Annukka Paju

Biochemistry and clinical role of trypsinogens and pancreatic secretory trypsin inhibitor.

Trypsinogens and PSTI/TATI/SPINK1 are expressed, usually together, at high levels by the pancreas but also by many other normal and malignant tissues. The present review describes studies on the expression and putative functions of trypsinogens and PSTI/TATI/SPINK1 in the human body. The clinical aspects are discussed, including the correlations between expression of trypsinogens and PSTI/TATI/SPINK1 in tissues, serum, and urine of patients with pancreatitis or cancer and clinicopathological characteristics, i.e., the roles of trypsinogens and PSTI/TATI/SPINK1 in spontaneous and hereditary pancreatitis, tumor progression, and prognosis.

Expression and characterization of trypsinogen produced in the human male genital tract.

Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon cancer and in vascular endothelial cells. In this study, we found that trypsinogen-1 and -2 are present at high concentrations (median levels, 0.4 and 0.5 mg/L, respectively) in human seminal fluid and purified them to homogeneity by immunoaffinity and anion exchange chromatography. Purified trypsinogen isoenzymes displayed a M(r) of 25 to 28 kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Most of the trypsinogen-1 purified from seminal fluid was enzymatically active whereas trypsinogen-2 occurred as the proform, which could be activated by enteropeptidase in vitro. Immunohistochemically, trypsinogen protein was detected in the human prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands, and testis. Expression of trypsinogen mRNA in the same organs was demonstrated by in situ hybridization. Trypsinogen mRNA was also detected in the prostate and seminal vesicles by reverse transcriptase-polymerase chain reaction and Northern blotting. Isolated trypsin was shown to activate the proenzyme form of prostate-specific antigen. These results suggest that trypsinogen isoenzymes found in seminal fluid are produced locally in the male genital tract and that they may play a physiological role in the semen.

MMP-9 Activation by Tumor Trypsin-2 Enhances in vivo Invasion of Human Tongue Carcinoma Cells

Various human cancer cells express tumor-associated trypsinogen-2 (TAT-2), which can efficiently activate matrix metalloproteinases (MMPs) in vitro. MMP-2 and MMP-9 are particularly associated with the invasive malignant potential of several tumors. To investigate the role of TAT-2 in tumor invasion, we overexpressed TAT-2 in two malignant human squamous cell carcinoma cell lines of tongue and in non-malignant human papilloma virus transformed gingival keratinocytes. The TAT-2 overexpression significantly increased the levels of active MMP-9 in the most malignant cell line. TAT-2-transfected cells intravasated (invaded blood vessels) up to 60% more efficiently than did the control cells in an in vivo chick embryo chorioallantoic membrane invasion model. This increased intravasation was almost completely abolished by a specific tumor-associated trypsin inhibitor (TATI). These results indicate that TAT-2 has a role in the invasive growth of tumors, either alone or in cascade with gelatinases, especially by generating active MMP-9.

Expression of Trypsinogen-1, Trypsinogen-2, and Tumor-Associated Trypsin Inhibitor in Ovarian Cancer Prognostic Study on Tissue and Serum

Purpose: The purpose is to study the prognostic significance of tissue expression of trypsinogen-1, trypsinogen-2, and tumor-associated trypsin inhibitor (TATI) and serum concentration of trypsinogen-2, trypsin-2-API (complex of trypsin-2 with α-1-proteinase inhibitor), and TATI in epithelial ovarian cancer. Experimental Design: Expression of trypsinogen-1, trypsinogen-2, and TATI was determined by immunohistochemistry with monoclonal antibodies in tissue sections of tumors from 119 patients with untreated primary epithelial ovarian cancer. Preoperative serum concentrations of trypsinogen-2, trypsin-2-API and TATI were analyzed using specific immunoassays. Results: Fifty-four percent of the tumors expressed trypsinogen-1, 45% expressed trypsinogen-2, and 30% expressed TATI. In patients with stage III and IV disease, TATI tissue expression (P = 0.002) and elevated TATI concentration in serum (P = 0.048) were associated with adverse cancer-specific and progression-free survival in univariate analysis. In multivariate analysis, TATI tissue expression (P = 0.005), tumor grade (P = 0.0001), histological type (P = 0.02), and stage (P = 0.0005) were independent prognostic factors for adverse cancer-specific survival and TATI tissue expression (P = 0.006) and grade (P = 0.0003) for progression-free survival. In multivariate analysis of all patients and those with advanced disease, serum trypsin-2-API concentration was an adverse prognostic factor for cancer-specific and progression-free survival, and it was independent of stage and histological type of the tumor (P ≤ 0.01). Conclusions: Tissue expression of TATI and an elevated preoperative serum concentration of trypsin-2-API are strong independent prognostic factors in advanced epithelial ovarian cancer. These results suggest that trypsin expression plays a role in the progression of ovarian cancer. TATI and trypsin-2-API are of potential use as an aid for stratification of randomized studies and for selecting treatment strategies.

Tumor associated trypsin inhibitor as a prognostic factor in renal cell carcinoma.

PURPOSE We analyzed the prognostic significance of pretreatment serum tumor associated trypsin inhibitor in renal cell carcinoma. MATERIALS AND METHODS Serum samples were obtained before surgery from 188 patients who underwent radical nephrectomy for renal cell carcinoma. Median followup of living patients was 8.5 years. Serum tumor associated trypsin inhibitor was measured by a time resolved immunofluorometric assay. Statistical analysis was performed using the Kaplan-Meier method, log rank and stratified log rank tests. RESULTS Preoperatively serum tumor associated trypsin inhibitor was elevated with a cutoff 16 microg/l in 48% of the patients with normal serum creatinine. The concentration in patients with cancer was significantly higher than in controls (p <0.0001). The serum level correlated with clinical stage and nuclear grade. Patients with an elevated level had significantly shorter survival time than those with a normal level (p = 0.005). Stratified log rank test demonstrated that tumor associated trypsin inhibitor was a prognostic factor independent of stage and grade in all patients as well as in those with nonmetastatic disease. CONCLUSIONS Increased preoperative serum tumor associated trypsin inhibitor was associated with poor survival in renal cell carcinoma. The serum level was an independent prognostic variable. Preoperative serum tumor associated trypsin inhibitor appears to be a useful predictive factor that may be used to identify patients at increased risk of aggressive disease.

Mutations N34S and P55S of the SPINK1 gene in patients with chronic pancreatitis or pancreatic cancer and in healthy subjects: A report from Finland

Objective Mutations in the Kazal type 1 serine protease inhibitor (SPINK1) gene have recently been associated with chronic pancreatitis (CP), an established risk factor for pancreatic cancer. The aim of this study was to investigate the frequency of the SPINK1 gene mutations (N34S and P55S) in patients with CP, or pancreatic cancer, and in healthy subjects in Finland. Material and methods The N34S and P55S mutations were determined by PCR amplification followed by solid-phase minisequencing in 116 patients with CP and in 188 with pancreatic cancer. In patients with CP, alcohol was the aetiological factor in 87 (75%), pancreas divisum in 4 (3%), gallstones in 5 (5%) and 20 patients (17%) had an idiopathic disease; 459 healthy individuals were enrolled as controls. Results The frequency of the N34S mutation was significantly higher in patients with CP (14/116, 12%) than in controls (12/459, 2.6%) (p<0.0001). There was no difference in the frequency of the P55S mutation between patients with CP (1/116, 0.9%) and controls (6/459, 1.3%). The N34S mutation was present in 9 (10%) out of 87 patients with alcoholic CP, and in 5 (25%) patients with idiopathic CP. No SPINK1 mutations were found in patients with CP caused by anatomical variations or gallstones. Among the 188 patients with a pancreatic malignant tumour, the N34S mutation was present in 7 cases (3.7%). The frequency of the N34S mutation in healthy controls in this study was significantly higher than earlier reported in other countries (p=0.03). Conclusions The SPINK1 N34S mutation was significantly associated with an increased risk of CP. The association of the N34S mutation with alcoholic CP was marginally stronger than in earlier studies, whereas in the Finnish population in general, this mutation was significantly more frequent than reported elsewhere.

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.

Expression of the free β-subunit of human chorionic gonadotropin in renal cell carcinoma: Prognostic study on tissue and serum

Expression of the free β‐subunit of human chorionic gonadotropin (hCGβ) in malignant tumors is frequently associated with aggressive disease. We have shown previously that the pretreatment serum concentration of hCGβ is an independent prognostic variable in patients with renal cell carcinoma (RCC). We now compare the serum levels with the expression of hCGβ antigen and mRNA in tumor tissue and studied whether these are associated with the clinical outcome. Serum samples were collected before surgery from patients with RCC (n = 256) and from 84 apparently healthy controls. HCGβ in serum was measured by a time‐resolved immunofluorometric assay. Tissue expression was detected by immunohistochemical staining of a tissue microarray (TMA) comprising 229 samples, and in selected cases by reverse transcription polymerase chain reaction (RT‐PCR) of hCGβ mRNA (n = 20) from tumor tissue. The prognostic value of hCGβ in serum and tissue and the association with usual clinicopathological variables was analyzed by the Kaplan‐Meier method, the log‐rank test, Cox multiple hazard regression, Mann‐Whitney U‐test or Kruskal‐Wallis test. The serum concentrations of hCGβ were increased in 27% of the RCC patients and patients with increased hCGβ levels had significantly shorter survival time than those with levels below the median (cut‐off 1.2 pmol l−1, p = 0.0044). HCGβ antigen was detected in 15% (35 of 229) of the tumors by immunohistochemistry, and hCGβ mRNA in 8 of 20 samples (40%) by RT‐PCR. Tissue positivity for hCGβ antigen was not associated significantly with mRNA expression, serum concentrations of hCGβ or survival. In multivariate analysis tumor stage, grade, size and serum hCGβ were independent prognostic variables. The serum concentration of hCGβ is an independent prognostic variable in RCC. Tissue expression of hCGβ detected by immunohistochemistry occurs in 15% of RCCs but it is not significantly associated with prognosis. Expression at the mRNA level seems to be associated with other predictors of adverse outcome.

New insights into juvenile parotitis

Aim: We enquired about the possibility of a familial trend in juvenile parotitis and evaluated the role of SPINK1 mutations in juvenile parotitis.

Food and Nutrient Intake and Nutritional Status of Finnish Vegans and Non-Vegetarians

Background Vegetarian and vegan diets have become more popular among adolescents and young adults. However, few studies have investigated the nutritional status of vegans, who may be at risk of nutritional deficiencies. Objective To compare dietary intake and nutritional status of Finnish long-term vegans and non-vegetarians. Methods Dietary intake and supplement use were estimated using three-day dietary records. Nutritional status was assessed by measuring biomarkers in plasma, serum, and urine samples. Vegans’ (n = 22) data was compared with those of sex- and age-matched non-vegetarians (n = 19). Results All vegans adhered strictly to their diet; however, individual variability was marked in food consumption and supplementation habits. Dietary intakes of key nutrients, vitamins B12 and D, were lower (P < 0.001) in vegans than in non-vegetarians. Nutritional biomarker measurements showed lower concentrations of serum 25-hydroxyvitamin D3 (25(OH)D3), iodine and selenium (corrected for multiple comparisons, P < 0.001), Vegans showed more favorable fatty acid profiles (P < 0.001) as well as much higher concentrations of polyphenols such as genistein and daidzein (P < 0.001). Eicosapentaenoic acid proportions in vegans were higher than expected. The median concentration of iodine in urine was below the recommended levels in both groups. Conclusions Long-term consumption of a vegan diet was associated with some favorable laboratory measures but also with lowered concentrations of key nutrients compared to reference values. This study highlights the need for nutritional guidance to vegans.