Pancreatic Secretory Trypsin Inhibitor Research Articles

Pancreatic Secretory Trypsin Inhibitor (SPINK1) Gene Mutation in Patients with Acute Alcohol Pancreatitis (AAP) Compared to Healthy Controls and Heavy Alcohol Users without Pancreatitis

Only 3-5% of heavy alcohol users develop acute alcohol pancreatitis (AAP). This suggests that additional triggers are required to initiate the inflammatory process. Genetic susceptibility contributes to the development of AAP, and SPINK1 mutation is a documented risk factor. We investigated the prevalence of the SPINK1(N34S) mutation in patients with AAP compared to heavy alcohol users who had never suffered an episode of pancreatitis. Blood samples for the mutational analysis from patients with first episode (n = 60) and recurrent AAP (n = 43) and from heavy alcohol users without a history of AAP (n = 98) as well as from a control population (n = 1914) were obtained. SPINK1 mutation was found in 8.7% of the patients with AAP. The prevalence was significantly lower in healthy controls (3.4%, OR 2.72; 1.32-5.64) and very low in alcoholics without pancreatitis (1.0%, OR 9.29; 1.15-74.74). In a comparison adjusted for potential cofounders between AAP patients and alcoholics, SPINK1 was found to be an independent marker for AAP. The prevalence of the SPINK1 mutation is overrepresented in AAP patients and very low in alcoholics without pancreatitis. This finding may play a role in understanding the variable susceptibility to AAP found in heavy alcohol users.

The Clinical Utility of Soluble Serum Biomarkers in Autoimmune Pancreatitis: A Systematic Review.

Autoimmune pancreatitis (AIP) is a rare etiological type of chronic pancreatitis. The clinical and radiological presentation of AIP often resembles that of pancreatic cancer. Identifying non-invasive markers for their early distinction is of utmost importance to avoid unnecessary surgery or a delay in steroid therapy. Thus, this systematic review was conducted to revisit all current evidence on the clinical utility of different serum biomarkers in diagnosing AIP, distinguishing AIP from pancreatic cancer, and predicting disease course, steroid therapy response, and relapse. A systematic review was performed for articles published up to August 2021 by searching electronic databases such as MEDLINE, Web of Science, and EMBASE. Among 5123 identified records, 92 studies were included in the qualitative synthesis. Apart from immunoglobulin (Ig) G4, which was by far the most studied biomarker, we identified autoantibodies against the following: lactoferrin, carboanhydrase II, plasminogen-binding protein, amylase-α2A, cationic (PRSS1) and anionic (PRSS2) trypsinogens, pancreatic secretory trypsin inhibitor (PSTI/SPINK1), and type IV collagen. The identified novel autoantigens were laminin 511, annexin A11, HSP-10, and prohibitin. Other biomarkers included cytokines, decreased complement levels, circulating immune complexes, N-glycan profile changes, aberrant miRNAs expression, decreased IgA and IgM levels, increased IgE levels and/or peripheral eosinophil count, and changes in apolipoprotein isoforms levels. To our knowledge, this is the first systematic review that addresses biomarkers in AIP. Evolving research has recognized numerous biomarkers that could help elucidate the pathophysiological mechanisms of AIP, bringing us closer to AIP diagnosis and its preoperative distinction from pancreatic cancer.

Chronic Pancreatitis: The True Pathogenic Culprit within the SPINK1 N34S-Containing Haplotype Is No Longer at Large.

A diverse range of loss-of-function variants in the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor) has been identified in patients with chronic pancreatitis (CP). The haplotype harboring the SPINK1 c.101A>G (p.Asn34Ser or N34S) variant (rs17107315:T>C) is one of the most important heritable risk factors for CP as a consequence of its relatively high prevalence worldwide (population allele frequency ≈ 1%) and its considerable effect size (odds ratio ≈ 11). The causal variant responsible for this haplotype has been intensively investigated over the past two decades. The different hypotheses tested addressed whether the N34S missense variant has a direct impact on enzyme structure and function, whether c.101A>G could affect pre-mRNA splicing or mRNA stability, and whether another variant in linkage disequilibrium with c.101A>G might be responsible for the observed association with CP. Having reviewed the currently available genetic and experimental data, we conclude that c.-4141G>T (rs142703147:C>A), which disrupts a PTF1L-binding site within an evolutionarily conserved HNF1A-PTF1L cis-regulatory module located ∼4 kb upstream of the SPINK1 promoter, can be designated as the causal variant beyond reasonable doubt. This case illustrates the difficulties inherent in determining the identity of the causal variant underlying an initially identified disease association.

A mouse testis serine protease, TESP1, as the potential SPINK3 receptor protein on mouse sperm acrosome.

Serine protease inhibitor Kazal type 3 (SPINK3) from mouse seminal vesicles is a Kazal-type trypsin inhibitor. It has been shown to bind to the sperm acrosome and modify sperm activity by influencing the sub-cellular Ca2+ influx. Previously, SPINK3 was reported to suppress in vitro sperm capacitation. However, under natural coitus, SPINK3 is removed from the mouse acrosome in the female reproductive tract, leading to successful fertilisation. Identification of the SPINK3 binding partner becomes essential to develop a contraceptive that works by prolonging the binding of SPINK3 to the sperm acrosome. We identified the SPINK3 receptor by using recombinant SPINK3 (rSPINK3). Testicular serine protease 1 (TESP1) was identified as the receptor for SPINK3 by 2D gel electrophoresis coupled with western blot analysis. To authenticate TESP1 as the receptor for SPINK3, sperm cells were incubated with TESP1 peptide antibody followed by determining the intracellular [Ca2+]i concentration by flow cytometry using Fluo-3 AM as a calcium probe. Furthermore, the 3D structures of SPINK3 and TESP1 were predicted by homology modelling (Schrodinger suite) using the crystal structure of pancreatic secretory trypsin inhibitor (PDB ID-1TGS) and human prostasin (PDB ID-3DFJ) as templates. The modelled protein structures were validated and subjected to molecular dynamics simulation (MDS) using GROMACS v5.0.5. Protein-protein docking was performed using HDOCK and the complex was validated by MDS. The results predicted that SPINK3 and TESP1 had strong binding affinity, with a dock score of -430.70 and 14 hydrogen bonds as key active site residues. If the binding affinity between SPINK3 and TESP1 could be increased, the SPINK3-TESP1 association will be prolonged, which will be helpful in the development of a male contraceptive.

Possibilities of the early diagnosis and prognosis of complicated clinical forms of chronic pancreatitis

AIM: This study aimed to improve the methods for the diagnosis of complicated clinical forms of chronic pancreatitis (CP) by evaluating the clinical significance of the polymorphisms of the genes of cationic trypsinogen (PRSS1), pancreatic secretory trypsin inhibitor (SPINK1), transmembrane regulator of cystic fibrosis (CFTR), and alcohol dehydrogenase (ADH) in patients with complicated and uncomplicated forms of CP.
 MATERIALS AND METHODS: The study was carried out on the clinical base of the Department of Hospital Surgery, Ryazan State Medical University, Center for Surgery of Liver, Pancreas, and Biliary Tract in Ryazan in 20142019. A total of 108 patients of both genders aged 2565 years were examined. Of these patients, 38 were surgically treated for complicated CP, 20 had complicated CP without surgery, and 50 had uncomplicated CP (control group). A comparative clinical study with the control group of patients was performed, and the genotype was simultaneously determined on days 1 and 10 under controlled laboratory parameters. DNA was isolated from the leukocytes of the whole blood by using a DNA-expressing blood reagent (OOO NPF Litekh, Russia) for further analysis.
 RESULTS: No polymorphism of cationic trypsinogen PRSS1 gene and cystic fibrosis-2 CFTR2 gene was found. The predictive value of these polymorphisms was insignificant. For the polymorphism of CFTR1 cystic fibrosis-1 gene, the odds ratio was 0.444, but this finding was not significant. Among patients with the complicated clinical forms of CP, mutations were observed in the PRSS1 cationic trypsinogen gene (c2 = 6.453, p = 0.012) and ADH (c2=14.176, p = 0.001). Conversely, they were not detected in the CFTR-1 gene (c2 = 0.873, p = 0.351), CFTR-2 (c2 was not determined), and SPINK1 (c2 = 0.873, p = 0.351). The polymorphisms of the ADH and PRSS1 genes of cationic trypsinogen were associated with more evident structural changes in the parenchyma and ductal system of the pancreas. They also had a higher likelihood of complications, severe disease course, and a lower efficiency of conservative treatment. The polymorphism of the ADH gene increased the risk of the development of the cystic form of CP (c2 = 5.898, p = 0.016).
 CONCLUSION: The polymorphism of ADH and cationic trypsinogen genes should be determined and used for the complex diagnosis of CP to specify indications for the surgical treatment of patients with CP.

Roles of Autophagy and Pancreatic Secretory Trypsin Inhibitor in Trypsinogen Activation in Acute Pancreatitis.

The focus of the review is on roles of autophagy and pancreatic secretory trypsin inhibitor (PSTI), an endogenous trypsin inhibitor, in trypsinogen activation in acute pancreatitis. Acute pancreatitis is a disease in which tissues in and around the pancreas are autodigested by pancreatic digestive enzymes. This reaction is triggered by the intrapancreatic activation of trypsinogen. Autophagy causes trypsinogen and cathepsin B, a trypsinogen activator, to colocalize within the autolysosomes. Consequently, if the resultant trypsin activity exceeds the inhibitory activity of PSTI, the pancreatic digestive enzymes are activated, and they cause autodigestion of the acinar cells. Thus, autophagy and PSTI play important roles in the development and suppression of acute pancreatitis, respectively.

Pancreatic secretory trypsin inhibitor reduces multi-organ injury caused by gut ischemia/reperfusion in mice.

Intestinal ischemia/reperfusion (I/R) injury occurs during transplantation, mesenteric arterial occlusion, trauma and shock, causing systemic inflammation, multiple organ dysfunction and high mortality. Pancreatic secretory trypsin inhibitor (PSTI), a serine protease inhibitor expressed in gut mucosa may function as a mucosal protective/repair peptide. We examined whether PSTI affected mesenteric I/R-induced injury. Hypoxia/normoxia (H/N) caused 50% drop in cell viability of AGS, RIE1 and Caco-2 cells but PSTI (10 μg/ml) given prior- or during-hypoxic period improved survival by 50% (p<0.01). Similarly, Caco-2 monolayers exposed to H/N had 300% increase in transepithelial permeability, PSTI truncated this by 50% (p<0.01). Mice underwent mesenteric I/R by clamping jejunum, causing severe mucosal injury, increased apoptotic markers and 3-fold increases in plasma IL-6, IL1β, TNFα, and tissue lipid peroxidation (MDA) and inflammatory infiltration (MPO) levels. Lungs showed similar significant injury and inflammatory infiltrate markers. Smaller increases in MDA and MPO were seen in kidney & liver. PSTI (20 mg/kg) reduced all injury markers by 50–80% (p<0.01). In vitro and in vivo studies showed PSTI reduced pro-apoptotic Caspase 3, 9 and Baxα levels, normalised Bcl2 and caused additional increases in HIF1α, VEGF and Hsp70 above rises caused by I/R alone (all p<0.01). PSTI also prevented reduction of tight junction molecules ZO1 and Claudin1 (all p<0.01) but did not affect increased ICAM-1 caused by I/R in gut or lung. PSTI may be a useful clinical target to prevent I/R injury.

A Case of Recurrent Pancreatitis Associated With Pancreas Divisum and a Mutation of the Pancreatic Secretory Trypsin Inhibitor, Kazal Type 1 (SPINK1) Gene

A Case of Recurrent Pancreatitis Associated With Pancreas Divisum and a Mutation of the Pancreatic Secretory Trypsin Inhibitor, Kazal Type 1 (SPINK1) Gene

Abstract 124: The soluble factor SPINK1 is a biomarker of therapeutically damaged TME that confers acquired resistance and an exploitable molecular target in human cancer medicine

Abstract Clinical efficacy of most anticancer treatments is frequently compromised by the development of cancer resistance arising as either a consequence of innate adaptation of cancer cells, or acquired fitness conferred by extracellular factors generated by the surrounding tumor microenvironment (TME). Of note, stromal cells in the TME become overtly senescent upon genotoxic treatment and develop a senescence-associated secretory phenotype (SASP), the latter characterized by secretion of numerous soluble factors that are responsible for disease exacerbation. We recently disclosed significant upregulation of the serine protease inhibitor Kazal type I (SPINK1) in human prostate stroma after chemotherapy of prostate cancer (PCa) patients. SPINK1, originally reported as a pancreatic secretory trypsin inhibitor, prevents pancreatic tissues from protease degradation with expression normally restricted to gastrointestinal (GI) tract organs. However, the influence of SPINK1 as a TME-derived soluble factor on cancer progression remains unexplored. In this study, we defined the expression modality and biological roles of SPINK1 in a therapy-damaged and functionally activated TME under therapeutic pressure. Distinct from a recent finding that revealed the involvement of a regulatory circuit comprising HNF4G and HNF1A in the PCa-GI transcriptome, we found that SPINK1 expression in human stromal cells is regulated by alternative transcriptional factors including NF-κB and C/EBP in response to chemotherapy. TME-released SPINK1 can significantly enhance the aggressiveness of PCa cells including accelerated proliferation, increased migration, elevated invasion and more importantly, enhanced chemoresistance. SPINK1 triggers a typical epithelial-mesenchymal transition in cancer cells, a process mediated by EGFR/PI3K/Akt and MAPK/Erk signaling. Further, SPINK1 remarkably reprograms PCa cells by inducing a cancer stem cell (CSC)-like phenotype and promoting transdifferentiation of cancer cells toward neovasculature. As supporting evidence, our preclinical studies suggested that SPINK1 expression in the prostate TME substantially promoted cancer survival and disease progression. Clinical investigation revealed increased expression of SPINK1 in the stroma of multiple organ types including the prostate, lung and breast of cancer patients after chemotherapy, implying the systemic induction of SPINK1 by anticancer agents. Thus, our study demonstrates that SPINK1 is a soluble biomarker of therapeutically damaged TME and represents a potentially exploitable molecular target in human cancer clinics. Citation Format: Yu Sun. The soluble factor SPINK1 is a biomarker of therapeutically damaged TME that confers acquired resistance and an exploitable molecular target in human cancer medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 124.

Prevalence of Pancreatic Secretory Trypsin Inhibitor (SPINK1) gene mutation in patients with acute alcohol pancreatitis (AAP) compared to alcoholics and healthy controls

Prevalence of Pancreatic Secretory Trypsin Inhibitor (SPINK1) gene mutation in patients with acute alcohol pancreatitis (AAP) compared to alcoholics and healthy controls

An immunocapture-LC-MS-based assay for serum SPINK1 allows simultaneous quantification and detection of SPINK1 variants.

Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420Da peptide produced by the pancreas, but also by several other tissues and many tumors. Some mutations of the SPINK1 gene, like the one causing amino acid change N34S, have been shown to confer susceptibility to recurrent or chronic pancreatitis. Detection of such variants are therefore of clinical utility. So far SPINK1 variants have been determined by DNA techniques. We have developed and validated an immunocapture-liquid chromatography-mass spectrometric (IC-LC-MS) assay for the detection and quantification of serum SPINK1, N34S-SPINK1, and P55S-SPINK1. We compared this method with a time-resolved immunofluorometric assay (TR-IFMA) for serum samples and primer extension analysis of DNA samples. We used serum and DNA samples from patients with acute pancreatitis, renal cell carcinoma, or benign urological conditions. With the help of a zygosity score calculated from the respective peak areas using the formula wild-type (wt) SPINK1/(variant SPINK1 + wt SPINK1), we were able to correctly characterize the heterozygotes and homozygotes from the samples with DNA information. The score was then used to characterize the apparent zygosity of the samples with no DNA characterization. The IC-LC-MS method for SPINK1 was linear over the concentration range 0.5-1000μg/L. The limit of quantitation (LOQ) was 0.5μg/L. The IC-LC-MS and the TR-IFMA assays showed good correlation. The median zygosity score was 1.00 (95% CI 0.98-1.01, n = 11), 0.55 (95% CI 0.43-0.61, n = 14), and 0.05 (range 0.04-0.07, n = 3) for individuals found to be wt, heterozygous, and homozygous, respectively, for the N34S-SPINK1 variant by DNA analysis. When DNA samples are not available, this assay facilitates identification of the N34S- and P55S-SPINK1 variants also in archival serum samples.

Effects of Calcium Channel Blockers on Trypsinogen Activation and Severity of Cerulein-induced Acute Pancreatitis in Rats

Acute pancreatitis is an inflammatory disease of the pancreas associated with high mortality but without a specific therapy. Intrapancreatic protease activation appears to be an early event in the development of acute pancreatitis but its association with pathogenesis has not been established. Recently it has been demonstrated that an enormous rise in the cytoplasmic Ca2+ levels of pancreatic acinar cells could be associated with the development of acute pancreatitis, thereby suggesting that Ca2+ channel could be a potential target for treatment. In this study, the protective effects of calcium channel blockers such as 2-aminoethoxydiphenyl borate, dantrolene and verapamil were investigated in cerulein-induced acute pancreatitis in vivo. Rats were divided into five groups. Group 1 (control), group 2 (cerulein, 100 µg/kg), group 3 (cerulein+ 2-aminoethoxydiphenyl borate, mg/kg), group 4 (cerulein+dantrolene, 10 mg/kg) and group 5 (cerulein+verapamil, 2.5 mg/kg). Activities of amylase, lipase, cathepsin B, pancreatic secretory trypsin inhibitor and trypsin and levels of trypsinogen activation peptide and trypsinogen were measured addition to histological examination of the sections of pancreas. The results showed that cerulein increased the amylase, lipase and trypsin activities and trypsinogen activation peptide levels significantly. Administration of calcium channel blockers significantly protected the pancreas from histological damage (p<0.05). The results of this study demonstrated that calcium channel blockers can mitigate early protease activation and pancreas injury. It was suggested regulation of calcium channels would be relevant to maintain pancreatic acinar cell homeostasis and further research is necessary to understand the protective effects of calcium channel blockers.

Common Genetic Variants in Trypsin Regulating Genes Are Associated with AsparAginase-Associated Pancreatitis in Children with Acute Lymphoblastic Leukemia: A Ponte Di Legno Toxicity Working Group Study

Background: Asparaginase-associated pancreatitis (AAP) is a well-known toxicity of childhood acute lymphoblastic leukemia (ALL) therapy. Recent multi-trial group phenotyping of 465AAP caseshas documented severe complications to AAP, including 8% risk of needing assisted mechanical ventilation, 26% risk of developing pancreatic pseudocysts and 9% risk of developing persisting diabetes (Wolthers et al. Lancet Oncology, 2017) . Investigation of host genome variation associated with AAP has been limited by varying phenotype definition, inclusion criteria and small study sizes.Objectives and Methods: To investigate genetic variants associated with risk of developing AAP, this genome-wide association study reports data on 1544 children (1.0−17.9 years) from 10 ALL trial groups treated with ALL from January 2000−January 2016. The Ponte di Legno toxicity working group consensus definition (Schmiegelow et al. Lancet Oncology, 2016) was used to diagnose AAP: At least two of i) amylase, pancreatic amylase, or pancreatic lipase >3x upper normal limit (UNL), ii) abdominal pain, iii) imaging compatible with AAP. Controls included children treated for ALL with verified completion of intended asparaginase therapy, 78% of whom (1024/1320) received at least 8 injections of PEG-asparaginase without developing AAP. Germline DNA obtained after clinical remission was genotyped on Illumina Infinium Omni2.5exome-8 BeadChip arrays. Association analyses were done in PLINK and annotation in Ensembl.Results: Of 1564 patients passing genotype quality control, 244 had AAP. 205 of 244 (84%) of cases and 1185/1320 (90%) of controls were of European ancestry. Median age was 8.1 years (IQR 4.3−13.1) and 5 (IQR: 3−9) for cases and controls, respectively. After filtering, 1401908 single nucleotide polymorphisms (SNPs) with a minor allele frequency above 1% were analyzed. In logistic regression analysis, adjusting for age and ancestry, the variant rs62228256 (reference allele=C, minor allele=T (C>T)) on 20q13.2 had the strongest association to AAP (OR=3.75; 95% CI 2.33−6.04; p=5.2∙10-8). rs62228256 is located in a non-coding region without known regulatory effects. rs13228878 (A>G; OR=0.61; 95% CI 0.5−0.76; p=7.1∙10-6) and rs10273639 (C>T; OR=0.62; 95% CI 0.5−0.77; p =1.1∙10-5) were among the top 30 SNPs most significantly associated to AAP. They are in high linkage disequilibrium (R2=0.94) and located in the PRSS1-PRSS2 locus on chromosome 7. The rs13228878 A risk allele was not associated with level of amylase (p=0.1) or lipase (p=0,68) at diagnosis of AAP, age at diagnosis of AAP (p=0.63), or risk of pseudocysts (p=0.78). Using identical diagnostic criteria for pancreatitis, the major C allele in rs10273639 has been associated with pancreatitis risk in adults (Whitcomb et al. Nature Genetics, 2012; Masson et al. Gut, 2017) with identical risk allele and similar odds ratios. PRSS1 and PRSS2 encode cationic and anionic trypsinogen, respectively. rs10273639 is an expressive quantitative locus for PRSS1 and the C risk-variant is associated with elevated expression of trypsinogen in pancreatic tissue. Gain of function mutations in PRSS1, known from hereditary pancreatitis, lead to increased autoactivation, increased intra-acinar trypsin levels, and increased risk of auto-digestion leading to pancreatitis. Further investigation of previously validated SNPs known to regulate trypsin activation gave the following results for associations with AAP; rs17107315 in pancreatic secretory trypsin inhibitor (SPINK1; OR=2.87; 95% CI 1.36−5.8; p=4∙10-3), rs10436957 in chymotrypsin C (CTRC ; OR=0.69; 95% CI 0.53−0.89; p=5∙10-3) and rs4409525 in Claudin-2 (CLDN2 ; OR=1.41; 95% CI 1.08−1.83; p=1∙10-2). In total, 207 out of 244 cases were homozygous for the risk allele in rs13228878 (n=104), rs17107315 (n=1), rs10436957 (n=165) and/or rs4409525 (n=16). However, no significant additive effect of having more than one risk allele was found.Conclusion: Children who develop AAP possess the same pancreatitis risk variants as adults with non-asparaginase associated pancreatitis. This shared genetic disposition may facilitate research into pathogenesis and identification of effective interventions towards AAP. DisclosuresNo relevant conflicts of interest to declare.

In silico prioritization and further functional characterization of SPINK1 intronic variants

BackgroundSPINK1 (serine protease inhibitor, kazal-type, 1), which encodes human pancreatic secretory trypsin inhibitor, is one of the most extensively studied genes underlying chronic pancreatitis. Recently, based upon data from qualitative reverse transcription-PCR (RT-PCR) analyses of transfected HEK293T cells, we concluded that 24 studied SPINK1 intronic variants were not of pathological significance, the sole exceptions being two canonical splice site variants (i.e., c.87 + 1G > A and c.194 + 2T > C). Herein, we employed the splicing prediction tools included within the Alamut software suite to prioritize the ‘non-pathological’ SPINK1 intronic variants for further quantitative RT-PCR analysis.ResultsAlthough our results demonstrated the utility of in silico prediction in classifying and prioritizing intronic variants, we made two observations worth noting. First, we established that most of the prediction tools employed ignored the general rule that GC is a weaker donor splice site than the canonical GT site. This finding is potentially important because for a given disease gene, a GC variant donor splice site may be associated with a milder clinical manifestation. Second, the non-pathological c.194 + 13T > G variant was consistently predicted by different programs to generate a new and viable donor splice site, the prediction scores being comparable to those for the physiological c.194 + 2T donor splice site and even higher than those for the physiological c.87 + 1G donor splice site. We do however provide convincing in vitro evidence that the predicted donor splice site was not entirely spurious.ConclusionsOur findings, taken together, serve to emphasize the importance of functional analysis in helping to establish or refute the pathogenicity of specific intronic variants.

Polymorphism of genes for alcoholdehydrogenase, cysticfibrosis, pancreatic secretory trypsin inhibitor, a cationic trypsinogen in patients with chronic pancreatitis

The study involved 63 patients with a verified diagnosis of chronic pancreatitis. In the 1st group included 31 patients with a complicated clinical forms of chronic pancreatitis, in 2st group included 32 patients with uncomplicated forms. The study analyzed the polymorphisms of genes PRSS1 (mutation R122H) - gene for cationic trypsingene, CFTR1 (mutation del508) - the gene for cystic fibrosis-1, CFTR2 (mutation Gly542Ter) - the gene for cystic fibrosis-2, SPINK1 (mutation N34S) - gene for pancreatic secretory trypsin inhibitor, gene ADH (ADH1B * 2). The study found a statistically significant correlation between ADH gene polymorphism and the risk of complicated forms of chronic pancreatitis.

Emerging Roles of SPINK1 in Cancer.

Tumor-associated trypsin inhibitor (TATI) was originally isolated from the urine of a patient with ovarian cancer. It was later shown to be produced by many other tumors and several normal tissues. It had earlier been isolated from the pancreas and was hence called pancreatic secretory trypsin inhibitor (PSTI). It belongs to a family of protease inhibitors presently called serine peptidase inhibitor Kazal type (SPINK). In the SPINK family TATI/PSTI is SPINK1, which is the name used in this review. In addition to being a protease inhibitor, SPINK1 also acts as an acute-phase reactant and a growth factor. Furthermore, it has been shown to modulate apoptosis. Overexpression of SPINK1 predicts an unfavorable outcome in several cancers and determination of SPINK1 in serum can be used to identify patients at increased risk of aggressive disease. Thus serum SPINK1 can be used as a prognostic tumor marker. Because SPINK1 acts as a growth factor and an inhibitor of apoptosis in some cancers, it has also been suggested that it can be a therapeutic target in cancer. However, because SPINK1 is the major physiological inhibitor of trypsin, inhibition of SPINK1 may increase the risk of pancreatitis. Taking into account the many functions of SPINK1, assessing the role of SPINK1 in cancer has several potentially important clinical applications ranging from a biomarker to a potential new target for cancer therapy.

Human cationic trypsinogen but not serine peptidase inhibitor, Kazal type 1 variants increase the risk of type 1 autoimmune pancreatitis.

Autoimmune pancreatitis (AIP) is a distinct disease entity. Whether the genes involved in pancreatic acinar cell injury, cationic trypsinogen gene (protease, serine, 1 [trypsin 1] [PRSS1]) and the pancreatic secretory trypsin inhibitor gene (serine peptidase inhibitor, Kazal type 1 [SPINK1]), are associated with AIP remains to be explored. Genetic analyses of PRSS1 variants (exon 2 and 3) and SPINK1 variants (exon 1, 2, and 3) including the intronic areas in 118 patients with AIP and 200 control subjects were performed by direct DNA sequencing. Clinical features including imaging, histology, serology, response to steroid, and extra-pancreatic organ involvement in AIP patients with and without variants were compared. A total of 19 PRSS1 variants and one SPINK1 variant were identified in 20 (16.9%) out of 118 AIP patients. They included one K92N, nine R116C, seven T137M, one C139S, and one C139F of PRSS1 and one 2(IVS3 + 2) of SPINK1. No PRSS1 or SPINK1 variant was identified in the control group. Patients with PRSS1 variants had an increased risk of AIP with odds ratio 22.37 (95% confidence interval: 2.96-168.8, P = 0.003) and higher frequency of serum IgG4 above 280 mg/dL. Using immunosuppressive agent and PRSS1 variant were predictors of less disease relapse in univariate analysis. Presence of PRSS1 variants was the only negative predictor for disease relapse in multivariate analysis. We found a significantly higher frequency of PRSS1 variants in AIP patients than in geographically and ethnically matched control subjects. PRSS1 variants are associated with less disease relapse in AIP.

Abstract 244: SPINK1 insufficiency induces impaired autophagy resulting in chronic pancreatitis

Abstract Background&amp; Objective: Several studies reported that chronic pancreatitis is a risk factor for pancreatic ductal adenocarcinoma (PDAC). Serine protease inhibitor Kazal type 1 (SPINK1), also known as pancreatic secretory trypsin inhibitor (PSTI), was originally isolated from the pancreas. In mice, the homologous gene is designated as Spink3. SPINK1/Spink3 covalently can bind to activated trypsin within the pancreatic acinar cells to form an inactive and stable complex and to prevent acute pancreatitis, a major inflammatory disorder of the pancreas. The mutations of SPINK1 gene are associated with various forms of idiopathic pancreatitis. We previously showed that deletion of Spink3 causes pancreatitis-like changes in the mouse. A careful molecular and pathological analysis of this genetically engineered mouse model will reveal the chronic of nflammation in the pancreas. The aim of this study was to rescue the Spink3-/- phenotype by generating Spink3-/- mice with knocked-in SPINK1, and to analyze molecular mechanisms that initiate pancreatitis or drive its progression from acute to chronic disease in this genetic model. Methods: We placed SPINK1 minigene (KI) into mouse Spink3 locus, using the Cre-loxP technology and generated chimeric mice (Spink3KI/+). Spink3KI/+ mice were crossed to Spink3+/- mice, thus generating Spink3KI/-. Mice were followed up to 8 weeks old. Time-dependent changes in pancreatic histology, stellate cell ctivation, and serum amylase were measured. Results: Spink3KI/KI mice showed no abnormalities in pancreas or any other organ during the 8 weeks age of observation, indicating that human SPINK1 can rescue Spink3 deficiency in physiological condition. Spink3KI/- mice showed slight growth etardation. The pancreas of Spink3KI/- mice at birth contained normal acinar cells. The Spink3KI/- mice time-dependently developed pathologic features of chronic tissue damage, including loss of acinar cells, intralobular fibrosis with activated pancreatic stellate cells. Interlobular ducts were dilated and contained protein plugs, which resembled chronic pancreatitis in human. In the pancreas of Spink3KI/- mice, we found that trypsin activity increased, and large vacuoles, derived from autophagy, appeared in acinar cells. p62, which is a selective substrate for autophagy aggregated in acinar cells, suggested that autophagy is impaired in Spink3KI/- acinar cells. Conclusions: We demonstrated that SPINK1 insufficiency caused chronic pancreatitis in vivo. Impaired autophagy and high trypsin activity may contribute to cause chronic pancreatitis in human.We established chronic pancreatitis model in human SPINK1 knockin mice and identified shared pathological alterations in human and mouse chronic pancreatitis.Thus,this mouse model provides a suitable tool to analyze the oncogenesis from chronic pancreatitis. Citation Format: Kazuya Sakata, Masaki Ohmuraya, Katsunobu Taki, Daisuke Hashimoto, Satoshi Ida, Hidetoshi Nitta, Hiromitu Hayashi, Akira Chikamoto, Tooru Beppu, Hideo Baba. SPINK1 insufficiency induces impaired autophagy resulting in chronic pancreatitis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 244. doi:10.1158/1538-7445.AM2014-244

Identification of a Novel Frame-Shift Mutation in PRSS1 Gene in Han Patients with Autoimmune Pancreatitis

To detect mutations of trypsinogen gene (PRSS1) in patients with autoimmune pancreatitis (AIP) and to determine the underlying pathogenesis. DNA sequencing was used to detect full-length of PRSS1, cystic fibrosis transmembrane conductance regulator (CFTR), and pancreatic secretory trypsin inhibitor (SPINK1) genes mutations in an AIP family and a sporadic case and 520 normal controls. Furthermore, a mutant-expressing system was constructed for functional confirmation. For the first time, we report a deletion mutation at exon 2 of PRSS1 gene (IVS 2 +56_60 del CCCAG) which encoded a truncated PRSS1 protein without trypsinogen activation peptide (TAP). Vitro functional study suggested the identified mutation would result in loss of PRSS1 activity. Mutant trypsinogen activated at a faster rate than wild-type trypsinogen in the autoactivation experiment. Histopathologic examination revealed the ratio of IgG4/IgG-positive plasma cells exceeded 0.455 in pancreas, and the patients responded to glucocorticoids. PRSS1: IVS 2 +56_60 del CCCAG is a noval mutant which may contribute to AIP pathogenesis.

Identification of Serpinb6b as a Species-specific Mouse Granzyme A Inhibitor Suggests Functional Divergence between Human and Mouse Granzyme A

The granzyme family serine proteases are key effector molecules expressed by cytotoxic lymphocytes. The physiological role of granzyme (Gzm) A is controversial, with significant debate over its ability to induce death in target cells. Here, we investigate the natural inhibitors of GzmA. We employed substrate phage display and positional proteomics to compare substrate specificities of mouse (m) and human (h) GzmA at the peptide and proteome-wide levels and we used the resulting substrate specificity profiles to search for potential inhibitors from the intracellular serpin family. We identified Serpinb6b as a potent inhibitor of mGzmA. Serpinb6b interacts with mGzmA, but not hGzmA, with an association constant of 1.9 ± 0.8 × 10(5) M(-1) s(-1) and a stoichiometry of inhibition of 1.8. Mouse GzmA is over five times more cytotoxic than hGzmA when delivered into P815 target cells with streptolysin O, whereas transfection of target cells with a Serpinb6b cDNA increases the EC50 value of mGzmA 13-fold, without affecting hGzmA cytotoxicity. Unexpectedly, we also found that Serpinb6b employs an exosite to specifically inhibit dimeric but not monomeric mGzmA. The identification of an intracellular inhibitor specific for mGzmA only indicates that a lineage-specific increase in GzmA cytotoxic potential has driven cognate inhibitor evolution.