults without the Pi*Z mutation (controls), all without pre-existing liver disease. We collected data on demographic parameters, comorbidities, lung- and liver-related health, and blood samples for laboratory analysis. Liver fibrosis was assessed non-invasively via the serum tests Aspartate Aminotransferase to Platelet Ratio Index and HepaScore and via transient elastography. Liver steatosis was determined via transient elastography-based controlled attenuation parameter. We performed histologic analyses of livers from transgenic mice that overexpress the AATD-associated Pi*Z variant. Results Serum levels of liver enzymes were significantly higher in Pi*ZZ carriers vs controls. Based on non-invasive tests for liver fibrosis, significant fibrosis was suspected in 20%–36% of Pi*ZZ carriers, whereas signs of advanced fibrosis were 9- to 20-fold more common in Pi*ZZ carriers compared to non-carriers. Male sex; age older than 50 years; increased levels of alanine aminotransferase, aspartate aminotransferase, or γ-glutamyl transferase; and low numbers of platelets were associated with higher liver fibrosis burden. We did not find evidence for a relationship between lung function and liver fibrosis. Controlled attenuation parameter ≥280 dB/m, suggesting severe steatosis, was detected in 39% of Pi*ZZ carriers vs 31% of controls. Carriers of Pi*ZZ had lower serum concentrations of triglyceride and low- and very-low-density lipoprotein cholesterol than controls, suggesting impaired hepatic secretion of lipid. Livers from Pi*Z-overexpressing mice had steatosis and down-regulation of genes involved in lipid secretion. Conclusions In studies of AATD adults with the Pi*ZZ mutation, and of Pi*Z-overexpressing mice, we found evidence of liver steatosis and impaired lipid secretion. We identified factors associated with significant liver fibrosis in patients, which could facilitate hepatologic assessment and counseling of individuals who carry the Pi*ZZ mutation. ClinicalTrials.gov Number NCT02929940 .

The intraperitoneal application of lipopolysaccharide (LPS) alone or in combination with other hepatotoxins is an experimental model for inducing systemic and hepatic inflammation in rodents applied worldwide. The endotoxin is recognized by the LPS-binding protein. This complex binds together with the lymphocyte antigen 96 (MD2) and the pattern-recognition receptor CD14 to members of the toll-like receptor family. The activated receptor complex in turn transduces signals to well characterized intracellular cascades that result in a multifaceted network of intracellular responses ending in inflammation. The most prominent among these is the activation of the NF-κB pathway and the production of a multitude of inflammatory cytokines. Although the application of LPS is in general easy to perform, unintended variations in preparation of the injection solution or in handling of the animals might affect the reproducibility or the outcome of a specific experiment. Here, we present a well-standardized protocol that allows for an induction of highly reproducible acute hepatic inflammation in mice. Furthermore, examples of appropriate readouts for the resulting inflammatory response are given.

The intravenous injection of the plant lectin concanavalin A (ConA) is a widely used model for acute immune-mediated hepatitis in mice. In contrast to several other models for acute hepatic damage, ConA-induced injury is primarily driven by the activation and recruitment of T cells to the liver. Hence, the ConA model has unique features with respect to its pathogenesis and important similarities to immune-mediated hepatitis in humans, such as autoimmune hepatitis, acute viral hepatitis or distinct entities of drug toxicity leading to immune activation. However, the ConA model has considerable variability, depending on the preparation of the compound, genetic background of the mice, sex, age and microbial environment of the animal facility barrier. This standard operating procedure (SOP) comprises a detailed protocol for the ConA application, including preparation of ConA working solution, handling of the animals, choice of the appropriate conditions and endpoints, as well as efficient dose-finding.

The chemokine receptor CXCR4 is crucial in neointima formation which is a problem after stent implantation. Drug-eluting stents reduce the restenosis risk but delayed re-endothelialization promotes late stent thrombosis. We compared CXCR4 antagonism with sirolimus treatment in preventing neointima formation after arterial injury in mice. Apolipoprotein E -/- mice after carotid wire injury were given a CXCR4 antagonist (POL5551 (P), a Protein Epitope Mimetic (PEM)) or sirolimus (n=6-8/group). The drugs were administered continuously (P: 2 and 20 mg/kg/d; sirolimus: 1.25 mg/kg/d; osmotic pumps) or intermittently (P: 20 mg/kg; IP, once daily) for 28 days. The neointimal area, smooth muscle cell (SMC) content, macrophage content, and endothelial coverage were quantified by planimetry from injured arteries stained with Elastica van Giesson, or after immunostaining for α-smooth muscle actin, Mac-2, and von Willebrand Factor, respectively. Peripheral Sca-1 + /Lin - smooth muscle progenitor cells (SPCs) were measured by flow cytometry. Treatment with sirolimus as well as with a continuous (C) or intermittent (I), high (H) or low (L) dose (D) of POL5551 (P) reduced the neointimal area compared to corresponding vehicle (Sirolimus: 69%, CHD-P: 70%, CLD-P: 63%, IHD-P: 53%). The relative neointimal content of macrophages increased by sirolimus application (70%). In contrast, the macrophage content decreased by CHD-P and IHD-P application (57% and 37%, respectively). However, CLD-P application did not alter the macrophage content. Sirolimus, CHD-P, and IHD-P highly diminished the neointimal SMC content (78%, 85%, and 67%, respectively), while CLD-P treatment did not. SPC increase in the circulation 1 day post injury was prevented by sirolimus, CHD-P, and IHD-P, but not by CLD-P. The endothelial coverage was not reduced by P, but significantly by sirolimus (14%). In conclusion, CXCR4 antagonism by POL5551 is equally effective as sirolimus in reducing neointima formation after arterial injury in mice. It might be more beneficial than sirolimus because it does not impair re-endothelialization and leads to a more stable lesional phenotype. These results indicate that POL5551 might be a promising alternative for restenosis prevention after stent implantation.