Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

A simple and effective genotyping workflow for rapid detection of CRISPR genome editing.

Genetically engineered mouse models play a pivotal role in the modeling of diseases, exploration of gene functions, and the development of novel therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing technology has revolutionized the process of developing such models by enabling precise genome modifications of the multiple interested genes simultaneously. Following genome editing, an efficient genotyping methodology is crucial for subsequent characterization. However, current genotyping methods are laborious, time-consuming, and costly. Here, using targeting the mouse trypsinogen genes as an example, we introduced common applications of CRISPR-Cas9 editing and a streamlined cost-effective genotyping workflow for CRISPR-edited mouse models, in which Sanger sequencing is required only at the initial steps. In the F0 mice, we focused on identifying the presence of positive editing by PCR followed by Sanger sequencing without the need to know the exact sequences, simplifying the initial screening. In the F1 mice, Sanger sequencing and algorithms decoding were used to identify the precise editing. Once the edited sequence was established, a simple and effective genotyping strategy was established to distinguish homozygous and heterozygous status by PCR from tail DNA. The genotyping workflow applies to deletions as small as one nucleotide, multiple-gene knockout, and knockin studies. This simplified, efficient, and cost-effective genotyping shall be instructive to new investigators who are unfamiliar with characterizing CRISPR-Cas9-edited mouse strains.NEW & NOTEWORTHY This study presents a streamlined, cost-effective genotyping workflow for clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) edited mouse models, focusing on trypsinogen genes. It simplifies initial F0 mouse screening using PCR and Sanger sequencing without needing exact sequences. For F1 mice, precise editing is identified through Sanger sequencing and algorithm decoding. The workflow includes a novel PCR strategy for distinguishing homozygous and heterozygous statuses in subsequent generations, effective for small deletions, multiple-gene knockouts, and knockins.

Secretagogue-induced pancreatitis in mice devoid of chymotrypsin.

The serine protease chymotrypsin protects the pancreas against pancreatitis by degrading trypsinogen, the precursor to the digestive protease trypsin. Taking advantage of previously generated mouse models with either the Ctrb1 gene (encoding chymotrypsin B1) or the Ctrl gene (encoding chymotrypsin-like protease) disrupted, here we generated the novel Ctrb1-del × Ctrl-KO strain in the C57BL/6N genetic background, which harbors a naturally inactivated Ctrc gene (encoding chymotrypsin C). The newly created mice are devoid of chymotrypsin, yet the animals develop normally, breed well, and show no spontaneous phenotype, indicating that chymotrypsin is dispensable under laboratory conditions. When given cerulein, the Ctrb1-del × Ctrl-KO strain exhibited markedly increased intrapancreatic trypsin activation and more severe acute pancreatitis, relative to wild-type C57BL/6N mice. After the acute episode, Ctrb1-del × Ctrl-KO mice spontaneously progressed to chronic pancreatitis, whereas C57BL/6N mice recovered rapidly. The cerulein-induced pancreas pathology in Ctrb1-del × Ctrl-KO mice was highly similar to that previously observed in Ctrb1-del mice; however, trypsin activation was more robust and pancreatitis severity was increased. Taken together, the results confirm and extend prior observations demonstrating that chymotrypsin safeguards the pancreas against pancreatitis by limiting pathologic trypsin activity. In mice, the CTRB1 isoform, which constitutes about 90% of the total chymotrypsin content, is responsible primarily for the anti-trypsin defenses and protection against pancreatitis; however, the minor isoform CTRL also contributes to an appreciable extent.NEW & NOTEWORTHY Chymotrypsins defend the pancreas against the inflammatory disorder pancreatitis by degrading harmful trypsinogen. This study demonstrates that mice devoid of pancreatic chymotrypsins are phenotypically normal but become sensitized to secretagogue hyperstimulation and exhibit increased intrapancreatic trypsin activation, more severe acute pancreatitis, and rapid progression to chronic pancreatitis. The observations confirm and extend the essential role of chymotrypsins in pancreas health.

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.

Why cystic fibrosis newborn screening programs have failed to meet original expectations thus far.

This Commentary summarizes what the author has learned in 46 years of research on newborn screening (NBS) for cystic fibrosis (CF) combined with healthcare and public health practice. The original expectation was that screening for this relatively common, life-threatening genetic disorder would lead to consistently timely diagnoses in the neonatal period and be equitable. Unfortunately, this ambitious goal has not been achieved in the USA despite the availability of an excellent, although imperfect, 2-tiered screening test employing immunoreactive trypsinogen (IRT) and DNA analysis for pathogenic variants in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR). In fact, variations in the quality of NBS programs, inconsistencies in their operations, and disparities in outcomes have been prominent features. The causes include leadership challenges and deficiencies among both CF centers and NBS labs; failures to form effective partnerships among CF centers and with NBS programs; relatively rapid implementation after 2005 with variable quality planning; misunderstandings and erroneous dogma about CF; data limitations regarding IRT, especially cutoff values, and CFTR genetics; tolerance of suboptimal protocols and false negative results; problems in dried blood spot collections plus a lack of transparency and national oversight; partial lack of readiness, qualifications, funding and/or willingness to innovate with floating IRT cutoffs and DNA/CFTR analyses; follow up challenges/deficiencies impairing timeliness, including sweat testing limitations; and published guidelines that are more descriptive than sufficiently critical and directive. But the lessons learned through uniquely intensive CF NBS research have been enlightening and guided the U.S. Cystic Fibrosis Foundation to nationwide quality improvement initiatives.

Mouse model of PRSS1 p.R122H-related hereditary pancreatitis highlights context-dependent effect of autolysis-site mutation.

Mutation p.R122H in human cationic trypsinogen (PRSS1) is the most frequently identified cause of hereditary pancreatitis. The mutation blocks protective degradation of trypsinogen by chymotrypsin C (CTRC), which involves an obligatory trypsin-mediated cleavage at Arg122. Previously, we found that C57BL/6N mice are naturally deficient in CTRC, and trypsinogen degradation is catalyzed by chymotrypsin B1 (CTRB1). Here, we used biochemical experiments to demonstrate that the cognate p.R123H mutation in mouse cationic trypsinogen (isoform T7) only partially prevented CTRB1-mediated degradation. We generated a novel C57BL/6N mouse strain harboring the p.R123H mutation in the native T7 trypsinogen locus. T7R123H mice developed no spontaneous pancreatitis, and severity parameters of cerulein-induced pancreatitis trended only slightly higher than those of C57BL/6N mice. However, when treated with cerulein for 2 days, more edema and higher trypsin activity was seen in the pancreas of T7R123H mice compared to C57BL/6N controls. Furthermore, about 40% of T7R123H mice progressed to atrophic pancreatitis in 3 days, whereas C57BL/6N animals showed full histological recovery. Taken together, the observations indicate that mutation p.R123H inefficiently blocks chymotrypsin-mediated degradation of mouse cationic trypsinogen, and modestly increases cerulein-induced intrapancreatic trypsin activity and pancreatitis severity. The findings support the notion that the pathogenic effect of the PRSS1 p.R122H mutation in hereditary pancreatitis is dependent on its ability to defuse chymotrypsin-dependent defenses.

Classification of PRSS1 variants responsible for chronic pancreatitis: An expert perspective from the Franco-Chinese GREPAN study group.

BACKGROUND: PRSS1 was the first reported chronic pancreatitis (CP) gene. The existence of both gain-of-function (GoF) and gain-of-proteotoxicity (GoP) pathological PRSS1 variants, together with the fact that PRSS1 variants have been identified in CP subtypes spanning the range from monogenic to multifactorial, has made the classification of PRSS1 variants very challenging. METHODS: All currently reported PRSS1 variants (derived primarily from two databases) were manually reviewed with respect to their clinical genetics, functional analysis and population allele frequency. They were classified by variant type and pathological mechanism within the framework of our recently proposed ACMG/AMP guidelines-based seven-category system. RESULTS: The total number of distinct germline PRSS1 variants included for analysis was 100, comprising 3 copy number variants (CNVs), 12 5' and 3' variants, 19 intronic variants, 5 nonsense variants, 1 frameshift deletion variant, 6 synonymous variants, 1 in-frame duplication, 3 gene conversions and 50 missense variants. Based upon a combination of clinical genetic and functional analysis, population data and in silico analysis, we classified 26 variants (all 3 CNVs, the in-frame duplication, all 3 gene conversions and 19 missense) as "pathogenic", 3 variants (missense) as "likely pathogenic", 5 variants (four missense and one promoter) as "predisposing", 13 variants (all missense) as "unknown significance", 2 variants (missense) as "likely benign", and all remaining 51 variants as "benign". CONCLUSIONS: We describe an expert classification of the 100 PRSS1 variants reported to date. The results have immediate implications for reclassifying many ClinVar-registered PRSS1 variants as well as providing optimal guidelines/standards for reporting PRSS1 variants.

Evaluation of specificity and sensitivity of IRT/IRT protocol in the cystic fibrosis newborn screening program: 6-year experience of three tertiary centers.

We aimed to evaluate cutoff values of immunoreactive trypsinogen (IRT)/IRT and determine relationship between IRT values and clinical characteristics of children with cystic fibrosis (CF). This study is cross-sectional study. Data of children with positive newborn screening (NBS) between 2015 and 2021 were evaluated in three pediatric pulmonology centers. Age at admission, sex, gestational age, presence of history of meconium ileus, parental consanguinity, sibling with CF, and doll-like face appearance, first and second IRT values, sweat chloride test, fecal elastase, fecal fat, biochemistry results, and age at CF diagnosis were recorded. Sensitivity and specificity of IRT cutoff values were evaluated. Of 815 children with positive NBS, 58 (7.1%) children were diagnosed with CF. Median values of first and second IRT were 157.2 (103.7-247.6) and 113.0 (84.0-201.5) µg/L. IRT values used in current protocol, sensitivity was determined as 96.6%, specificity as 17.2% for first IRT, and 96.6% sensitivity, 20.5% specificity for second IRT. Positive predictive value (PPV) was determined as 7.1%. When cutoff value for first IRT was estimated as 116.7 µg/L, sensitivity was 69.0% and specificity was 69.6%, and when cutoff value was set to 88.7 µg/L for second IRT, sensitivity was 69.0% and specificity was 69.0%. Area under curve was 0.757 for first and 0.763 for second IRT (p < 0.001, p < 0.001, respectively). PPV was calculated as 4.3%.    Conclusion: Although sensitivity of CF NBS is high in our country, its PPV is significantly lower than expected from CF NBS programs. False-positive NBS results could have been overcome by revising NBS strategy. What is Known: • Although immunoreactive trypsinogen elevation is a sensitive test used in cystic fibrosis newborn screening, its specificity is low. • In countries although different algorithms are used, all strategies begin with the measurement of immunoreactive trypsinogen in dried blood spots. What is New: • In our study, it was shown that use of the IRT/IRT protocol for cystic fibrosis newborn screening is not sufficient for the cut-off values determined by the high number of patients. • Newborn screening strategy should be reviewed to reduce false positive newborn screening results.

Plasma biomarkers TAP, CPA1, and CPA2 for the detection of pancreatic injury in rat: the development of a novel multiplex IA-LC-MS/MS assay and biomarker performance evaluation.

Drug-induced pancreatic injury (DIPI) is an issue seen in drug development both in nonclinical and clinical contexts. DIPI is typically monitored by measurement of lipase and/or amylase, however, both enzymes lack sensitivity and specificity. Although candidate protein biomarkers specific to pancreas exist, antibody-based assay development is difficult due to their small size or the rapid cleavage by proteolytic enzymes released during pancreatic injury. Here we report the development of a novel multiplexed immunoaffinity-based liquid chromatography mass spectrometric assay (IA-LC-MS/MS) for trypsinogen activation peptide (TAP) and carboxypeptidases A1 and A2 (CPA1, CPA2). This method is based on the enzymatic digestion of the target proteins, immunoprecipitation of the peptides with specific antibodies and LC-MS/MS analysis. This assay was used to detect TAP, CPA1, and CPA2 in 470 plasma samples collected from 9 in-vivo rat studies with pancreatic injury and 8 specificity studies with injury in other organs to assess their performance in monitoring exocrine pancreas injury. The TAP, CPA1, and CPA2 response was compared to histopathology, lipase, amylase and microRNA217. In summary, TAP, CPA1, and CPA2 proteins measured in rat plasma were sensitive and specific biomarkers for monitoring drug-induced pancreatic injury; outperforming lipase and amylase both by higher sensitivity of detection and by sustained increases in plasma observed over a longer time period. These protein-based assays and potentially others under development, are valuable tools for use in nonclinical drug development and as future translatable biomarkers for assessment in clinical settings to further improve patient safety.

Acid ceramidase targeting pyruvate kinase affected trypsinogen activation in acute pancreatitis.

BACKGROUND: Acute pancreatitis is the sudden inflammation of the pancreas. Severe cases of acute pancreatitis are potentially fatal and have no specific treatment available. Premature trypsinogen activation could initiate acute pancreatitis. However, the mechanism underlying premature trypsinogen activation is not fully understood. METHODS: In this research, a primary pancreatic acinar cell or mouse acute pancreatitis model was constructed. The effect of acid ceramidase (ASAH1), which is responsible for sphingosine production, was investigated in trypsinogen activation in vitro and in vivo. Meanwhile, the proteins regulating ASAH1 or binding to sphingosine were also detected by co-immunoprecipitation followed by mass spectrometry. RESULTS: The results showed that ASAH1 increased in acute pancreatitis. Increased ASAH1 promoted the activation of trypsinogen and cathepsin B. On the contrary, ASAH1 downregulation inhibited trypsinogen and cathepsin B. Meanwhile, ASAH1 regulated the activity of trypsin and cathepsin B through sphingosine. Additionally, E3 ligase Mind bomb homolog 1 (MIB1) decreased in acute pancreatitis resulting in the decreased binding between MIB1 and ASAH1. Exogenous MIB1 diminished the elevation in trypsin activity induced by acute pancreatitis inducer. ASAH1 increased owing to the inhibition of the proteasome degradation by MIB1. In acute pancreatitis, sphingosine was found to bind to pyruvate kinase. Pyruvate kinase activation could reduce trypsinogen activation and mitochondrial reactive oxygen species (ROS) production induced by sphingosine. CONCLUSIONS: In conclusion, during the process of acute pancreatitis, MIB1 downregulation led to ASAH1 upregulation, resulting in pyruvate kinase inhibition, followed by trypsinogen activation.

Immunoassay for trypsinogen-4.

Trypsin has been identified as a pancreatic protease comprising three isoenzymes, trypsin-1, -2, and -3. However, the gene for trypsinogen-3, PRSS3, also gives rise to additional variants, trypsinogen-4A and B, which differ from trypsinogen-3 only with respect to the leader-peptide part, and when activated are identical to trypsin-3. The unique overlapping leader peptides of trypsinogen-4A and B allowed us to develop a specific sandwich-type immunofluorometric assay that detects both these isoforms, but not trypsinogen-3 or activated trypsinogen-4. We measured the concentrations of trypsinogen-4 in various cell line lysates and bile of primary sclerosing cholangitis patients. Lysates of cell lines MDA-MB-231 and PC-3, and astrocytes contained trypsinogen-4, while the conditioned media from these cells did not, suggesting that trypsinogen-4, lacking a classical signal sequence, is not secreted from the cells. Interestingly, 5.7% of the 212 bile samples analyzed contained measurable (>2.4 µg/l) trypsinogen-4. In conclusion, we have established a specific assay for trypsinogen-4 and demonstrated that trypsinogen-4 can be found in biological samples. However, the clinical utility of the assay remains to be established.

A rare PRSS1 p.S127C mutation is associated with chronic pancreatitis and causes misfolding-induced ER-stress.

BACKGROUND: /Objectives: Sequence variants in several genes have been identified as being associated with an increased inherited risk to develop chronic pancreatitis (CP). In a genetic survey of a CP patient we identified in the PRSS1gene a new c.380C > G sequence variation, giving rise to a non-synonymous p.S127C mutation. Functional studies were performed to analyze the associated pathophysiology of the variant. METHODS: Following generation of an expression vector for the new PRSS1 variant we compared its expression, secretion and catalytic activity with already known PRSS1 risk variants in HEK 293T cells. The intracellular protein accumulation and induction of endoplasmic reticulum (ER)-stress was analyzed. RESULTS: Prediction tool analysis indicated a probably deleterious effect of the p.S127C variant on protein function which was confirmed by detection of a secretion defect in HEK293T cells leading to intracellular protein accumulation. While protein misfolding was associated with reduced trypsin activity, the increased expression of BIP and presence of spliced XBP1 indicated that the p.S127C variant induces ER stress and activates the UPR signaling pathway. CONCLUSIONS: The disease mechanism of the PRSS1 p.S127C variant involves defective protein secretion and the induction of ER-stress due to accumulation of presumably misfolded trypsinogen within the ER. The new variant should be considered disease-causing with an incomplete penetrance. Our results confirm that in addition to dysregulated trypsin-activity or reduced fluid secretion, ER-stress induction is an important trigger for acinar cell damage and the development of recurrent or chronic pancreatic inflammation.

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.

Clinical findings of patients with cystic fibrosis according to newborn screening results.

BACKGROUND: Cystic fibrosis (CF) is a lethal recessive genetic disease caused by loss of function associated with mutations in the CF trans-membrane conductance regulator. It is highly prevalent (approximately 1 in 3,500) in Caucasians. The aim of this study was to compare demographic and clinical features, diagnostic tests, treatments, and complications of patients with CF whose newborn screening (NBS) with twice-repeated immune reactive trypsinogen testing was positive, normal, and not performed. METHODS: In this study, 359 of all 1,488 CF patients recorded in the CF Registry of Turkey in 2018, who had been born through the process of NBS, were evaluated. Demographic and clinical features were compared in patients diagnosed with positive NBS (Group 1), normal (Group 2), or without NBS (Group 3). RESULTS: In Group 1, there were 299 patients, in Group 2, there were 40 patients, and in Group 3, there were 20 patients. Among all patients, the median age at diagnosis was 0.17 years. The median age at diagnosis was higher in Groups 2 and 3 than in Group 1 (P = 0.001). Fecal elastase results were higher in Group 2 (P = 0.033). The weight z-score was lower and chronic Staphylococcus aureus infection was more common in Group 3 (P = 0.017, P = 0.004, respectively). CONCLUSIONS: Frequency of growth retardation and chronic S. aureus infection can be reduced with an early diagnosis using NBS. In the presence of clinical suspicion in patients with normal NBS, further analyses such as genetic testing should be performed, especially to prevent missing patients with severe mutations.

Inactivation of mesotrypsin by chymotrypsin C prevents trypsin inhibitor degradation.

Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in Km , it digested ß-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.

Evolutionary expansion of polyaspartate motif in the activation peptide of mouse cationic trypsinogen limits autoactivation and protects against pancreatitis.

The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation and facilitates activation by enteropeptidase. This evolutionary mechanism protects the pancreas from premature trypsinogen activation while allowing physiological activation in the gut lumen. Inborn mutations that disrupt the tetra-aspartate motif cause hereditary pancreatitis in humans. A subset of trypsinogen paralogs, including the mouse cationic trypsinogen (isoform T7), harbor an extended penta-aspartate motif (P2-P6) in their activation peptide. Here, we demonstrate that deletion of the extra P6 aspartate residue (D23del) increased the autoactivation of T7 trypsinogen threefold. Mutagenesis of the P6 position in wild-type T7 trypsinogen revealed that bulky hydrophobic side chains are preferred for maximal autoactivation, and deletion-induced shift of the P7 Leu to P6 explains the autoactivation increase in the D23del mutant. Accordingly, removal of the P6 Leu by NH2-terminal truncation with chymotrypsin C reduced the autoactivation of the D23del mutant. Homozygous T7D23del mice carrying the D23del mutation did not develop spontaneous pancreatitis and severity of cerulein-induced acute pancreatitis was comparable with that of C57BL/6N controls. However, sustained stimulation with cerulein resulted in markedly increased histological damage in T7D23del mice relative to C57BL/6N mice. Furthermore, when the T7D23del allele was crossed to a chymotrypsin-deficient background, the double-mutant mice developed spontaneous pancreatitis at an early age. Taken together, the observations argue that evolutionary expansion of the polyaspartate motif in mouse cationic trypsinogen contributes to the natural defenses against pancreatitis and validate the role of the P6 position in autoactivation control of mammalian trypsinogens.NEW & NOTEWORTHY Unwanted autoactivation of the digestive protease trypsinogen can result in pancreatitis. The trypsinogen activation peptide contains a polyaspartate motif that suppresses autoactivation. This study demonstrates that evolutionary expansion of these aspartate residues in mouse cationic trypsinogen further inhibits autoactivation and enhances protection against pancreatitis.

The soymilk diet: A previously unknown etiology of acute pancreatitis.

We present a case of daily, large ingestions of soymilk that likely led to acute pancreatitis. Soybean contains trypsin inhibitor that when ingested will reduce the activity of trypsin in the intestine. A decrease in intestinal proteolytic activity removes the negative feedback on the pancreatic acinar cells, leading to an inappropriate increase in intrapancreatic trypsin secretion. When trypsin activation exceeds the capacity of pancreatic secretory trypsin inhibitor, the subsequent cascade of events can lead to acute pancreatitis.

Dopamine D2 receptor activator quinpirole protects against trypsinogen activation during acute pancreatitis via upregulating HSP70.

Trypsinogen activation is the hallmark of acute pancreatitis (AP) independent of intra-acinar NF-κB activation and inflammation. We previously found that dopamine (DA) receptor 2 (DRD2) activation controls inflammation during AP via PP2A-dependent NF-κB activation. In this study, we sought to examine whether DRD2 signaling mediates trypsinogen activation and the underlying mechanisms. Pancreatic acinar cells were stimulated with cholecystokinin-8 in vitro. AP was induced by intraperitoneal injections of caerulein and LPS or l-arginine. Pancreatitis severity was assessed biochemically and histologically. We found that activation of DRD2 by quinpirole, a potent DRD2 agonist, resulted in the reduction of trypsinogen activation and the upregulation of HSP70 in vitro and in vivo. Mechanistically, we found that quinpirole induced dephosphorylation of heat shock factor 1 (HSF1), a master transcription factor of HSP70, leading to increased nuclear translocation of HSF1 in a PP2A-dependent pathway. Furthermore, DRD2 activation restored lysosomal pH and, therefore, maintained lysosomal cathepsin B activity in a HSP70-dependent manner. VER155008, a potent HSP70 antagonist, abolished the protective effects observed with DRD2 activation in vitro and in two experimental models of AP. Our data showed that besides controlling NF-κB activation, DRD2 activation prevented trypsinogen activation during acute pancreatitis via PP2A-dependent upregulation of HSP70 and further support that DRD2 agonist could be a promising therapeutic strategy for treating AP.NEW & NOTEWORTHY The current study demonstrated that activation of DRD2 by quinpirole protects against trypsinogen activation in the in vitro and in vivo setting of acute pancreatitis by upregulating HSP70 and restoring lysosomal degradation via a PP2A-dependent manner, therefore leading to reduced pancreatic injury. These findings provide a new mechanistic insight on the protective effect of DRD2 activation in acute pancreatitis.

Genetics, Cell Biology, and Pathophysiology of Pancreatitis.

Since the discovery of the first trypsinogen mutation in families with hereditary pancreatitis, pancreatic genetics has made rapid progress. The identification of mutations in genes involved in the digestive protease-antiprotease pathway has lent additional support to the notion that pancreatitis is a disease of autodigestion. Clinical and experimental observations have provided compelling evidence that premature intrapancreatic activation of digestive proteases is critical in pancreatitis onset. However, disease course and severity are mostly governed by inflammatory cells that drive local and systemic immune responses. In this article, we review the genetics, cell biology, and immunology of pancreatitis with a focus on protease activation pathways and other early events.

Generation and characterization of murine monoclonal antibodies against immunoreactive trypsinogen for newborn screening of cystic fibrosis.

Cystic fibrosis (CF) is a multisystem disorder that reduces quality of life and survival in affected individuals. In newborns, the release of pancreatic enzymes into the blood raises the levels of immunoreactive trypsinogen (IRT), the main marker for CF screening, which is detected in dried blood samples on filter paper by immunoenzymatic assays. In Cuba, CF has an estimated incidence of 1/9862 live births and should be included in the national basic newborn screening (NBS) panel given its benefits in terms of nutrition, lung function and survival. The Immunoassay Center develops and produces diagnostic kits allowing the establishment of large-scale NBS programs for inherited metabolic disorders in Cuba and other Latin American countries. IRT-specific monoclonal antibodies (MAbs) obtained at the Immunoassay Center are essential for developing an affordable immunoassay for IRT to support CF NBS in our low-income country. An immunization scheme with trypsinogen-1 originated two IgG1-producing murine hybridomas. 4C9C9 and 4C9E11 MAbs recognized different determinants on both trypsin-1 and trypsin-2 molecules. Both antibodies identified conformational epitopes on the molecule of trypsin-1 and of its zymogen. As 4C9E11 MAb cross-reacted with proteins structurally and functionally related to trypsinogen, it was used as revealing antibody in a sandwich-type UMELISA® assay for IRT determination with 4C9C9 MAb for capture. This combination, aside from detecting several commercially available trypsins, adequately quantified IRT from dried blood samples on filter paper of newborns. The evaluation of the assay's accuracy yielded percentage recoveries ranging 93.3-109.2% for commercial controls. The properties of the studied MAbs demonstrate their suitability for being used in a sandwich-type UMELISA® assay for the CF NBS in Cuba.