Eija Tukiainen

High Mobility Group Box 1 Protein as a Marker of Hepatocellular Injury in Human Liver Transplantation

High mobility group box 1 protein (HMGB1), a cytokine actively secreted by phagocytes and passively released from necrotic cells, is an inflammatory mediator in experimental hepatic ischemia/reperfusion injury. We characterized its expression in human liver transplantation. In 20 patients, in addition to systemic samples, blood was drawn from portal and hepatic veins during and after reperfusion to assess changes within the graft. Plasma HMGB1, tumor necrosis factor α (TNF‐α), and interleukin‐6 (IL‐6) levels were measured, and HMGB1 immunohistochemistry was performed on biopsies taken before and after reperfusion. Plasma HMGB1 was undetectable before reperfusion, and levels in systemic circulation peaked after graft reperfusion. At portal declamping, HMGB1 levels were substantially higher in the caval effluent [188 (80‐371) ng/mL] than in portal venous blood [0 (0‐3) ng/mL, P < 0.001]. HMGB1 release from the graft continued thereafter. HMGB1 levels were not related to TNF‐α or IL‐6 levels. HMGB1 expression was up‐regulated in biopsies taken after reperfusion (P = 0.020), with intense hepatocyte and weak neutrophil staining. HMGB1 levels in hepatic venous blood correlated with graft steatosis (r = 0.497, P = 0.03) and peak postoperative alanine aminotransferase levels (r = 0.588, P = 0.008). Our results indicate that HMGB1 originates from the graft and is a marker of hepatocellular injury in human liver transplantation. Liver Transpl 14:1517–1525, 2008.

Pancreatic Secretory Trypsin Inhibitor (SPINK1) Gene Mutations in Patients With Acute Pancreatitis

Objectives: Mutations in the secretory trypsin inhibitor (SPINK1) gene have been found to be associated with hereditary and chronic pancreatitis. There are no previous reports on SPINK1 mutations in patients with acute pancreatitis (AP). Methods: The study population consists of 371 patients with AP, of which 207 patients had mild and 164 had a severe form of the disease. The etiologies of AP were identified. Four hundred fifty-nine blood donors served as controls. SPINK1 N34S and P55S mutations were detected by minisequencing and confirmed by direct sequencing. Results: The N34S mutation was found in 29 (7.8%) of the patients and in 12 (2.6%) of the controls (P < 0.0001, Fisher exact test). There was no difference in the frequency of the P55SS mutation between the groups. A majority of the patients (n = 229; 61.7%) had alcohol-induced AP. The frequency of the N34S mutation was higher in the subgroups of severe AP (15/164; 9.1%) and alcohol-induced AP (21/229; 9.2%), but the differences were not statistically significant. No differences in age at admission and number of attacks of AP were observed between the groups. Conclusion: SPINK1 N34S mutation enhances the susceptibility of AP.

Acute liver failure after valproate exposure in patients with POLG1 mutations and the prognosis after liver transplantation.

Patients with mutations in the POLG1 gene encoding mitochondrial DNA polymerase gamma have an increased risk of valproate‐induced liver failure. POLG1 mutations are common, and these patients often suffer from intractable seizures. The role of liver transplantation in the treatment of patients with mitochondrial diseases has been controversial. We studied valproate‐induced liver failure associated with POLG1 mutations and the prognosis for these patients after liver transplantation. POLG1 was analyzed in blood DNA, mitochondrial DNA (mtDNA) was quantified in liver samples, and clinical data were collected. Five patients with valproate‐induced liver failure associated with POLG1 mutations were retrospectively identified. Three patients were previously suspected to have Wilsons disease. Four patients with homozygous p.W748S and p.E1143G mutations had mtDNA depletion in the liver. One of these patients died before anticipated transplantation; the other 3 patients with liver transplantation have survived 4 to 19 years. Two patients have presented with occasional epileptic seizures, and 1 patient has been seizure‐free for 11 years. One patient with a heterozygous p.Q1236H mutation (but without mtDNA depletion in the liver) died suddenly 2 years after liver transplantation. In conclusion, the POLG1 mutation status and the age at presentation of valproate‐induced liver failure can affect the prognosis after liver transplantation. A heterozygous POLG1 p.Q1236H mutation was related to valproate‐induced liver failure without mtDNA depletion, whereas patients homozygous for POLG1 p.W748S and p.E1143G mutations had mtDNA depletion. An analysis of the POLG1 gene should be performed for all patients with suspected mitochondrial disease before the introduction of valproate therapy, and treatment with valproic acid should be avoided in these patients. Liver Transpl 20:1402–1412, 2014.

Complement activation during liver transplantation-special emphasis on patients with atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy often caused by mutations in complement factor H (CFH), the main regulator of alternative complement pathway. Because CFH is produced mainly by the liver, combined liver–kidney transplantation is a reasonable option in treatment of patients with severe aHUS. We studied complement activation by monitoring activation markers during liver transplantation in two aHUS patients treated extensively with plasma exchange and nine other liver transplantation patients. After the reperfusion, a clear increase in all the activation markers except C4d was observed indicating that the activation occurs mainly through the alternative pathway. Concentration of SC5b‐9 was higher in the hepatic than the portal vein indicating complement activation in the graft. Preoperatively and early during the operation, the aHUS patients showed highest C3d concentrations but otherwise their activation markers were similar to the other patients. In the other patients, correlation was found between perioperative SC5b‐9 concentration and postoperative alanine aminotransferase and histological changes. This study explains why supply of normal CFH by extensive plasma exchange is beneficial before combined liver–kidney transplantation of aHUS patients. Also the results suggest that perioperative inhibition of the terminal complement cascade might be beneficial if enhanced complement activation is expected.

Circulating levels of a soluble form of receptor for advanced glycation end products and high-mobility group box chromosomal protein 1 in patients with acute pancreatitis.

Objectives: To study in patients with acute pancreatitis (AP) the plasma soluble form of the receptor for advanced glycation end products (sRAGE) and high-mobility group box chromosomal protein 1 (HMGB1) levels, followed-up for 12 days after hospitalization, in relation to the occurrence of organ failure and mortality. Methods: Thirty-eight patients with severe AP and organ failure (grade 2). A control group (127 patients) consisted of 38 patients with severe AP without organ failure (grade 1) and 89 patients with mild AP (grade 0). Plasma samples for determination of HMGB1 and sRAGE levels were collected on admission and on days 1 and 2, days 3 and 4, and days 7 and 12 after admission. Results: The median of the highest sRAGE levels was higher in grade 2 patients (472 pg/mL; interquartile range [IQR], 259-912) than in grade 0 plus grade 1 patients (349 pg/mL; IQR, 209-544; P = 0.024). Among the patients with detectable HMGB1, the median of the highest HMGB1 levels was 117 ng/mL (IQR, 56-212; n = 24) in grade 2 patients and 87 ng/mL (IQR, 54-161; n = 62) in grade 0 plus grade 1 patients (P = 0.310). Conclusions: We demonstrate that sRAGE level, but not HMGB1 level, is significantly higher in AP patients who develop organ failure than in AP patients without organ failure who recover.

Polymorphisms of the Tnf, Cd14, and Hspa1b Genes in Patients With Acute Alcohol-induced Pancreatitis

Objectives: Genotype assessment has been suggested to be a tool for predicting disease severity in acute pancreatitis (AP). To study this hypothesis, we performed genotype analysis of tumor necrosis factor (TNF) −308 A/G, CD14 −159C/T, and HSPA1B +1267 A/G polymorphisms. Methods: This is a case-control association study of 397 patients with AP (214 of whom had an alcohol-induced AP) and 300 controls. The control group comprised 218 subjects with detailed data of alcohol consumption, 70 of whom were heavy drinkers (daily alcohol intake >40 g), and 92 blood donors. The severity of AP was determined according to the Atlanta classification. Genotyping was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-assisted genotyping method. Results: Major allele frequency in TNF gene was 0.87 for patients with AP and 0.86 for controls. For CD14, the gene major allele frequency was 0.60 for patients and 0.63 for controls. For HSPA1B, the major allele frequencies were 0.52 for patients and 0.49 for controls, respectively. The allele frequencies did not differ significantly between AP patients with organ failure and those with mild disease, patients with alcohol-induced AP, or those with biliary AP. The patients with septic infectious complications (n = 47) had genotype distribution no different from those with mild, uncomplicated disease (n = 245). Conclusions: The TNF, CD14, and HSPA1B polymorphisms studied seem not to play a role in determining the severity of AP or the risk of alcohol-induced AP and thus do not serve as a tool for predicting disease severity.

Disialotransferrin, determined by capillary electrophoresis, is an accurate biomarker for alcoholic cause of acute pancreatitis.

Objectives: Serum disialotransferrin is a specific marker of heavy alcohol consumption. We tested its accuracy and probability in detecting alcoholic cause of acute pancreatitis (AP). Methods: Blood samples from 271 consecutive AP patients, admitted to the Helsinki University Central Hospital emergency unit, were analyzed. Results: The median (range) disialotransferrin value was significantly higher (P = 0.001) in AP patients with alcoholic (n = 172) 1.6% (0.3%-14.4) than with biliary (n = 60) 0.7% (0.3%-1.3%) or other causes (n = 39) 0.8% (0.3%-4.1%). In receiver operating curve analysis, disialotransferrin, as a single analyte, was significantly (P = 0.001-0.0001) more accurate (area under the curve [AUC], 0.88; 95% confidence interval [CI], 0.84-0.92) in detecting alcoholic AP as compared with glutamyl transferase (AUC, 0.51; 95% CI, 0.45-0.57), aspartate aminotransferase (AUC, 0.57; 95% CI, 0.51-0.63), alanine aminotransferase (AUC, 0.63; 95% CI, 0.57-0.69), erythrocyte mean cell volume (AUC, 0.72; 95% CI, 0.67-0.78), amylase (AUC, 0.74; 95% CI, 0.67-0.78), C-reactive protein (AUC, 0.65; 95% CI, 0.59-0.71), and bilirubin (AUC, 0.55; 95% CI, 0.49-0.62). At a disialotransferrin cutoff of 1.2%, giving an 8% false-positive rate, the positive likelihood ratio was 8.47. Thus, a positive disialotransferrin test result, performed within 24 hours of admission, increased the probability of alcoholic AP from pretest 64% to posttest 94%.Abbreviations: AP - acute pancreatitis, DST - disialotransferrin, CDT - carbohydrate-deficient transferrin, CE - capillary electrophoresis, CI - confidence interval, CV - coefficient of variation, ERCP - endoscopic retrograde cholangiopancreaticography, HPLC - high-performance liquid chromatography, LR - likelihood ratio, NPV - negative predictive value, PPV - positive predictive value Conclusions: Disialotransferrin, determined by capillary electrophoresis, is accurate, simple, and a rapid single biomarker of the alcoholic cause of AP.

Relative Quantification of Several Plasma Proteins during Liver Transplantation Surgery

Plasma proteome is widely used in studying changes occurring in human body during disease or other disturbances. Immunological methods are commonly used in such studies. In recent years, mass spectrometry has gained popularity in high-throughput analysis of plasma proteins. In this study, we tested whether mass spectrometry and iTRAQ-based protein quantification might be used in proteomic analysis of human plasma during liver transplantation surgery to characterize changes in protein abundances occurring during early graft reperfusion. We sampled blood from systemic circulation as well as blood entering and exiting the liver. After immunodepletion of six high-abundant plasma proteins, trypsin digestion, iTRAQ labeling, and cation-exchange fractionation, the peptides were analyzed by reverse phase nano-LC-MS/MS. In total, 72 proteins were identified of which 31 could be quantified in all patient specimens collected. Of these 31 proteins, ten, mostly medium-to-high abundance plasma proteins with a concentration range of 50–2000 mg/L, displayed relative abundance change of more than 10%. The changes in protein abundance observed in this study allow further research on the role of several proteins in ischemia-reperfusion injury during liver transplantation and possibly in other surgery.

Thrombin Generation in vitro and in vivo, and Disturbed Tissue Factor Regulation in Patients with Acute Pancreatitis

Background: Being a central link between inflammation and coagulation, tissue factor (TF) and its inhibitor (TFPI) might be associated with the severity of acute pancreatitis (AP) and the development of organ failure (OF). Methods: The study comprises 9 severe AP patients with OF and 24 reference patients (11 mild AP and 13 severe AP without OF). Plasma samples were collected on admission. TF-induced thrombin generation in plasma samples was studied using the thrombogram method. In vivo thrombin generation was estimated by prothrombin fragment F1+2. Free and total TFPI levels were measured. To evaluate coagulation status the activated partial thromboplastin time, prothrombin time, platelet count, D-dimer, fibrinogen, antithrombin (AT) 3 and protein C (PC) were determined. Results: There was no significant difference in F1+2 levels between the patient groups. Patients with severe AP tended to show low platelet counts, PC and AT3 levels, and high D-dimer levels. In 11 patients the standard TF stimulation did not trigger thrombin generation in the thrombogram. All deaths occurred in these patients. Free TFPI levels and free/total TFPI ratios were significantly higher in these patients and in non-survivors. Conclusion: Failure of TF-initiated thrombin generation in the thrombogram assay explained by high levels of circulating free TFPI may be associated with OF and mortality in AP.

Hemostatic gene polymorphisms in severe acute pancreatitis.

Objective: Systemic inflammatory reaction in acute pancreatitis (AP) is associated with activation of the coagulation system. The prothrombotic component of the coagulation system, which may promote microvascular thrombosis and vital organ injury, is strengthened by genetic factors such as polymorphism of plasminogen activator inhibitor type 1 (PAI-1) and factor V Leiden (FVL) mutation. This prompted us to study the occurrence of FVL and PAI-1 4G/5G polymorphisms in patients with AP. Methods: This case control association study included 397 patients with AP and 310 controls. Severe AP was determined according to the Atlanta Classification. Genotyping was performed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry-assisted genotyping method. Results: Factor V Leiden was identified in 5 (3.3%) of 152 cases of severe AP and in 8 (3.3%) of 245 cases of mild AP. The prothrombotic PAI-1 4G allele frequency was 0.49 for patients with severe AP and 0.57 for patients with mild AP (P < 0.05). Patients with septic infectious complications (n = 47) and patients with organ failure (n = 55) had genotype distribution not different from those with mild, uncomplicated disease (n = 245). Conclusions: The results do not support the hypothesis that prothrombotic polymorphisms such as FVL mutation and PAI-1 4G/5G are associated with AP severity.