Hannu Koistinen - Academia.edu (original) (raw)

Papers by Hannu Koistinen

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with ... more Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with an increased risk of cholangiocarcinoma (CCA). Novel markers, to complement or replace CA19-9, are urgently needed for the screening of PSC-associated biliary neoplasia. Previous studies have suggested that serum trypsinogen-2 and human chorionic gonadotropin β-subunit (hCGβ) may serve as such markers. Using highly specific in-house immunoassays, we studied trypsin(ogen)-2 and -3, SPINK1 and hCGβ in bile samples of 214 patients, referred for endoscopic retrograde cholangiography. We found that biliary trypsinogen-2 was decreased (p = 0.027) and hCGβ was elevated (p < 0.001) in PSC patients who were diagnosed 1.6 years (median, range 0.1–8.8 years) later with CCA or in whom biliary dysplasia was observed at least twice in brush cytology (n = 11) as compared to PSC patients without CCA or repeated dysplasia (n = 171). The other studied markers did not show sign...

Livers

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with ... more Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with an increased risk of cholangiocarcinoma (CCA). Novel markers, to complement or replace CA19-9, are urgently needed for the screening of PSC-associated biliary neoplasia. Previous studies have suggested that serum trypsinogen-2 and human chorionic gonadotropin β-subunit (hCGβ) may serve as such markers. Using highly specific in-house immunoassays, we studied trypsin(ogen)-2 and -3, SPINK1 and hCGβ in bile samples of 214 patients, referred for endoscopic retrograde cholangiography. We found that biliary trypsinogen-2 was decreased (p = 0.027) and hCGβ was elevated (p < 0.001) in PSC patients who were diagnosed 1.6 years (median, range 0.1–8.8 years) later with CCA or in whom biliary dysplasia was observed at least twice in brush cytology (n = 11) as compared to PSC patients without CCA or repeated dysplasia (n = 171). The other studied markers did not show significant differences betwee...

International Journal of Molecular Sciences, 2021

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been s... more The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of β-endorphin and ...

Analytical and bioanalytical chemistry, 2018

Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da peptide produced by the... more Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da 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 w...

Clinical Chemistry, 2018

BACKGROUND Genetic association studies have reported single-nucleotide polymorphisms (SNPs) at ch... more BACKGROUND Genetic association studies have reported single-nucleotide polymorphisms (SNPs) at chromosome 19q13.3 to be associated with prostate cancer (PCa) risk. Recently, the rs61752561 SNP (Asp84Asn substitution) in exon 3 of the kallikrein-related peptidase 3 (KLK3) gene encoding prostate-specific antigen (PSA) was reported to be strongly associated with PCa risk (P = 2.3 × 10−8). However, the biological contribution of the rs61752561 SNP to PCa risk has not been elucidated. METHODS Recombinant PSA protein variants were generated to assess the SNP-mediated biochemical changes by stability and substrate activity assays. PC3 cell–PSA overexpression models were established to evaluate the effect of the SNP on PCa pathogenesis. Genotype-specific correlation of the SNP with total PSA (tPSA) concentrations and free/total (F/T) PSA ratio were determined from serum samples. RESULTS Functional analysis showed that the rs61752561 SNP affects PSA stability and structural conformation and ...

Molecular and Cellular Endocrinology, 2019

While human chorionic gonadotropin (hCG) appears to have an essential role in early pregnancy, it... more While human chorionic gonadotropin (hCG) appears to have an essential role in early pregnancy, it is controversial whether the hyperglycosylated form of hCG (hCG-h), which is the major hCG isoform during the first 4-5 weeks of pregnancy, is able to activate LH/hCG receptor (LHCGR). To address this, we utilized different extensively characterized hCG and hCGβ reference reagents, cell culture-and urine-derived hCG-h preparations, and an in vitro reporter system for LHCGR activation. The WHO hCG reference reagent (99/688) was found to activate LHCGR with an EC 50-value of 3.3 ± 0.6 pmol/L (n = 9). All three studied hCG-h preparations were also able to activate LHCGR, but with a lower potency (EC 50-values between 7.1 ± 0.5 and 14 ± 3 pmol/L, n = 5-11, for all P < 0.05 as compared to the hCG reference). The activities of commercial urinary hCG (Pregnyl) and recombinant hCG (Ovitrelle) preparations were intermediate between those of the hCG reference and the hCG-h. These results strongly suggest that the hCG-h is functionally similar to hCG, although it has lower potency for LHCGR activation. Whether this explains the reduced proportion of hCG-h to hCG reported in patients developing early onset pre-eclampsia or those having early pregnancy loss remains to be determined.

Environmental health perspectives, Oct 31, 2017

Observational studies have reported associations between maternal phthalate levels and adverse ou... more Observational studies have reported associations between maternal phthalate levels and adverse outcomes at birth and in the health of the child. Effects on placental function have been suggested as a biologic basis for these findings. We evaluated the effects of phthalates on placental function in vitro by measuring relevant candidate genes and proteins. Human trophoblast progenitor cells were isolated at 7-14 wk of pregnancy (two female and three male concepti), and villous cytotrophoblast cells (vCTBs) were isolated at 15-20 wk (three female and four male concepti). Cells were cultured in vitro with four phthalate metabolites and their combination at concentrations based on levels found previously in the urine of pregnant women: mono-n-butyl (MnBP, 200 nM), monobenzyl (MBzP, 3μM), mono-2-ethylhexyl (MEHP, 700 nM), and monoethyl (MEP, 1.5μM) phthalates. mRNA levels of CGA, CGB, PPARG, CYP19A1, CYP11A1, PTGS2, EREG, and the intracellular β subunit of human chorionic gonadotropin (hC...

Molecular oncology, Feb 28, 2017

The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (C... more The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N = 571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted in vitro studies using ...

Atlas of Genetics and Cytogenetics in Oncology and Haematology, 2017

Review on SPINK1, with data on DNA, on the protein encoded, and where the gene is implicated.

Molecular endocrinology (Baltimore, Md.), Jan 23, 2015

Recently several LH/hCG receptor-independent activities for human chorionic gondotropin (hCG) hav... more Recently several LH/hCG receptor-independent activities for human chorionic gondotropin (hCG) have been described, including activation of the TGF-β receptor (TGFβR) by hyperglycosylated hCG (hCG-h) and stimulation of trophoblast invasion. As the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, that act at very low concentrations, may be significant. Several commercial hCG preparation have been found to contain significant amounts of EGF, which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-β1. These hCG preparations were able to activate extracellular signal-regulated kinases 1/2 (ERK1/2) in JEG-3 choriocarcinoma cells or TGFβR in mink lung epithelial cells transfected with a reporter gene for TGFβR activation. No such activation was found with highly purified hCG or its free β-subunit (hCGβ), irrespective...

Cancer Research, 2013

Human chorionic gonadotropin (hCG) is a heterodimer consisting of an α- and a β-subunit (hCGα and... more Human chorionic gonadotropin (hCG) is a heterodimer consisting of an α- and a β-subunit (hCGα and hCGβ) that are non-covalently linked. Intact hCG, but not free subunits, bind to the LH/hCG receptor. Expression of hCG and hCGβ has been observed in several cancers. Most trophoblastic tumors of placental and germ cell origin produce hCG, while many non-trophoblastic tumors, including bladder, renal and gastrointestinal cancers, often produce hCGβ, and increased serum concentrations of these are associated with adverse prognosis. hCG has been found to stimulate trophoblast invasion. The stimulatory activity has especially been ascribed to a hyperglycosylated form of hCG (hCG-h) expressed in early pregnancy. hCG-h is also a major form of hCG produced by choriocarcinoma and by testicular cancers, although tumor-derived hCG-h is often glycosylated differently than that produced by the placenta. We aimed to compare different forms of hCG in their stimulatory activities on the invasion of J...

The Prostate, 2012

BACKGROUND. Proteolytically active prostate-specific antigen (PSA or kallikrein-related peptidase... more BACKGROUND. Proteolytically active prostate-specific antigen (PSA or kallikrein-related peptidase 3, KLK3) has been shown to exert antiangiogenic properties. High levels of PSA in prostatic tumors may thus slow down cancer progression by inhibiting angiogenesis. We hypothesize that factors stimulating the activity of PSA could be used to reduce prostate tumor growth. Using phage display, we have developed peptides C4 and B2 that stimulate the enzymatic activity of PSA. Our aim was to study whether these peptides enhance the antiangiogenic activity of PSA. METHODS. We used an in vitro angiogenesis assay where human umbilical vein endothelial cells (HUVECs) form tubular networks when they are grown on Matrigel. Proteolytically active PSA and peptides that stimulate the activity of PSA were added to the cells. Endothelial cell tube formation was quantified and expressed as an angiogenesis index. RESULTS. PSA reduced the angiogenesis index to 5050% of controls both in serum-containing and serum-free medium. The addition of peptide C4 or B2 together with PSA caused a significant further decrease in angiogenesis index to 5070% of that caused by PSA alone. A similar decrease in angiogenesis index was observed when PSA concentration was increased 2.4-fold of that used with peptides. CONCLUSIONS. The inhibitory effect of PSA on tube formation can be enhanced by the addition of peptides that stimulate the activity of PSA. This supports our hypothesis that stimulation of PSA activity can be used to reduce angiogenesis and thereby inhibit prostate tumor growth. Prostate

The Prostate, 2012

BACKGROUND. PSA is the most useful prostate cancer marker. However, its levels are increased also... more BACKGROUND. PSA is the most useful prostate cancer marker. However, its levels are increased also in some non-malignant conditions. In circulation, the majority of PSA is complexed with protease inhibitors, including a 1-antichymotrypsin (ACT). The proportion of the PSA-ACT complex is higher in patients with prostate cancer than in controls without cancer. The expression of ACT has been shown to be higher in prostate cancer than in benign prostatic hyperplasia. However, results regarding the extent which PSA forms complexes within the prostate and whether there are differences in complex formation between normal and malignant prostatic tissue are inconsistent and limited. METHODS. We studied complex formation of PSA secreted by cultured human prostate tissues and in the tissue by in situ proximity ligation assay (PLA). Free, total and active PSA, and the PSA-ACT complex were determined in tissue culture media by immunoassays, immunoblotting, and chromatographic methods. RESULTS. The majority of PSA in tissue culture medium was free and enzymatically active. However, a significant proportion (1.6 AE 0.5%) of immunoreactive PSA was found to be complexed with ACT. Complex formation was confirmed by in situ PLA, which showed more intense staining of PSA-ACT in cancers with Gleason grade 3 than in adjacent benign tissues from the same patients. CONCLUSIONS. These results show that PSA forms complexes already within the prostate and that PSA-ACT levels are increased in moderately differentiated prostate cancer tissue. This may explain, at least partially, why the ratio of serum PSA-ACT to total PSA is increased in prostate cancer. Prostate

Neuroscience, 2009

Trypsin and other trypsin-like serine proteases have been shown to play important roles in neural... more Trypsin and other trypsin-like serine proteases have been shown to play important roles in neural development, plasticity and neurodegeneration. Their activity is modulated by serine protease inhibitors, serpins. However, for human brain trypsin, trypsin-4, no brain-derived inhibitors have been described. Here, we report that nexin-1 inhibits trypsin-4, and forms stable complexes only with this trypsinisoenzyme. This result suggests that nexin-1 could modulate trypsin activity in brain where both nexin-1 and trypsin-4 are expressed.

The Journal of Urology, 1999

Journal of Peptide Science, 2007

Human glandular kallikrein (KLK2) is a highly prostate-specific serine protease, which is mainly ... more Human glandular kallikrein (KLK2) is a highly prostate-specific serine protease, which is mainly excreted into the seminal fluid, but part of which is also secreted into circulation from prostatic tumors. Since the expression level of KLK2 is elevated in aggressive tumors and it has been suggested to mediate the metastasis of prostate cancer, inhibition of the proteolytic activity of KLK2 is of potential therapeutic value. We have previously identified several KLK2-specific linear peptides by phage display technology. Two of its synthetic analogs, A R R P A P A P G (KLK2a) and G A A R F K V W W A A G (KLK2b), show specific inhibition of KLK2 but their sensitivity to proteolysis in vivo may restrict their potential use as therapeutic agents. In order to improve the stability of the linear peptides for in vivo use, we have prepared cyclic analogs and compared their biological activity and their structural stability. A series of cyclic variants with cysteine bridges were synthesized. Cyclization inactivated one peptide (KLK2a) and its derivatives, while the other peptide (KLK2b) and its derivatives remained active. Furthermore, backbone cyclization of KLK2b improved significantly the resistance against proteolysis by trypsin and human plasma. Nuclear magnetic resonance studies showed that cyclization of the KLK2b peptides does not make the structures more rigid. In conclusion, we have shown that backbone cyclization of KLK2 inhibitory peptides can be used to increase stability without losing biological activity. This should render the peptides more useful for in vivo applications, such as tumor imaging and prostate cancer targeting.

The Journal of Clinical Endocrinology & Metabolism, 2000

High serum levels of insulin-like growth factor I (IGF-I) and low levels of IGF-binding protein-3... more High serum levels of insulin-like growth factor I (IGF-I) and low levels of IGF-binding protein-3 (IGFBP-3) have been shown to correlate with increased prostate cancer risk. To evaluate this, IGF-I, IGFBP-3, and prostate-specific antigen (PSA) were measured in serum from 665 consecutive men (179 with prostate cancer), aged 55–67 yr, with elevated serum prostate-specific antigen (PSA; ≥4 μg/L) in a screening trial. Men in the highest quartile of IGF-I levels had an odds ratio (OR) for prostate cancer of 0.50 [95% confidence interval (CI) 0.26–0.97] when adjusting for serum IGFBP-3. IGFBP-3 itself was not significantly associated with prostate cancer risk (OR, 1.24; 95% CI, 0.68–2.24). Prostate volume was larger in men without than in those with prostate cancer (P < 0.001), and after adjustment for prostate volume, the negative association between serum IGF-I and prostate cancer risk was no longer significant (OR, 0.57; 95% CI, 0.28–1.16). In screen-positive men with elevated serum...

Clinical Chemistry, 2011

BACKGROUND Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin... more BACKGROUND Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin 1 and 2, trypsin 3 degrades pancreatic secretory trypsin inhibitor, which may lead to an excess of active trypsin and acute pancreatitis (AP). We developed an immunoassay for trypsinogen 3 and studied whether an assay of serum trypsinogen 3 is of clinical utility in the diagnosis of AP. METHODS Monoclonal antibodies were generated using recombinant human trypsinogen 3 as the antigen and used to establish a sandwich-type immunoassay. We analyzed serum trypsinogen 3 concentrations in 82 patients with AP and 63 patients with upper abdominal pain (controls). The reference interval was determined using serum samples from 172 apparently healthy individuals. RESULTS The measuring range of the trypsinogen 3 assay was 1.0–250 μg/L. Intra- and interassay CVs were <11%, and cross-reactivity with other trypsinogen isoenzymes was <0.1%. The median trypsinogen 3 concentration in serum from heal...

Clinical Chemistry, 2009

Background: Prostate specific antigen (PSA)–α1-protease inhibitor complex (PSA-API) is a minor fo... more Background: Prostate specific antigen (PSA)–α1-protease inhibitor complex (PSA-API) is a minor form of PSA in serum. It may be useful for prostate cancer (PCa) diagnosis, but its specific detection is hampered by nonspecific background. To avoid this, we developed an immunoassay for PSA-API based on proximity ligation. Methods: We used a monoclonal antibody (mAb) to total PSA (tPSA) to capture PSA, while using another anti-tPSA mAb together with an anti-API mAb as probes. We measured PSA-API by quantification of amplified DNA strands conjugated to the probes. We measured serum PSA-API in 84 controls and 55 men with PCa who had PSA concentrations of 4.0–10 μg/L. Results: The detection limit of the assay was 6.6 ng/L. The proportion of PSA-API to tPSA (%PSA-API) tended to be lower in men with PCa (2.8%) than without cancer (3.3%) but was not statistically significant (P = 0.363). When used alone, %PSA-API [area under the curve (AUC) 0.546] did not improve detection of PCa, whereas %fP...

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with ... more Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with an increased risk of cholangiocarcinoma (CCA). Novel markers, to complement or replace CA19-9, are urgently needed for the screening of PSC-associated biliary neoplasia. Previous studies have suggested that serum trypsinogen-2 and human chorionic gonadotropin β-subunit (hCGβ) may serve as such markers. Using highly specific in-house immunoassays, we studied trypsin(ogen)-2 and -3, SPINK1 and hCGβ in bile samples of 214 patients, referred for endoscopic retrograde cholangiography. We found that biliary trypsinogen-2 was decreased (p = 0.027) and hCGβ was elevated (p < 0.001) in PSC patients who were diagnosed 1.6 years (median, range 0.1–8.8 years) later with CCA or in whom biliary dysplasia was observed at least twice in brush cytology (n = 11) as compared to PSC patients without CCA or repeated dysplasia (n = 171). The other studied markers did not show sign...

Livers

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with ... more Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with an increased risk of cholangiocarcinoma (CCA). Novel markers, to complement or replace CA19-9, are urgently needed for the screening of PSC-associated biliary neoplasia. Previous studies have suggested that serum trypsinogen-2 and human chorionic gonadotropin β-subunit (hCGβ) may serve as such markers. Using highly specific in-house immunoassays, we studied trypsin(ogen)-2 and -3, SPINK1 and hCGβ in bile samples of 214 patients, referred for endoscopic retrograde cholangiography. We found that biliary trypsinogen-2 was decreased (p = 0.027) and hCGβ was elevated (p < 0.001) in PSC patients who were diagnosed 1.6 years (median, range 0.1–8.8 years) later with CCA or in whom biliary dysplasia was observed at least twice in brush cytology (n = 11) as compared to PSC patients without CCA or repeated dysplasia (n = 171). The other studied markers did not show significant differences betwee...

International Journal of Molecular Sciences, 2021

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been s... more The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of β-endorphin and ...

Analytical and bioanalytical chemistry, 2018

Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da peptide produced by the... more Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da 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 w...

Clinical Chemistry, 2018

BACKGROUND Genetic association studies have reported single-nucleotide polymorphisms (SNPs) at ch... more BACKGROUND Genetic association studies have reported single-nucleotide polymorphisms (SNPs) at chromosome 19q13.3 to be associated with prostate cancer (PCa) risk. Recently, the rs61752561 SNP (Asp84Asn substitution) in exon 3 of the kallikrein-related peptidase 3 (KLK3) gene encoding prostate-specific antigen (PSA) was reported to be strongly associated with PCa risk (P = 2.3 × 10−8). However, the biological contribution of the rs61752561 SNP to PCa risk has not been elucidated. METHODS Recombinant PSA protein variants were generated to assess the SNP-mediated biochemical changes by stability and substrate activity assays. PC3 cell–PSA overexpression models were established to evaluate the effect of the SNP on PCa pathogenesis. Genotype-specific correlation of the SNP with total PSA (tPSA) concentrations and free/total (F/T) PSA ratio were determined from serum samples. RESULTS Functional analysis showed that the rs61752561 SNP affects PSA stability and structural conformation and ...

Molecular and Cellular Endocrinology, 2019

While human chorionic gonadotropin (hCG) appears to have an essential role in early pregnancy, it... more While human chorionic gonadotropin (hCG) appears to have an essential role in early pregnancy, it is controversial whether the hyperglycosylated form of hCG (hCG-h), which is the major hCG isoform during the first 4-5 weeks of pregnancy, is able to activate LH/hCG receptor (LHCGR). To address this, we utilized different extensively characterized hCG and hCGβ reference reagents, cell culture-and urine-derived hCG-h preparations, and an in vitro reporter system for LHCGR activation. The WHO hCG reference reagent (99/688) was found to activate LHCGR with an EC 50-value of 3.3 ± 0.6 pmol/L (n = 9). All three studied hCG-h preparations were also able to activate LHCGR, but with a lower potency (EC 50-values between 7.1 ± 0.5 and 14 ± 3 pmol/L, n = 5-11, for all P < 0.05 as compared to the hCG reference). The activities of commercial urinary hCG (Pregnyl) and recombinant hCG (Ovitrelle) preparations were intermediate between those of the hCG reference and the hCG-h. These results strongly suggest that the hCG-h is functionally similar to hCG, although it has lower potency for LHCGR activation. Whether this explains the reduced proportion of hCG-h to hCG reported in patients developing early onset pre-eclampsia or those having early pregnancy loss remains to be determined.

Environmental health perspectives, Oct 31, 2017

Observational studies have reported associations between maternal phthalate levels and adverse ou... more Observational studies have reported associations between maternal phthalate levels and adverse outcomes at birth and in the health of the child. Effects on placental function have been suggested as a biologic basis for these findings. We evaluated the effects of phthalates on placental function in vitro by measuring relevant candidate genes and proteins. Human trophoblast progenitor cells were isolated at 7-14 wk of pregnancy (two female and three male concepti), and villous cytotrophoblast cells (vCTBs) were isolated at 15-20 wk (three female and four male concepti). Cells were cultured in vitro with four phthalate metabolites and their combination at concentrations based on levels found previously in the urine of pregnant women: mono-n-butyl (MnBP, 200 nM), monobenzyl (MBzP, 3μM), mono-2-ethylhexyl (MEHP, 700 nM), and monoethyl (MEP, 1.5μM) phthalates. mRNA levels of CGA, CGB, PPARG, CYP19A1, CYP11A1, PTGS2, EREG, and the intracellular β subunit of human chorionic gonadotropin (hC...

Molecular oncology, Feb 28, 2017

The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (C... more The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N = 571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted in vitro studies using ...

Atlas of Genetics and Cytogenetics in Oncology and Haematology, 2017

Review on SPINK1, with data on DNA, on the protein encoded, and where the gene is implicated.

Molecular endocrinology (Baltimore, Md.), Jan 23, 2015

Recently several LH/hCG receptor-independent activities for human chorionic gondotropin (hCG) hav... more Recently several LH/hCG receptor-independent activities for human chorionic gondotropin (hCG) have been described, including activation of the TGF-β receptor (TGFβR) by hyperglycosylated hCG (hCG-h) and stimulation of trophoblast invasion. As the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, that act at very low concentrations, may be significant. Several commercial hCG preparation have been found to contain significant amounts of EGF, which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-β1. These hCG preparations were able to activate extracellular signal-regulated kinases 1/2 (ERK1/2) in JEG-3 choriocarcinoma cells or TGFβR in mink lung epithelial cells transfected with a reporter gene for TGFβR activation. No such activation was found with highly purified hCG or its free β-subunit (hCGβ), irrespective...

Cancer Research, 2013

Human chorionic gonadotropin (hCG) is a heterodimer consisting of an α- and a β-subunit (hCGα and... more Human chorionic gonadotropin (hCG) is a heterodimer consisting of an α- and a β-subunit (hCGα and hCGβ) that are non-covalently linked. Intact hCG, but not free subunits, bind to the LH/hCG receptor. Expression of hCG and hCGβ has been observed in several cancers. Most trophoblastic tumors of placental and germ cell origin produce hCG, while many non-trophoblastic tumors, including bladder, renal and gastrointestinal cancers, often produce hCGβ, and increased serum concentrations of these are associated with adverse prognosis. hCG has been found to stimulate trophoblast invasion. The stimulatory activity has especially been ascribed to a hyperglycosylated form of hCG (hCG-h) expressed in early pregnancy. hCG-h is also a major form of hCG produced by choriocarcinoma and by testicular cancers, although tumor-derived hCG-h is often glycosylated differently than that produced by the placenta. We aimed to compare different forms of hCG in their stimulatory activities on the invasion of J...

The Prostate, 2012

BACKGROUND. Proteolytically active prostate-specific antigen (PSA or kallikrein-related peptidase... more BACKGROUND. Proteolytically active prostate-specific antigen (PSA or kallikrein-related peptidase 3, KLK3) has been shown to exert antiangiogenic properties. High levels of PSA in prostatic tumors may thus slow down cancer progression by inhibiting angiogenesis. We hypothesize that factors stimulating the activity of PSA could be used to reduce prostate tumor growth. Using phage display, we have developed peptides C4 and B2 that stimulate the enzymatic activity of PSA. Our aim was to study whether these peptides enhance the antiangiogenic activity of PSA. METHODS. We used an in vitro angiogenesis assay where human umbilical vein endothelial cells (HUVECs) form tubular networks when they are grown on Matrigel. Proteolytically active PSA and peptides that stimulate the activity of PSA were added to the cells. Endothelial cell tube formation was quantified and expressed as an angiogenesis index. RESULTS. PSA reduced the angiogenesis index to 5050% of controls both in serum-containing and serum-free medium. The addition of peptide C4 or B2 together with PSA caused a significant further decrease in angiogenesis index to 5070% of that caused by PSA alone. A similar decrease in angiogenesis index was observed when PSA concentration was increased 2.4-fold of that used with peptides. CONCLUSIONS. The inhibitory effect of PSA on tube formation can be enhanced by the addition of peptides that stimulate the activity of PSA. This supports our hypothesis that stimulation of PSA activity can be used to reduce angiogenesis and thereby inhibit prostate tumor growth. Prostate

The Prostate, 2012

BACKGROUND. PSA is the most useful prostate cancer marker. However, its levels are increased also... more BACKGROUND. PSA is the most useful prostate cancer marker. However, its levels are increased also in some non-malignant conditions. In circulation, the majority of PSA is complexed with protease inhibitors, including a 1-antichymotrypsin (ACT). The proportion of the PSA-ACT complex is higher in patients with prostate cancer than in controls without cancer. The expression of ACT has been shown to be higher in prostate cancer than in benign prostatic hyperplasia. However, results regarding the extent which PSA forms complexes within the prostate and whether there are differences in complex formation between normal and malignant prostatic tissue are inconsistent and limited. METHODS. We studied complex formation of PSA secreted by cultured human prostate tissues and in the tissue by in situ proximity ligation assay (PLA). Free, total and active PSA, and the PSA-ACT complex were determined in tissue culture media by immunoassays, immunoblotting, and chromatographic methods. RESULTS. The majority of PSA in tissue culture medium was free and enzymatically active. However, a significant proportion (1.6 AE 0.5%) of immunoreactive PSA was found to be complexed with ACT. Complex formation was confirmed by in situ PLA, which showed more intense staining of PSA-ACT in cancers with Gleason grade 3 than in adjacent benign tissues from the same patients. CONCLUSIONS. These results show that PSA forms complexes already within the prostate and that PSA-ACT levels are increased in moderately differentiated prostate cancer tissue. This may explain, at least partially, why the ratio of serum PSA-ACT to total PSA is increased in prostate cancer. Prostate

Neuroscience, 2009

Trypsin and other trypsin-like serine proteases have been shown to play important roles in neural... more Trypsin and other trypsin-like serine proteases have been shown to play important roles in neural development, plasticity and neurodegeneration. Their activity is modulated by serine protease inhibitors, serpins. However, for human brain trypsin, trypsin-4, no brain-derived inhibitors have been described. Here, we report that nexin-1 inhibits trypsin-4, and forms stable complexes only with this trypsinisoenzyme. This result suggests that nexin-1 could modulate trypsin activity in brain where both nexin-1 and trypsin-4 are expressed.

The Journal of Urology, 1999

Journal of Peptide Science, 2007

Human glandular kallikrein (KLK2) is a highly prostate-specific serine protease, which is mainly ... more Human glandular kallikrein (KLK2) is a highly prostate-specific serine protease, which is mainly excreted into the seminal fluid, but part of which is also secreted into circulation from prostatic tumors. Since the expression level of KLK2 is elevated in aggressive tumors and it has been suggested to mediate the metastasis of prostate cancer, inhibition of the proteolytic activity of KLK2 is of potential therapeutic value. We have previously identified several KLK2-specific linear peptides by phage display technology. Two of its synthetic analogs, A R R P A P A P G (KLK2a) and G A A R F K V W W A A G (KLK2b), show specific inhibition of KLK2 but their sensitivity to proteolysis in vivo may restrict their potential use as therapeutic agents. In order to improve the stability of the linear peptides for in vivo use, we have prepared cyclic analogs and compared their biological activity and their structural stability. A series of cyclic variants with cysteine bridges were synthesized. Cyclization inactivated one peptide (KLK2a) and its derivatives, while the other peptide (KLK2b) and its derivatives remained active. Furthermore, backbone cyclization of KLK2b improved significantly the resistance against proteolysis by trypsin and human plasma. Nuclear magnetic resonance studies showed that cyclization of the KLK2b peptides does not make the structures more rigid. In conclusion, we have shown that backbone cyclization of KLK2 inhibitory peptides can be used to increase stability without losing biological activity. This should render the peptides more useful for in vivo applications, such as tumor imaging and prostate cancer targeting.

The Journal of Clinical Endocrinology & Metabolism, 2000

High serum levels of insulin-like growth factor I (IGF-I) and low levels of IGF-binding protein-3... more High serum levels of insulin-like growth factor I (IGF-I) and low levels of IGF-binding protein-3 (IGFBP-3) have been shown to correlate with increased prostate cancer risk. To evaluate this, IGF-I, IGFBP-3, and prostate-specific antigen (PSA) were measured in serum from 665 consecutive men (179 with prostate cancer), aged 55–67 yr, with elevated serum prostate-specific antigen (PSA; ≥4 μg/L) in a screening trial. Men in the highest quartile of IGF-I levels had an odds ratio (OR) for prostate cancer of 0.50 [95% confidence interval (CI) 0.26–0.97] when adjusting for serum IGFBP-3. IGFBP-3 itself was not significantly associated with prostate cancer risk (OR, 1.24; 95% CI, 0.68–2.24). Prostate volume was larger in men without than in those with prostate cancer (P < 0.001), and after adjustment for prostate volume, the negative association between serum IGF-I and prostate cancer risk was no longer significant (OR, 0.57; 95% CI, 0.28–1.16). In screen-positive men with elevated serum...

Clinical Chemistry, 2011

BACKGROUND Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin... more BACKGROUND Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin 1 and 2, trypsin 3 degrades pancreatic secretory trypsin inhibitor, which may lead to an excess of active trypsin and acute pancreatitis (AP). We developed an immunoassay for trypsinogen 3 and studied whether an assay of serum trypsinogen 3 is of clinical utility in the diagnosis of AP. METHODS Monoclonal antibodies were generated using recombinant human trypsinogen 3 as the antigen and used to establish a sandwich-type immunoassay. We analyzed serum trypsinogen 3 concentrations in 82 patients with AP and 63 patients with upper abdominal pain (controls). The reference interval was determined using serum samples from 172 apparently healthy individuals. RESULTS The measuring range of the trypsinogen 3 assay was 1.0–250 μg/L. Intra- and interassay CVs were <11%, and cross-reactivity with other trypsinogen isoenzymes was <0.1%. The median trypsinogen 3 concentration in serum from heal...

Clinical Chemistry, 2009

Background: Prostate specific antigen (PSA)–α1-protease inhibitor complex (PSA-API) is a minor fo... more Background: Prostate specific antigen (PSA)–α1-protease inhibitor complex (PSA-API) is a minor form of PSA in serum. It may be useful for prostate cancer (PCa) diagnosis, but its specific detection is hampered by nonspecific background. To avoid this, we developed an immunoassay for PSA-API based on proximity ligation. Methods: We used a monoclonal antibody (mAb) to total PSA (tPSA) to capture PSA, while using another anti-tPSA mAb together with an anti-API mAb as probes. We measured PSA-API by quantification of amplified DNA strands conjugated to the probes. We measured serum PSA-API in 84 controls and 55 men with PCa who had PSA concentrations of 4.0–10 μg/L. Results: The detection limit of the assay was 6.6 ng/L. The proportion of PSA-API to tPSA (%PSA-API) tended to be lower in men with PCa (2.8%) than without cancer (3.3%) but was not statistically significant (P = 0.363). When used alone, %PSA-API [area under the curve (AUC) 0.546] did not improve detection of PCa, whereas %fP...