Establishment of trypsinogen-2 Amplification Luminescent Proximity Homogeneous Assay and its Application in Acute Pancreatitis.

We aim to develop an amplified luminescence proximity homogeneous assay (AlphaLISA) for quantification of trypsinogen-2 levels in human serum for the diagnosis of acute pancreatitis. Based on new amplified luminescence proximity homogeneity assay (AlphaLISA) method, carboxyl-modified donor and acceptor beads were coupled to capture and detection antibodies. A double antibody sandwich immunoassay was used to detect the concentration of trypsinogen-2 in serum. The method had good linearity (> 0.998). The intra - analysis precision was between 1.54% and 2.20% (< 10%), the inter-analysis precision was between 3.17% and 6.94% (< 15%), and the recovery was between 96.23% and 103.45%. The cross-reactivity of carbohydrate antigen 242 (CA242) and T-cell immunoglobulin mucin-3 (Tim-3) were 0.09% and 0.93%, respectively. The detection time only needed 15 min. The results of trypsinogen-2-AlphaLISA and time-resolved fluorescence immunoassay were consistent (ρ = 0.9019). In addition, serum trypsinogen-2 concentration in patients with acute pancreatitis [239.23 (17.83-807.58) ng/mL] was significantly higher than that in healthy controls [20.54 (12.10-39.73) ng/mL]. When the cut-off value was 35.38ng/mL, the sensitivity and specificity were 91.8% and 96.67%, and the positive detection rate was 91.80%. We have successfully established a trypsinogen-2-AlphaLISA method, which can promote the timely diagnosis of acute pancreatitis.

Noninvasive rapid urinary trypsinogen-2 dipstick test for early exclusion of post-endoscopic retrograde cholangiopancreatography pancreatitis within hours after endoscopic retrograde cholangiopancreatography: Clinical diagnosis and considerations.

Objective: Few reports have explored the application of urinary trypsinogen-2 measurement in the early diagnosis of post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis, and none have demonstrated the benefits of noninvasive testing. This study aimed to evaluate the clinical application of the rapid urinary trypsinogen-2 dipstick test (Nipro, Japan) compared with serum amylase and lipase levels for the early diagnosis of post-ERCP pancreatitis (PEP). Methods: A total of 100 consecutive patients (54 men and 46 women) who were admitted and underwent ERCP at Tokyo Medical University Hospital from August 2021 to December 2021 were recruited. All patients underwent rapid urinary trypsinogen-2 measurement using the dipstick test before and after ERCP. Measurements were taken 24 h before ERCP for pre-ERCP and 1-4 h after ERCP for post-ERCP. Additionally, serum amylase and lipase levels were measured at 8:00 a.m. of the day after ERCP (at least 8 h after ERCP), and their diagnostic abilities for PEP were compared and evaluated. Results: PEP occurred in 5/100 patients (5%). The sensitivity, specificity, positive predictive value, and negative predictive value of the dipstick test for diagnosing PEP were 100%, 83.2%, 23.8%, and 100%, respectively. These results were comparable to the diagnostic performance of serum amylase and lipase levels at 8:00 a.m. on the day after ERCP (at least 8 h after ERCP). However, false positives must be considered. Conclusion: The dipstick test may be useful in clinical practice as a noninvasive screening test for the early prediction of PEP.

Establishment and Clinical Application of a Highly Sensitive Time-Resolved Fluorescence Immunoassay for Tumor-Associated Trypsinogen-2.

To establish a rapid and highly sensitive assay for tumor-associated trypsinogen-2 (TAT-2) based on the time-resolved fluorescence immunoassay (TRFIA) and evaluate its potential clinical value in patients with lung cancer. The double-antibody sandwich method was used in detecting TAT-2 antigen concentrations, and two types of TAT-2 antibodies (coating antibodies and Eu3+ labeled antibodies) were used. A TAT-2-TRFIA method was then established, evaluated, and used in detecting the serum TAT-2 levels of healthy subjects and patients with lung cancer. The linear range of the TAT-2-TRFIA method was 1.53-300 ng/mL, the intra-assay coefficient of variation (CV) were between 1.67% and 8.42%, and the inter-assay CV were between 4.29% and 11.44%. The recovery rates of TAT-2-TRFIA were between 99.17% and 107.06%. The cross-reactivities of trypsin and T-cell immunoglobulin mucin 3 were 0.02% and 0.82%, respectively. The serum TAT-2 levels of patients with lung cancer were higher than those of healthy subjects (P < 0.001). Combined with TAT-2, the sensitivity and specificity of CEA and CA-125 for lung cancer improved significantly. Conclusion: We successfully established a highly sensitive TAT-2-TRFIA method, which was able to facilitate the timely diagnosis of lung cancer.

Effects of endoscopic submucosal dissection on post-operative early treatment effectiveness and serum TAT-2 and GP73 expression levels in patients with early gastric cancer.

The present study aimed to explore the effectiveness of endoscopic submucosal dissection (ESD) in the treatment of early gastric cancer (EGC) and its effect on serum tumor-associated trypsin-2 (TAT-2) and Golgi protein 73 (GP73) expression levels to provide a reference for the treatment of EGC. TAT-2 is a proteolytic target enzyme for tumor-associated trypsin inhibitor that has been previously reported to enhance invasion by promoting extracellular matrix degradation. GP73 is a novel type II Golgi membrane protein of unknown function that is expressed in the hepatocytes of patients with adult giant-cell hepatitis. A total of 161 patients with EGC treated at our hospital from April 2013 to February 2014 were selected as the study subjects. Among these, 86 patients underwent ESD (group A) and the remaining 75 underwent endoscopic mucosal resection (group B). Treatment effectiveness, incidence of complications and adverse reactions, operation time, intraoperative blood loss and length of hospital stay, as well as serum TAT-2 and GP73 expression levels, were compared between the two groups. The treatment effectiveness was significantly higher in group A than in group B (P<0.05). However, there was no significant inter-group difference in terms of incidence of complications/adverse reactions (P>0.05). After treatment, serum TAT-2 expression levels decreased in both groups (P<0.05) and serum TAT-2 expression levels were lower in group A than in group B (P<0.05). Furthermore, serum GP73 expression levels were significantly elevated in both groups (P<0.05). Kaplan-Meier survival analysis indicated no significant inter-group difference in the 5-year survival rate (P>0.05). In conclusion, ESD had a good therapeutic effect on EGC and is able to decrease serum TAT-2 expression levels and increase serum GP73 expression levels. The present study was registered into the Chinese Trials Registry (registration no. NCT02157534).

Urinary trypsinogen-2 level and local complications of acute pancreatitis.

BACKGROUND AND AIM: Local complications of acute pancreatitis (AP) carry risks of morbidity/mortality. This study aimed to assess whether urinary trypsinogen-2 levels and Bedside Index for Severity in Acute Pancreatitis (BISAP) score on admission predicted subsequent local complications. METHODS: One hundred and forty-four consecutive patients with AP were prospectively followed till 6 months after discharge. Urinary trypsinogen-2 levels were measured within 24 h of admission. Local complications (acute peripancreatic fluid collection, acute necrotic collection, pseudocyst, and walled-off necrosis) were diagnosed by abdominal computed tomography. Cut-off for trypsinogen-2 level was assessed using receiver operating characteristic curve, and predictors of local complications were analyzed by logistic regression. RESULTS: Thirty-seven (25.7%) patients developed local complications. Urinary trypsinogen-2 levels were significantly higher in patients with local complications compared with those without local complications (median [interquartile range], 3210 [620-9764.4] µg/L vs 627.3 [72.3-5895] µg/L, P = 0.006). Urinary trypsinogen-2 significantly outperformed BISAP score in predicting local complications (area under the receiver operating characteristic curve 0.65 [95% CI: 0.55-0.75] vs 0.48 [95% CI: 0.38-0.58], P = 0.005). At the optimal cut-off of 500 µg/L, the sensitivity, specificity, positive predictive value, and negative predictive value of trypsinogen-2 level were 78.4%, 45.8%, 33.3%, and 86.0%, respectively. Urinary trypsinogen-2 level > 500 µg/L was an independent predictor of local complications (adjusted odds ratio, 3.72; 95% CI: 1.42-9.76; P = 0.007). By contrast, BISAP score ≥ 3 and pleural effusion predicted organ failure but not local complications. CONCLUSION: In a prospective cohort, urinary trypsinogen-2 level > 500 µg/L independently predicted local complications of AP.

Usefulness of urinary trypsinogen-2 and trypsinogen activation peptide in acute pancreatitis: A multicenter study in Japan.

BACKGROUND: Rapid urinary trypsinogen-2 dipstick test and levels of urinary trypsinogen-2 and trypsinogen activation peptide (TAP) concentration have been reported as prognostic markers for the diagnosis of acute pancreatitis. AIM: To reconfirm the validity of all these markers in the diagnosis of acute pancreatitis by undertaking a multi-center study in Japan. METHODS: Patients with acute abdominal pain were recruited from 17 medical institutions in Japan from April 2009 to December 2012. Urinary and serum samples were collected twice, at enrollment and on the following day for measuring target markers. The diagnosis and severity assessment of acute pancreatitis were assessed based on prognostic factors and computed tomography (CT) Grade of the Japanese Ministry of Health, Labour, and Welfare criteria. RESULTS: A total of 94 patients were enrolled during the study period. The trypsinogen-2 dipstick test was positive in 57 of 78 patients with acute pancreatitis (sensitivity, 73.1%) and in 6 of 16 patients with abdominal pain but without any evidence of acute pancreatitis (specificity, 62.5%). The area under the curve (AUC) score of urinary trypsinogen-2 according to prognostic factors was 0.704, which was highest in all parameter. The AUC scores of urinary trypsinogen-2 and TAP according to CT Grade were 0.701 and 0.692, respectively, which shows higher than other pancreatic enzymes. The levels of urinary trypsinogen-2 and TAP were significantly higher in patients with extended extra-pancreatic inflammation as evaluated by CT Grade. CONCLUSION: We reconfirmed urinary trypsinogen-2 dipstick test is useful as a marker for the diagnosis of acute pancreatitis. Urinary trypsinogen-2 and TAP may be considered as useful markers to determine extra-pancreatic inflammation in acute pancreatitis.

Molecular functions of the iron-regulated metastasis suppressor, NDRG1, and its potential as a molecular target for cancer therapy.

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.

Endoscopic retrograde cholangiopancreatography with rendezvous cannulation reduces pancreatic injury.

AIM: To examine whether rendezvous endoscopic retrograde cholangiopancreatography (ERCP) is associated with less pancreatic damage, measured as leakage of proenzymes, than conventional ERCP. METHODS: Patients (n = 122) with symptomatic gallstone disease, intact papilla and no ongoing inflammation, were prospectively enrolled in this case-control designed study. Eighty-one patients were subjected to laparoscopic cholecystectomy and if intraoperative cholangiography suggested common bile duct stones (CBDS), rendezvous ERCP was performed intraoperatively (n = 40). Patients with a negative cholangiogram constituted the control group (n = 41). Another 41 patients with CBDS, not subjected to surgery, underwent conventional ERCP. Pancreatic proenzymes, procarboxypeptidase B and trypsinogen-2 levels in plasma, were analysed at 0, 4, 8 and 24 h. The proenzymes were determined in-house with a double-antibody enzyme linked immunosorbent assay. Pancreatic amylase was measured by an enzymatic colourimetric modular analyser with the manufacturer's reagents. All samples were blinded at analysis. RESULTS: Post ERCP pancreatitis (PEP) occurred in 3/41 (7%) of the patients cannulated with conventional ERCP and none in the rendezvous group. Increased serum levels indicating pancreatic leakage were significantly higher in the conventional ERCP group compared with the rendezvous ERCP group regarding pancreatic amylase levels in the 4- and 8-h samples (P = 0.0015; P = 0.03), procarboxypeptidase B in the 4- and 8-h samples (P < 0.0001; P < 0.0001) and trypsinogen-2 in the 24-hour samples (P = 0.03). No differences in these markers were observed in patients treated with rendezvous cannulation technique compared with patients that underwent cholecystectomy alone (control group). Post procedural concentrations of pancreatic amylase and procarboxypeptidase B were significantly correlated with pancreatic duct cannulation and opacification. CONCLUSION: Rendezvous ERCP reduces pancreatic enzyme leakage compared with conventional ERCP cannulation technique. Thus, laparo-endoscopic technique can be recommended with the ambition to minimise the risk for post ERCP pancreatitis.