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The proline-specific enzymes dipeptidyl peptidase 4 (DPP4), prolylcarboxypeptidase (PRCP), fibroblast activation protein α (FAP) and prolyl oligopeptidase (PREP) are known for their involvement in the immune system and blood pressure regulation. Only very limited information is currently available on their enzymatic activity and possible involvement in patients with sepsis and septic-shock. The activity of the enzymes was measured in EDTA-plasma of patients admitted to the intensive care unit (ICU): 40 septic shock patients (sepsis-2) and 22 ICU control patients after major intracranial surgery. These data were used to generate receiver operating characteristic (ROC) curves. A survival analysis (at 90 days) and an association study with other parameters was performed. PRCP (day 1) and PREP (all days) enzymatic activities were higher in septic shock patients compared to controls. In contrast, FAP and DPP4 were lower in these patients on all studied time points. Since large differences were found, ROC curves were generated and these yielded area under the curve (AUC) values for PREP, FAP and DPP4 of 0.88 (CI: 0.80-0.96), 0.94 (CI: 0.89-0.99) and 0.86 (CI: 0.77-0.95), respectively. PRCP had a lower predicting value with an AUC of 0.71 (CI: 0.58-0.83). A nominally significant association was observed between survival and the DPP4 enzymatic activity at day 1 (p<0.05), with a higher DPP4 activity being associated with an increase in survival. All four enzymes were dysregulated in septic shock patients. DPP4, FAP and PREP are good in discriminating between septic shock patients and ICU controls and should be further explored to see whether they are already dysregulated in earlier stages, opening perspectives for their further investigation as biomarkers in sepsis. DPP4 also shows potential as a prognostic biomarker. Additionally, the associations found warrant further research., Competing Interests: The authors have declared that no competing interests exist.

Background: Prolyl carboxypeptidase (PRCP) is involved in the regulation of body weight, likely by hydrolysing alpha-melanocyte-stimulating hormone and apelin in the hypothalamus and in the periphery. A link between PRCP protein concentrations in plasma and metabolic disorders has been reported. In this study, we investigated the distribution of circulating PRCP activity and assessed its relation with body weight and adipose tissue in obese patients and patients who significantly lost weight., Methods: PRCP activity was measured using reversed-phase high-performance liquid chromatography in different isolated blood fractions and primary human cells to investigate the distribution of circulating PRCP. PRCP activity was measured in serum of individuals (n = 75) categorized based on their body mass index (BMI < 25.0; 25.0-29.9; 30.0-39.9; ≥ 40.0 kg/m2) and the diagnosis of metabolic syndrome. Differences in serum PRCP activity were determined before and six months after weight loss, either by diet (n = 45) or by bariatric surgery (n = 24). Potential correlations between serum PRCP activity and several metabolic and biochemical parameters were assessed. Additionally, plasma PRCP concentrations were quantified using a sensitive ELISA in the bariatric surgery group., Results: White blood cells and plasma contributed the most to circulating PRCP activity. Serum PRCP activity in lean subjects was 0.83 ± 0.04 U/L and increased significantly with a rising BMI (p<0.001) and decreased upon weight loss (diet, p<0.05; bariatric surgery, p<0.001). The serum PRCP activity alteration reflected body weight changes and was found to be positively correlated with several metabolic parameters, including: total, abdominal and visceral adipose tissue. Plasma PRCP concentration was found to be significantly correlated to serum PRCP activity (0.865; p<0.001). Additionally, a significant decrease (p<0.001) in plasma PRCP protein concentration (mean ± SD) before (18.2 ± 3.7 ng/mL) and 6 months after bariatric surgery (15.7 ± 2.7 ng/mL) was found., Conclusion: Our novel findings demonstrate that white blood cells and plasma contributed the most to circulating PRCP activity. Additionally, we have shown that there were significant correlations between serum PRCP activity and various metabolic parameters, and that plasma PRCP concentration was significantly correlated to serum PRCP activity. These novel findings on PRCP activity in serum support further investigation of its in vivo role and involvement in several metabolic diseases., Competing Interests: The authors have declared that no competing interests exist.

Chronic low back pain (CLBP) is one of the most common medical conditions, ranking as the greatest contributor to global disability and accounting for huge societal costs based on the Global Burden of Disease 2010 study. Large genetic and -omics studies provide a promising avenue for the screening, development and validation of biomarkers useful for personalized diagnosis and treatment (precision medicine). Multicentre studies are needed for such an effort, and a standardized and homogeneous approach is vital for recruitment of large numbers of participants among different centres (clinical and laboratories) to obtain robust and reproducible results. To date, no validated standard operating procedures (SOPs) for genetic/-omics studies in chronic pain have been developed. In this study, we validated an SOP model that will be used in the multicentre (5 centres) retrospective "PainOmics" study, funded by the European Community in the 7th Framework Programme, which aims to develop new biomarkers for CLBP through three different -omics approaches: genomics, glycomics and activomics. The SOPs describe the specific procedures for (1) blood collection, (2) sample processing and storage, (3) shipping details and (4) cross-check testing and validation before assays that all the centres involved in the study have to follow. Multivariate analysis revealed the absolute specificity and homogeneity of the samples collected by the five centres for all genetics, glycomics and activomics analyses. The SOPs used in our multicenter study have been validated. Hence, they could represent an innovative tool for the correct management and collection of reliable samples in other large-omics-based multicenter studies.

Tumor metastasis is the leading cause of cancer death. In the metastatic process, EMT is a unique phenotypic change that plays an important role in cell invasion and changes in cell morphology. Despite the clinical significance, the mechanism underlying tumor metastasis is still poorly understood. Here we report a novel mechanism by which secreted plasma glutamate carboxypeptidase(PGCP) negatively involves Wnt/β-catenin signaling by DKK4 regulation in liver cancer metastasis. Pathway analysis of the RNA sequencing data showed that PGCP knockdown in liver cancer cell lines enriched the functions of cell migration, motility and mesenchymal cell differentiation. Depletion of PGCP promoted cell migration and invasion via activation of Wnt/β-catenin signaling pathway components such as phospho-LRP6 and β-catenin. Also, addition of DKK4 antagonized the Wnt/β-catenin signaling cascade in a thyroxine (T4)-dependent manner. In an in vivo study, metastatic nodules were observed in the lungs of the mice after injection of shPGCP stable cell lines. Our findings suggest that PGCP negatively associates with Wnt/β-catenin signaling during metastasis. Targeting this regulation may represent a novel and effective therapeutic option for liver cancer by preventing metastatic activity of primary tumor cells.

MicroRNAs (miRNAs) are short non-coding RNAs involved in biological and pathological processes of every cell type, including liver cells. Transcribed from specific genes, miRNA precursors are processed in the cytoplasm into mature miRNAs and as part of the RNA-induced silencing complex (RISC) complex binds to messenger RNA (mRNA) by imperfect complementarity. This leads to the regulation of gene expression at a post-transcriptional level. The function of a number of different miRNAs in fibrogenesis associated with the progression of chronic liver disease has recently been elucidated. Furthermore, miRNAs have been shown to be both disease-and tissue-specific and are stable in the circulation, which has led to increasing investigation on their utility as biomarkers for the diagnosis of chronic liver diseases, including those in children. Here, we review the current knowledge on the biogenesis of microRNA, the mechanisms of translational repression and the use of miRNA as circulatory biomarkers in chronic paediatric liver diseases including cystic fibrosis associated liver disease, biliary atresia and viral hepatitis B., Competing Interests: The authors declare no conflict of interest.

Unlabelled: Aim/Purpose of the Study: Activation of the renin-angiotensin system leading to increased angiotensin-(1-7) (Ang-(1-7)) and decreased angiotensin 2 (Ang 2) levels may be a new therapeutic approach to reduce acute lung injury. Prolylcarboxypeptidase (PRCP) and prolyloligopeptidase (PREP) are capable of hydrolyzing Ang 2 into Ang-(1-7). However, their relation with circulating Ang 2 levels after lung ischemia-reperfusion injury (LIRI) has never been explored. This study determines whether the activity and expression of PRCP and PREP in plasma and lung tissue is related to circulating Ang 2 levels in a murine model of LIRI., Materials and Methods: LIRI in Swiss mice (6 animals per group) was induced by temporary left lung hilar clamping (1 h) followed by 0, 1 or 24 h of reperfusion. Animals in the sham group received thoracotomy only. PRCP activity was measured via RP-HPLC, PREP activity using a fluorogenic substrate and plasma Ang 2 levels via ELISA. Western blotting was used to determine the PRCP and PREP protein expression profiles in left lung tissue., Results: Plasma Ang 2 levels significantly rise after lung ischemia and remain increased after 1 h and 24 h of reperfusion compared to the sham group. While a significant decrease in plasma PREP activity was found after 24 h of reperfusion, a transient increase in plasma PRCP activity was observed after ischemia. However, no correlation with plasma Ang 2 levels could be demonstrated. The activity profiles of PRCP and PREP and the protein expression of PRCP in the lung tissues remained unchanged after LIRI., Conclusions: LIRI causes a dysregulation of circulating Ang 2 levels and plasma PREP activity, although no direct link between both phenomena could be shown. The activity profile of pulmonary PRCP and PREP was not significantly changed after LIRI, which implies a minor role for local PRCP and PREP in the ischemic lung itself.

This review considers the basic metallocarboxypeptidases of human blood and their role in coagulologic disorders. In includes information on the history of the discovery and biological characteristics of potential enzymes-regulators of the fibrinolytic process: carboxypeptidase U and carboxypeptidase N. Certain attention is paid to the biochemical mechanisms and the main modern concepts of the antifibrinolytic effects of these enzymes.

Prolyl carboxypeptidase (PRCP) is an enzyme associated with cerebrovascular risk factors such as hypertension, diabetes mellitus, obesity and hyperlipidemia. We aim to evaluate the relation between serum PRCP activity and severity, evolution and outcome of acute ischemic stroke. We used a specific RP-HPLC activity assay to measure PRCP activity in serum of 50 stroke patients at admission, and at 24 h, 72 h and 7 days after stroke onset to assess correlations with stroke severity based on the National Institutes of Health Stroke scale score (NIHSS), infarct volume on brain MRI scan, stroke outcome based on the modified Rankin scale (mRS) and mortality at 3 months after stroke. The average PRCP activity in serum decreased significantly the first 24 h after stroke onset and returned to baseline values at day 7. High NIHSS scores and infarct volumes at admission were related with a more pronounced decrease of PRCP in the first 24 h after stroke (ΔPRCP24, r = 0.31, P < 0.05; r = 0.30, P < 0.05). In addition, patients who displayed a more pronounced decrease in PRCP levels during the first 24 h after stroke were more likely to be institutionalized upon discharge (n = 21) (ΔPRCP24 ± SD, 0.05 ± 0.10 U/L vs. 0.17 ± 0.14 U/L, P = 0.001). The decrease in PRCP levels in the first 24 h after stroke onset is associated with stroke severity and an unfavourable short-term stroke outcome.

Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45 years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis-Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65 ± 0.02 and 0.72 ± 0.03 U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP's role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Blood carboxypeptidases play an important role in the regulation of fibrinolysis. We have proposed here the method for the assay of blood carboxypeptidase activity associated with coagulation/fibrinolysis using the natural substrate fibrin and the detection of basic amino acids arginine and lysine as products in the conditions close to those in vivo. Plasma samples from 15 patients with arterial hypertension were investigated. The coagulation and subsequent fibrinolysis were initiated by addition of standard doses of thrombin and tissue plasminogen activator, respectively. Arginine and lysine concentrations before, during, and after completion of fibrinolysis were determined using HPLC. The parameters of fibrinolysis were evaluated by clot turbidity assay. Fibrinolysis led to a large and significant increase in concentrations of arginine and lysine in the incubation mixture by 101 and 81%, respectively. The duration of fibrinolysis initiation significantly correlated to the degree of increase of these amino acids: r(s) = -0.733 and -0.761 for arginine and lysine, respectively (p < 0.05). The rates of amino acids liberation during fibrinolysis demonstrate different pattern: arginine generation had two maximums: at the beginning of clot lysis and at his end, whereas the liberation of lysine occurred mainly at the middle of fibrinolysis. Thus, the carboxypeptidase activity associated with fibrinolysis can be considered as a local source of the essential aminoacids.

Background: Prolylcarboxypeptidase (PRCP) (angiotensinase C) has 3 major targets, angiotensin II, prekallikrein, and α-melanocyte stimulating hormone(1-13). The truncation of the latter leads to loss in appetite regulation and obesity in experimental animals. The objectives of this study were to purify PRCP from a native source, establish a sensitive immunoassay for PRCP, and relate plasma PRCP concentrations to signs and symptoms of obesity, diabetes mellitus, and cardiovascular dysfunction., Methods: Purification of PRCP from human neutrophils and establishment of a sensitive ELISA was carried out with the use of samples from study participants. Three cohorts were studied: healthy individuals (n = 40); a chest pain cohort (Fast Assessment of Thoracic Pain by Neural Networks) (n = 165); and a community-based cohort [Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS)] (n = 1004)., Results: PRCP was purified to homogeneity. Mean (SD) plasma concentrations in healthy individuals were 12.9 (3.2) μg/L and were increased in patients with chest pain and in patients with obesity and/or diabetes mellitus (P < 0.0001). In the PIVUS cohort the concentrations were related to several measures of arterial plaque formation, thickness of arterial intima media and posterior wall of the heart (P = 0.04-0.000005); the Framingham score (r = 0.14, P < 0.0001); and concentrations of C-reactive protein (r = 0.16, P < 0.0001) and N-terminal pro B-type natriuretic peptide (r = -0.13, P < 0.0001)., Conclusions: Plasma concentrations of PRCP may be used to reflect metabolic conditions in individuals with obesity and diabetes mellitus. The associations of PRCP concentrations with signs of cardiovascular dysfunction and cardiovascular abnormalities suggest a pivotal role of the enzyme in disease.

Objective: To detect the changes of leukotriene E4(LTE4), prostaglandin D2(PGD2), carboxypeptidase A3(CPA3) and platelet activating factor (PAF) in guinea pigs died from anaphylactic shock., Methods: Guinea pigs were used for establishing anaphylactic shock models. The levels of LTE4, PGD2 and CPA3, and PAF were detected in urine, plasma, and brain tissues with ELISA kit, respectively. The significant biomarkers were selected comparing with control group. The changes of PGD2, CPA3 and PAF in the guinea pigs at time zero, 12 and 24 hours after death were observed and compared respectively. The effect of platelet activating factor acetylhydrolase (PAF-AH) to PAF in guinea pig brain was examined and compared., Results: There were no statistically differences of LTE4 levels in urine observed between experimental group and control group. The levels of CPA3, PGD2 and PAF in the experimental group were significantly higher than that in the control group at 0 h. The levels of PAF at 12 and 24 hours after anaphylactic shock were significantly higher than that in the control group. The levels of PAF decreased significantly after pretreatment with PAF-AH., Conclusion: LTE4 in urine cannot be selected as a biomarker to determine the anaphylactic shock. PGD2 and CPA3 in plasma, and PAF in brain tissue may be used as biomarkers to determine the anaphylactic shock. PAF-AH may be potentially useful for clinical treatment of anaphylactic shock.

Objective: To evaluate the roles of plasma mast cell carboxypeptidase and chymase in the diagnosis of allergic diseases by measuring the contents of both in children., Methods: A total of 59 children with allergic diseases and 53 healthy children were recruited into the study. Plasma levels of mast cell carboxypeptidase and chymase were measured using ELISA., Results: The plasma levels of mast cell carboxypeptidase and chymase in children with allergic children were 1.089 ± 0.752 ng/mL and 0.905(0.375-2.318) ng/mL, respectively, which were significantly higher than those in healthy children [0.593 ± 0.380 ng/mL and 0.454 (0.097-1.077) ng/mL respectively; P<0.05]. There was a significantly positive correlation between plasma mast cell carboxypeptidase and chymase levels in children with allergic diseases (r=0.684, P<0.01)., Conclusions: Plasma levels of mast cell carboxypeptidase and chymase increase in children with allergic diseases, suggesting that mast cell carboxypeptidase and chymase may serve as the indexes for the diagnosis of allergic diseases.

Serine proteases play a central role in defense against pathogens by regulating processes such as blood clotting, melanization of injured surfaces, and proteolytic activation of signaling pathways involved in innate immunity. Here, we present the functional characterization of the Drosophila serine protease Sp7 (CG3006) by inducible RNA interference. We show that Sp7 is constitutively expressed in blood cells during embryonic and larval stages. Silencing of the gene impairs the melanization reaction upon injury. Our data demonstrate that Sp7 is required for phenoloxidase activation and its activity is restricted to a subclass of blood cells, the crystal cells. Transcriptional up-regulation of Sp7 was observed after clean, septic injury and in flies expressing an activated form of Toll; however, mutations in the Toll or the IMD pathway did not abolish expression of Sp7, indicating the existence of other regulatory pathways and/or independent basal transcription.

Angiotensin-converting enzyme 2 (ACE2) is a newly discovered, membrane-bound aminopeptidase responsible for the production of vasodilatory peptides such as angiotensin 1-7 (Ang 1-7). Thus, ACE2 is important in counteracting the adverse, vasoconstrictor effects of angiotensin II (Ang II). The objective of the present study was to clone and characterize a constitutively secreted form of ACE2 as a prelude to an investigation into its therapeutic potential in hypertension. A truncated form of ACE2 was cloned into a lentiviral vector behind the human elongation factor 1 alpha promoter (lenti-shACE2). Transfection experiments demonstrated that secreted human ACE2 (shACE2) was secreted constitutively into the medium. The kinetic properties of shACE2 were comparable to the human recombinant enzyme (rACE2). Transduction of human coronary artery endothelial cells and rat cardiomyocytes with lenti-shACE2 showed a significant secretion of the enzyme into the medium compared to its native, membrane-bound homolog (human ACE2 [hACE2]). In addition, systemic administration of lenti-shACE2 into neonatal rats resulted in a eightfold increase in ACE2 activity in the serum above control values. These observations establish that lenti-shACE2 can be used to transduce cardiovascularly relevant cells for the secretion of functional ACE2 enzyme both in vitro and in vivo. Collectively, these results set the stage for the use of these vectors to investigate the consequences of ACE2 over-expression in the pathogenesis of hypertension.

Objective: To explore the protective effect of propofol on liver during hepatic ischemia/reperfusion injury (HIRI) and its mechanisms in patients undergoing liver surgery., Methods: Eighteen patients who were scheduled for selective hepatic surgery were randomly divided into control group (n=9) and propofol treatment group (n=9). Changes of several parameters in plasma and effects of propofol on them were observed before liver ischemia, at end of ischemia and at reperfusion for 25 minutes, parameters of which included superoxide dismutase (SOD), xanthine oxidase (XO), lipid peroxide (LPO) and alanine aminotransferase (ALT) activity, and the ultrastructure changes in liver tissue were observed under electron microscope at 25 minutes after reperfusion., Results: SOD activity decreased remarkably (P<0.01); XO activity, LPO concentration and ALT value increased significantly (P<0.01) during HIRI, and there were abnormal changes of the hepatic ultrastructure at 25 minutes after reperfusion. Afer treatment with propofol, the variation of all parameters were alleviated markedly (P<0.05 and P<0.01)., Conclusion: Propofol has protective effects on HIRI by reducing oxygen free radical level and inhibiting lipid peroxidation after hepatic ischemia/reperfusion in patients undergoing liver cancer surgery.

Procarboxypeptidase B (also known as thrombin-activatable fibrinolysis inhibitor) is a recently described plasma zymogen known to be activated by thrombin in plasma. Carboxy-terminal lysine residues from partially degraded fibrin are important for the binding and activation of plasminogen, and carboxypeptidase B, an active form of procarboxypeptidase B, has been shown to inhibit fibrinolysis by eliminating these residues. The present paper investigates the effects of carboxypeptidase B inhibitors, DL-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGPA) and potato-derived carboxypeptidase inhibitor (CPI), on tissue factor (TF)-induced microthrombosis in rats. Intravenous injection of MGPA (3 mg/kg and higher) or CPI (0.3 mg/kg and higher) after microthrombi formation dramatically attenuated TF-induced glomerular fibrin deposition with an increase in plasma levels of D-dimer. These results indicate that carboxypeptidase B inhibitors can enhance endogenous fibrinolysis and reduce thrombi in the TF-induced microthrombosis model after systemic administration even after thrombi formation., (Copyright 2003 Elsevier Science B.V.)

RT-nested PCR has been introduced as a highly specific and sensitive assay method to detect the prostate-specific membrane antigen (PSM) mRNA in peripheral blood. However, appreciable percentages of false-positive cases have been reported. Additionally, primer sets reported previously could not discriminate between PSM and PSM', an alternatively spliced variant, mRNA. These isoforms can be produced from a single gene. Switches in alternative splicing patterns are often controlled with strict cell-type or developmental-stage specificity. Therefore, it is most important to discriminate between PSM mRNA and PSM' mRNA. Using our highly specific primer sets, PSM mRNA was detected in 3 of 24 peripheral blood samples of normal male volunteers (12.5%) and was not detected in peripheral blood of 11 normal female volunteers. PSM' mRNA was detected in 5 of 24 peripheral blood samples of normal male volunteers (20.8%) and in 4 of 11 of normal female volunteers (36.4%). PSM' mRNA induced false-positive results, it is important for genetic diagnosis of prostate cancer to discriminate between PSM and PSM' using our primer sets with high specificity. The advances in the uniquely designed primer sets may allow researchers to detect a real PSM mRNA without PSM' mRNA.

The major functions of plasminogen (Plg) in fibrinolysis and cell migration depend on its binding to carboxy-terminal lysyl residues. The ability of plasma carboxypeptidase B (pCPB) to remove these residues suggests that it may act as a suppressor of these Plg functions. To evaluate this role of pCPB in vivo, homozygote pCPB-deficient mice were generated by homologous recombination, and the resulting pCPB(-/-) mice, which were viable and healthy, were mated to Plg(-/-) mice. Plg(+/-) mice show intermediate levels of fibrinolysis and cell migration compared with Plg wild-type and deficient mice, reflecting the intermediate levels of the Plg antigen in their plasma. Differences in Plg-dependent functions between pCPB(+/+), pCPB(+/-), and pCPB(-/-) mice were then analyzed in a Plg(+/-) background. In a pulmonary clot lysis model, fibrinolysis was significantly increased in mice with partial (pCPB(+/-)) or total absence (pCPB(-/-)) of pCPB compared with their wild-type counterparts (pCPB(+/+)). In a thioglycollate model of peritoneal inflammation, leukocyte migration at 72 hours increased significantly in Plg(+/-)/pCPB(+/-) and Plg(+/-)/pCPB(-/-) compared with their wild-type counterparts. These studies demonstrate a definitive role of pCPB as a modulator of the pivotal functions of Plg in fibrinolysis and cell migration in vivo.

Many researchers have investigated the expressions of candidates for a suitable reverse transcription nested polymerase chain reaction (RT-PCR) marker. But typically biomarkers often have false-positive results. We assessed whether epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSM) could be detected in 28 different types of normal human sources. Using RT-nested PCR assay, EGFR mRNA was also detected in various types of normal tissue, including pancreas, prostate and uterus. CEA was detected in various types of normal tissue, including prostate, uterus, bladder and spleen. PSM mRNA was also detected in various types of normal tissue, including kidney, liver, skeletal muscle, spleen, bladder and ovary. We report here that the expression of these biomarkers in normal cells might have induced false-positives, and that further enhancement of sensitivity might compromise specificity. Conversely, these biomarkers can be utilized for attempts to define micrometastases in various types of tumors whose cells express these tissue-specific genes.

Background: Carboxypeptidase B from the pancreatic gland may exist in three different molecular and immunoreactive forms: the proenzyme, the active enzyme, and the activation peptide., Aims: To investigate levels of procarboxypeptidase B (proCAPB) and its activation peptide in serum in acute pancreatitis to test the accuracy of these two variables as markers for the diagnosis of acute pancreatitis and for prediction of pancreatic necrosis. To elucidate whether leakage of proenzymes and activation of proenzymes reflect two different pathophysiological events in acute pancreatitis., Methods: Sera from patients with acute pancreatitis (n=85) and acute abdominal pain of non-pancreatic origin (n=53) were analysed for proCAPB and its activation peptide. Patients with pancreatitis were divided into necrotising (n=33) and oedematous attacks (n=52) using contrast enhanced computed tomography. Accuracy was determined using receiver operating characteristic curve analysis., Results: Immunoreactive carboxypeptidase B activation peptide (ir-CAPAP) concentration in serum on admission was 0.7 nmol/l (0-18.1) in patients with oedematous pancreatitis compared with 5.8 nmol/l (1.9-34) in patients with later development of pancreatic necrosis. Elevated levels of the activation peptide on admission correlated with an accuracy of 92% to later development of pancreatic necrosis. Ir-proCAPB concentration in serum on admission was 16.0 nmol/l (1.4-50.5) in all patients with acute pancreatitis versus 0.3 nmol/l (0-3.6) in patients with non-pancreatic acute abdominal disorders. Cases with oedematous pancreatitis had ir-proCAPB levels of 15.4 nmol/l (1.4-50.5) versus 19.1 nmol/l (2.7-36.1) in cases with later development of pancreatic necrosis. Measurement of the proenzyme can thus be useful for the diagnosis of acute pancreatitis (accuracy 99%) but levels did not correlate with later development of pancreatic necrosis (accuracy 56%)., Conclusion: Leakage of proenzymes occurs in acute pancreatitis, irrespective of severity, while development of pancreatic necrosis occurs only when there is activation of the proenzymes.

Purpose: We report a prospective study examining the ability of preoperative nested reverse transcriptase polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) to predict pathologic stage and biochemical recurrence in patients with clinically localized prostate cancer treated with radical prostatectomy., Patients and Methods: One hundred forty-one patients were entered onto the study. Preoperative evaluation included clinical T stage, serum PSA, biopsy Gleason score, and serum RT-PCR for PSA/PSM. Univariate and multivariate logistic regression models, Kaplan-Meier estimates, and Cox proportional hazards modeling were used to identify predictors of pathologic stage and biochemical failure., Results: Seventy-three patients (51.8%) were RT-PCR positive for PSA, PSM, or both. In the multivariate logistic regression model, only initial PSA was an independent predictor of pathologic stage as defined by organ-confined disease (odds ratio [OR], 1.06; 95% confidence interval [CI], 1.00 to 1.13; P =.026) or organ-/specimen-confined disease (OR, 1.09; 95% CI, 1.02 to 1.16; P =.009). Overall Kaplan-Meier biochemical relapse-free survival (bRFS) was 85% at 59 months. Multivariate analysis of predictors for bRFS with the Cox proportional hazards model indicated that only initial PSA (OR, 1.05; 95% CI, 1.02 to 1.09; P =.004) and biopsy Gleason score (OR, 3.57; 95% CI, 1.37 to 9.58; P =.009) were independent predictors of biochemical failure. RT-PCR status did not predict pathologic stage or biochemical failure. Repeat analysis excluding 27 patients who received preoperative androgen-deprivation therapy did not change the results., Conclusion: Combined nested RT-PCR for PSA and PSM is not an independent predictor of pathologic stage or biochemical failure in patients with localized prostate cancer undergoing radical prostatectomy. This assay has no clinical utility in this patient population.

Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as procarboxypeptidase U or plasma procarboxypeptidase B, is a relatively recently described plasma glycoprotein synthesised in the liver. It can be activated into active enzyme TAFIa (carboxypeptidase U or plasma carboxypeptidase B) by a complex of thrombin/thrombomodulin. TAFIa can potentially inhibit fibrinolysis by removing carboxyterminal lysine residues from partially degraded fibrin, decreasing plasminogen binding on the surface of fibrin, which thereby results in a decrease of the fibrinolytic activity. Since TAFI represents a connection between coagulation and fibrinolysis, it can be expected that TAFI levels are altered in different thrombotic and hemorrhagic diseases. Thrombin generation is increased in patients with activated protein C (APC) resistance, while it has been shown that APC has profibrinolytic effect. Therefore, changes in TAFI level should be found in patients with APC resistance due to factor V Leiden (FV Leiden) mutation. TAFI antigen (including TAFI, TAFIa and the inactive form TAFIai) and TAFI activity were determined in 17 female patients heterozygous for FV Leiden mutation while 13 healthy volunteers were controls. No statistically significant difference in levels of TAFI antigen was observed. TAFI activity was significantly reduced in APC resistance patients compared to control (P=.018). The nondifference in TAFI antigen, together with the decrease of TAFI activity level, can be explained by activation of TAFI to TAFIa and shifting of equilibrium towards an increase of the latter. This can be an indirect proof that TAFIa is increased in patients with APC resistance due to FV Leiden mutation, indicating that downregulation of fibrinolysis can be an additional risk factor for thrombosis in these patients.

Carboxypeptidases (CP) in plasma and sera serve as regulators of anaphylatoxins such as C3a and C5a. The activity of CP can be measured by determining hippuric acid after cleavage of the small synthetic substrate hippuryl-L-arginine. Although a colorimetric assay is convenient for determining hippuric acid generated by CP, we noticed that some anticoagulants, such as citrate, interfere with the color development of the reagents used. EDTA and heparin provide an appropriate value. EGTA used as anticoagulant also provides an appropriate value. Therefore, concentration of citrate in samples should be controlled to be constant for background subtraction.

Prostate cancer remains the most common cancer type in men in the United States. Efforts are increasing to evaluate and to discover diagnostic and therapeutic markers for prostate cancer patients. One of these, prostate-specific membrane antigen (PSMA), is a transmembrane protein highly expressed in all types of prostatic tissue, especially cancer. The radio-immunoconjugate form of the anti-PSMA monoclonal antibody (mAb) 7E11, known as the ProstaScint scan, is currently being used to diagnose prostate cancer metastasis and recurrence. Early promising results from various Phase I and II trials have utilized PSMA as a therapeutic target. Recently, PSMA expression in endothelial cells of tumor-associated neovasculature has been described. PSMA's possible role in malignant angiogenesis newly expands the realm of its possible beneficial uses, especially as new anti-PSMA mAbs continue to be developed and refined.

Intense efforts of research are made for developing antitumor vaccines that stimulate T cell-mediated immunity. Tumor cells specifically express at their surfaces antigenic peptides presented by MHC class I and recognized by CTL. Tumor antigenic peptides hold promise for the development of novel cancer immunotherapies. However, peptide-based vaccines face two major limitations: the weak immunogenicity of tumor Ags and their low metabolic stability in biological fluids. These two hurdles, for which separate solutions exist, must, however, be solved simultaneously for developing improved vaccines. Unfortunately, attempts made to combine increased immunogenicity and stability of tumor Ags have failed until now. Here we report the successful design of synthetic derivatives of the human tumor Ag Melan-A/MART-1 that combine for the first time both higher immunogenicity and high peptidase resistance. A series of 36 nonnatural peptide derivatives was rationally designed on the basis of knowledge of the mechanism of degradation of Melan-A peptides in human serum and synthesized. Eight of them were efficiently protected against proteolysis and retained the antigenic properties of the parental peptide. Three of the eight analogs were twice as potent as the parental peptide in stimulating in vitro Melan-specific CTL responses in PBMC from normal donors. We isolated these CTL by tetramer-guided cell sorting and expanded them in vitro. The resulting CTL efficiently lysed tumor cells expressing Melan-A Ag. These Melan-A/MART-1 Ag derivatives should be considered as a new generation of potential immunogens in the development of molecular anti-melanoma vaccines.

Background: Nested reverse-transcriptase polymerase chain reaction (nested rt-PCR) for the detection of mRNA of prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) in peripheral blood samples (PB) and bone marrow biopsies (BM) can assess the extraprostatic growth of prostate cells., Materials and Methods: Nested rt-PCR for PSA and PSMA at PB and BM was performed (a) at diagnosis, (b) after 6 months from the initiation of combined androgen blockade [(CAB); triptorelin 3.75 mg, i.m., q28 days plus flutamide 250 mg, per os, tid] and, (c) at progression to androgen refractory stage in 28 patients with newly-diagnosed stage D2 prostate cancer., Results: At diagnosis, all patients were found to be rt-PCR positive for PSA and PSMA at BM while 7 (25%) were rt-PCR negative for PSA and PSMA at PB. Nine out of 21 patients with rt-PCR-positive status have converted to rt-PCR-negative status at PB during CAB while they remained rt-PCR-positive at BM. The rt-PCR-negative status at PB during CAB was associated with progression-free survival >12 months (p=0.029). At progression to androgen refractory stage all but 2 patients were rt-PCR-positive at PB for PSA and PSMA, among them 3 who were rt-PCR-negative at diagnosis., Conclusion: Our data suggest that conversion to rt-PCR-negative status at PB for PSA and PSMA during objective clinical response to CAB is associated with long progression-free survival in stage D2 disease.

The lack of a sensitive immunoassay for quantitating serum prostate-specific membrane antigen (PSMA) hinders its clinical utility as a diagnostic/prognostic biomarker. An innovative protein biochip immunoassay was used to quantitate and compare serum PSMA levels in healthy men and patients with either benign or malignant prostate disease. PSMA was captured from serum by anti-PSMA antibody bound to ProteinChip arrays, the captured PSMA detected by surface-enhanced laser desorption/ionization mass spectrometry, and quantitated by comparing the mass signal integrals to a standard curve established using purified recombinant PSMA. The average serum PSMA value for prostate cancer (623.1 ng/ml) was significantly different (P < 0.001) from that for benign prostate hyperplasia (117.1 ng/ml) and the normal groups (age <50, 272.9 ng/ml; age >50, 359.4 ng/ml). These initial results suggest that serum PSMA may be a more effective biomarker than prostate-specific antigen for differentiating benign from malignant prostate disease and warrants additional evaluation of the surface-enhanced laser desorption/ionization PSMA immunoassay to determine its diagnostic utility.

Until now, no molecular parameter has been available for predicting the metastatic potential of prostate tumours, which leaves their outcome uncertain despite an apparent benign histology or early stage. Abnormal expression of adhesion molecules, such as E-cadherin, can be contributing factors for increased invasiveness and metastatic potential. Histological analysis for E-cadherin expression was carried out on paraffin-embedded tumour tissues. Tumour metastatic potential was indirectly evaluated by detecting circulating prostate cells (CPC), using reverse transciptase-polymerase chain reaction (RT-PCR) and prostate-specific membrane antigen (PSMA) as a target. Patients were followed-up for a median of 14 months (range 10--19 months) after surgery with serum prostate-specific antigen (PSA) level measurement. Interestingly, 23 of 44 localised tumours exhibited aberrant E-cadherin expression. Prior to primary surgery, PSMA RT-PCR detected the spread of prostate cells to the blood in 24 patients. Statistical analysis showed that abnormal E-cadherin expression in the tumours was the only variable that was independently correlated with prostate cell dissemination in the blood (P<0.0001). In logistic regression analysis, abnormal E-cadherin expression was a significant independent predictor for a later biological relapse. This impaired adhesion status was clearly correlated with a haematogenous spread of the primary tumour cells. It could therefore be an objective way to restrict the indications for radical surgery to patients not presenting with this feature.

By decreasing plasminogen binding to fibrin surface, the thrombin activatable fibrinolysis inhibitor (TAFI) has been hypothesized to constitute an early marker for atherothrombotic diseases. Previous studies have shown that plasma TAFI levels exhibit a high interindividual variability that is only poorly explained by lifestyle factors. Several polymorphisms of the TAFI gene have been described, and a combination of a C+1542G substitution in the 3' untranslated region and an Ala147Thr amino acid change has been shown to explain 60% of TAFI variability in a sample of unrelated individuals. A segregation-linkage analysis was performed to determine whether these polymorphisms are directly involved in the genetic regulation of TAFI levels, or whether they are only markers in linkage disequilibrium (LD) with unmeasured TAFI-linked quantitative trait loci (QTLs). The sample consisted of 97 healthy nuclear families from the Stanislas Cohort. The C+1542G and Ala147Thr polymorphisms were in complete negative LD, with minor allele frequencies of 0.27 and 0.28, respectively. Results of the segregation-linkage analysis provided evidence of two TAFI-linked QTLs in LD with the two measured polymorphisms, which would explain 78% of the TAFI variance, as compared with 55% explained by the C+1542G and the Ala147Thr polymorphisms combined. The two putative QTLs would have minor allele frequencies of 0.45 and 0.32, respectively. The hypothesis that one of the measured polymorphisms is one of the QTLs was rejected. The putative QTLs also did not seem compatible with the other TAFI gene polymorphisms that we have previously described. More extensive sequencing of the TAFI gene is necessary to identify the functional variants.

Background: Cardiovascular disease (CVD) is a major cause of death in renal transplant recipients (RTR). Suppression of fibrinolysis plays a role in the progression of atherosclerosis. Accelerated progression of atherosclerosis and fibrinolytic system suppression has been observed in RTR. Despite many years of intensive research, the reason for impaired fibrinolysis in this patient population is not fully understood. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a recently discovered glycoprotein combining coagulation and fibrinolysis. This study was conducted to evaluate concentrations of TAFI, markers of thrombin generation, endothelial injury, and some standard laboratory parameters in RTR receiving triple immunosuppressive drug regimen., Methods: The study was performed in 29 stable, non-diabetic kidney transplant recipients treated with cyclosporin A, azathioprine, and prednisone and in 18 age- and sex-matched healthy volunteers. Soluble thrombomodulin (sTM), prothrombin fragments F1+2 (F1+2), thrombin--antithrombin complexes (TAT), plasmin--antiplasmin complexes (PAP), and TAFI were measured with commercially available kits., Results: The RTR group had significantly higher plasma levels of TAT, F1+2, sTM and TAFI than the healthy volunteers. There were no differences in PAP concentrations between the two groups. Plasma sTM correlated inversely with creatinine clearance, body mass index, haemoglobin, and albumin. Plasma TAT level was positively associated with total cholesterol. TAFI antigen influenced negatively PAP antigen concentration., Conclusions: On the basis of our research, we concluded that elevated circulating TAFI antigen might be a new link in the pathogenesis of impaired fibrinolysis in RTR, and thus atherosclerosis progression. In the patient group there is also evidence of endothelial injury, followed by secondary activation of the coagulation cascade. Hypercholesterolaemia in RTR is associated with enhanced thrombin activity.

In 1988, Hendricks et al. first reported on the presence of carboxypeptidase U (U refers to the unstable nature of the enzyme) in human serum. One decade later, the importance of carboxypeptidase U (CPU) in the regulation of fibrin clot dissolution is well documented. CPU circulates in plasma as an inactive zymogen, proCPU, that is converted to its active form during coagulation and fibrinolysis. CPU cleaves off C-terminal lysine residues exposed on fibrin partially degraded by the action of plasmin. Because these C-terminal lysine residues are important for upregulating the fibrinolytic rate, CPU thus slows down fibrinolysis.

A prospective cohort study was performed in 50 patients with dengue haemorrhagic fever (DHF) to determine the potential role of the contact activation system and factor XI activation (intrinsic pathway) in the coagulation disorders in DHF. To establish whether TAFI (thrombin-activatable fibrinolysis inhibitor) was involved in the severity of the coagulation disorders, the TAFI antigen and activity levels were also determined. Markers of contact activation (kallikrein--C1-inhibitor complexes), the intrinsic pathway of coagulation (factor XIa--C1-inhibitor complexes) and TAFI were measured and correlated to thrombin generation markers (thrombin--anti-thrombin complexes (TAT), prothrombin fragment 1+2 (F1+2)) and a marker for fibrinolysis [plasmin--alpha 2--anti-plasmin complexes (PAP)]. Activation of the intrinsic pathway of coagulation was clearly demonstrated by elevated levels of factor XIa--C1-inhibitor complexes, without evidence of contact activation, reflected by undetectable kallikrein--C1-inhibitor complexes. Both TAFI antigen and activity levels were decreased in all patients, which may contribute to the severity of bleeding complications in DHF because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis. These findings in a human viral infection model are in accordance with earlier findings in bacterial sepsis.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described carboxypeptidase that is potentially involved in the regulation of fibrinolysis by decreasing plasminogen binding to the fibrin surface. This role makes the TAFI gene a good candidate in atherothrombotic diseases. The great interindividual variability of plasma TAFI antigen levels is poorly explained by lifestyle characteristics, thus suggesting its genetic determination. To test this hypothesis, the promoter and the 3'-untranslated region of the TAFI gene were screened for polymorphisms, and their contribution to the variability of plasma TAFI antigen levels was evaluated. Seven new polymorphisms are described, 5 in the promoter (C-2599G, -2345 2G/1G, A-1690G, G-1102T, and G-438A) and 2 in the 3'UTR (C+1542G and T+1583A). All these polymorphisms were in strong linkage disequilibrium with each other and with the previously described Ala147Thr polymorphism. They generated 4 main haplotypes, accounting for 80% of all observed haplotypes. In univariate analyses, all polymorphisms were associated with plasma TAFI Ag levels and, individually, contributed to a large fraction of plasma TAFI Ag levels, ranging from 20% to 52%. In a stepwise regression analysis including all polymorphisms, several combinations remained significantly and independently associated with plasma TAFI Ag levels: C+1542G associated with Ala147Thr, T+1583A, or -2345 2G/1G explaining 61.6%, 60.2%, and 58.1% of the variance, respectively. These findings clearly demonstrate that circulating levels of TAFI are strongly determined by polymorphic variations in the promoter and the 3'UTR of the TAFI gene. (Blood. 2001;97:2053-2058)

Recently, a new inhibitor of fibrinolysis was described. This inhibitor downregulated fibrinolysis after it was activated by thrombin, and was therefore named TAFI (thrombin-activatable fibrinolysis inhibitor; EC 3.4.17.20). TAFI turned out to be identical to previously described proteins, procarboxypeptidase U, procarboxypeptidase R, and plasma procarboxypeptidase B. In this overview, the protein will be referred to as TAFI. TAFI is a procarboxypeptidase and a member of the family of metallocarboxypeptidases. These enzymes are circulating in plasma and are present in several tissues such as pancreas. In this review, we will describe the properties of basic carboxypeptidases with the emphasis on the role of TAFI in coagulation and fibrinolysis. It cannot be ruled out, however, that TAFI has other, yet undefined, functions in biology.

Human brain carboxypeptidase B (HBCPB) is a novel brain protease that processes native brain beta-amyloid precursor protein (APP) in vitro. Immunoblot analysis of human serum and cerebrospinal fluid (CSF) using anti C14-module antibody, which recognizes the C-terminal peptide unique to HBCPB, detected the 30 and 40 kDa immunoreactive bands. Analysis of HBCPB prepared from both serum and CSF demonstrated proteolytic activities for brain APP. Protease inhibitor spectrum analysis also supports that these bands correspond to the mature form and and prepro form of HBCPB, respectively. As is the case in brain parenchyma, the prepro-form is dominant in CSF. In serum, however, the majority of HBCPB exists in the mature form, possibly due to an abundant trypsin-like proteolytic activity in serum. HBCPB expressed in serum and CSF, therefore, may have a significance as a peripheral marker of the brain protease, which participates in APP processing in human brain.

Cysteine proteinases cathepsin (Cath) B and L and their endogenous inhibitors stefin (Stef) A and B concentrations were measured using a quantitative immunosorbent assay (ELISA; KRKA d.d., Novo mesto, Slovenia) in serum samples from 35 patients with primary and 7 patients with recurrent squamous cell carcinoma of the head and neck (SCCHN), obtained at diagnosis (Serum no.1) and after therapy (Serum no. 2), and compared to sera from 30 (Stef B, 90) healthy volunteers. A significantly higher Stef A (P = 0.005) and lower Stef B (P < 0.001) concentrations were measured in patients' Serum no.1 than in controls, and the levels of Caths B and L and Stef A were found to be significantly elevated in Serum no.1 as compared to Serum no. 2 (P = 0.045, P = 0.041 and P = 0.024, respectively). The time of Serum no.2 collection did not influence the concentration of either Caths or Stefs in these samples, and no correlation was observed with the established prognostic factors for any of the parameters studied. Patients with subsequently diagnosed recurrent disease had a significantly lower Cath L concentration than those without evidence of relapse during follow up (P = 0.05). The risk of disease recurrence and SCCHN-related death correlated significantly with low Cath L serum levels (P = 0.012, P = 0.006). The serum levels of Cath B, Stef A and Stef B did not influence significantly the probability of survival.

The importance of carboxypeptidase U as a novel regulator of the fibrinolytic rate has attracted a lot of interest recently. In the present work, an ELISA was developed using polyclonal antibodies raised against recombinant proCPU, expressed in DON cells. The assay determines the antigen concentration of the zymogen of carboxypeptidase U, procarboxypeptidase U, in human citrated plasma or EDTA plasma. No interference is observed with plasma carboxypeptidase N. The assay is very reproducible (within-run: 4.6% CV, between-run: 6.8% CV). In a group of 479 healthy individuals the mean proCPU antigen concentration is 13.4 microg/ml (SD 2.5 microg/ml). A good correlation is found with the functional procarboxypeptidase U assay described earlier (r = 0.82, p < 0.0001) (Schatteman K, Goossens F, Scharpé S, Neels H, Hendriks D Clin Chem 1999: 45: 807-813). The significant correlation between the proCPU antigen concentration and the 50% clot lysis time stresses its importance as a player in fibrinolysis control.

Procarboxypeptidase U (proCPU) is the plasma precursor of carboxypeptidase U (CPU, carboxypeptidase R. plasma carboxypeptidase B or activated thrombin-activatable fibrinolysis inhibitor, TAFIa). CPU removes C-terminal lysine residues that act as plasminogen binding sites from partially degraded fibrin, thereby down-regulating plasminogen activation and fibrinolysis. The present study was carried out as a pilot study to examine whether the plasma proCPU concentration is related to the presence of coronary artery disease (CAD) and/or to levels of established risk indicators for CAD, in a case-control study of 110 men requiring coronary artery bypass grafting (CABG) because of stable angina pectoris. The preoperative plasma proCPU level in the CABG patients was significantly higher than in population-based controls (1029 +/- 154 vs. 974 +/- 140 U/L, p <0.05). In addition, in a subset of the patients (n = 31 ) the proCPU concentration, which was significantly lower on the third postoperative day (-17 +/- 10%), had increased significantly on the sixth day (+14 +/- 12%) after surgery, compared with the preoperative level. In both patients and controls, proCPU concentration was strongly and positively associated with factor VII amidolytic activity and protein C activity, suggesting a common mechanism modulating the plasma levels of these proteins. Otherwise, statistically significant correlations with proCPU were group-specific. In the patients, proCPU correlated significantly with plasma fibrinogen and protein S. In the controls, proCPU correlated significantly with concentrations of cholesterol in plasma. VLDL and LDL. In addition, proCPU correlated significantly with C-reactive protein and haptoglobin levels in the controls only, indicating that also inflammatory mechanisms are involved in the regulation of plasma proCPU. These results suggest that a mechanism exists by which fibrinolytic function is impaired in a manner that is likely to result in more stable fibrin deposits and increase the risk of precocious CAD as well as early occlusion of venous bypass grafts.

Objectives: To report a survey of blood-based RNAs obtained from common groups of control and renal cancer patients for expression of both MN/CA9 and prostate-specific membrane antigen (PSMA) messenger RNAs., Methods: Reverse transcription polymerase chain reaction (RT-PCR) assays for MN/CA9 and PSMA were performed on RNAs extracted from 81 blood samples (59 patients with renal cancer, 7 with benign tumors, and 15 control volunteers). The results of these assays were statistically analyzed to determine whether a positive result (individually or combined) correlates with any tumor characteristics., Results: Neither MN/CA9 nor PSMA amplification products were detected in the RNAs from peripheral blood samples of the 15 control volunteers and from the 7 patients with benign renal tumor (sensitivity 100%). MN/CA9 alone was detected in 11 (19%) of 59 samples and PSMA alone in 12 (20%) of 59 samples from patients with renal cancer. PSMA positivity was significantly correlated with vascular invasion of the primary tumor. Expression of one or both of these molecular tumor markers was detected in 21 (36%) of 59 renal cancer patients. When combined, the results of the MN/CA9 and PSMA RT-PCR tests were found to be highly associated with vascular invasion in nephrectomy specimens (sensitivity 67%, specificity 77%, odds ratio = 6.89, P = 0.002)., Conclusions: Combination of RT-PCR assays for MN/CA9 and PSMA provides a sensitive blood test for molecular detection of clear cell carcinoma of the kidney and its potential for vascular invasion. Further testing of this assay will be required to evaluate its efficacy in the diagnosis, screening, and follow-up of patients with kidney cancer.

Background: We tested the ability of the nested reverse transcription polymerase chain reaction (RT-PCR) assay to detect signs of biochemical recurrence of prostate cancer in the lymph nodes and peripheral blood of patients with pT3N0 prostate cancer., Methods: Using lymph nodes and pre- and postoperative peripheral blood dissected from 30 patients with pT3N0 prostate cancer treated by radical prostatectomy, we used RT-PCR for prostate-specific membrane antigen (PSM) and serum prostate-specific antigen (PSA) to determine the presence of prostate cancer. Results of the nested RT-PCR assay were compared with pathological stages and biochemical recurrence., Results: Two of 13 patients with capsular penetration (15%), 6 of 10 patients with invasion of seminal vesicles (60%), and 3 of 7 patients with a positive surgical margin (43%) were RT-PCR-positive for PSM and/or PSA in the lymph nodes. Results of preoperative RT-PCRs of peripheral blood for PSM and for PSA significantly differed between positive and negative results of RT-PCR in lymph nodes (P < 0.001 and P < 0.001, respectively). Results of postoperative RT-PCRs of peripheral blood for PSM and for PSA also significantly different between positive and negative results of RT-PCR in lymph nodes (P = 0.011 and P = 0.001, respectively). Nine of 11 patients with positive nested RT-PCR for PSM and/or PSA in the lymph nodes (82%) experienced biochemical recurrence. Significant difference in Kaplan-Meier recurrence-free actuarial curves was noted between patients who nested positive and negative on RT-PCR in the lymph nodes, pre- and postoperative peripheral blood, biopsy and prostatectomy Gleason score, and preoperative PSA values. In multivariate analysis, biopsy and prostatectomy Gleason score (P = 0.026, P = 0.020, respectively), pre- and postoperative RT-PCR for PSM in peripheral blood (P = 0.030 and P = 0.040, respectively), and RT-PCR for PSM in lymph nodes (P = 0.035) were independent prognostic factors., Conclusions: Nested RT-PCR assay of the lymph nodes or peripheral blood significantly predicted biochemical recurrence after surgery. It may help identify patients at risk for recurrence and progression of prostate cancer., (Copyright 2000 Wiley-Liss, Inc.)

Carboxypeptidase R (EC 3.4.17.20; CPR) and carboxypeptidase N (EC 3. 4.17.3; CPN) cleave carboxyl-terminal arginine and lysine residues from biologically active peptides such as kinins and anaphylatoxins, resulting in regulation of their biological activity. Human proCPR, also known as thrombin-activatable fibrinolysis inhibitor, plasma pro-carboxypeptidase B, and pro-carboxypeptidase U, is a plasma zymogen activated during coagulation. CPN, however, previously termed kininase I and anaphylatoxin inactivator, is present in a stable active form in plasma. We report here the isolation of mouse proCPR and CPN cDNA clones that can induce their respective enzymatic activities in culture supernatants of transiently transfected cells. Potato carboxypeptidase inhibitor can inhibit carboxypeptidase activity in culture medium of mouse proCPR-transfected cells. The expression of proCPR mRNA in murine liver is greatly enhanced following LPS injection, whereas CPN mRNA expression remains unaffected. Furthermore, the CPR activity in plasma increased 2-fold at 24 h after LPS treatment. Therefore, proCPR can be considered a type of acute phase protein, whereas CPN is not. An increase in CPR activity may facilitate rapid inactivation of inflammatory mediators generated at the site of Gram-negative bacterial infection and may consequently prevent septic shock. In view of the ability of proCPR to also inhibit fibrinolysis, an excess of proCPR induced by LPS may contribute to hypofibrinolysis in patients suffering from disseminated intravascular coagulation caused by sepsis.

Prostate-specific membrane antigen (PSMA) is a 100-kDa transmembrane glycoprotein identified by the monoclonal antibody 7E11-C5.3 from the human prostate tumor cell line LNCaP. Because of its significant upregulation in androgen refractory and metastatic prostate cancers, PSMA may be a useful prognostic biomarker and a target for developing novel therapeutic strategies. However, the lack of abundant pure PSMA protein and the low efficacy in immunoaffinity isolation from LNCaP cells have hampered the development of clinical assays. In order to obtain a renewable and reliable source of pure antigen, we used the baculovirus/insect cell system to express and purify a recombinant PSMA. A recombinant baculovirus containing a 6x histidine-tagged PSMA gene was generated, from which rPSMA was expressed and purified using cobalt-chelating affinity chromatography. The purity and correct molecular size of rPSMA were demonstrated by gel electrophoresis and mass spectrometry. Glycosidase digestions showed that the oligosaccharides on rPSMA are primarily N-linked high-mannose type. Although the glycosylation is different from the native PSMA, it did not affect the immunoreactivity of rPSMA to antibodies specific for either the intra- or the extracellular domains of PSMA. Finally, the purified rPSMA was successfully used to develop a quantitative PSMA immunoassay using the novel ProteinChip surface-enhanced laser desorption/ionization mass spectrometry technology., (Copyright 2000 Academic Press.)

The Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently described inhibitor of fibrinolysis. The physiological variations of plasma TAFI antigen are not well known. We studied TAFI antigen values in healthy populations with a commercially available kit from Milan Analytica (Switzerland). Broad range of TAFI antigen values (from 41% to 259%) was found in a population of 249 healthy individuals. Gender as well as pregnancy did not influence mean values of TAFI antigen. There was a positive correlation between TAFI antigen and age in female (r = 0.28; p <0.05) but not in male populations. Mean TAFI antigen value of a black African male group [mean +/- SD (range): 87 +/- 23 (39-144%)] was significantly lower than the one of age matched Caucasian men [114 +/- 34 (52-259%)] (p <0.0001). TAFI antigen values were very stable within individuals, they did not significantly vary on day time or at several months period. Thus, in contrast to large inter-individual variations, TAFI antigen levels are quite stable within individuals.

Objective: To investigate the relationships between serum and urinary molecular markers (MM) used to monitor osteoarthritis., Design: Forty osteoarthritis patients had blood and urine collected at baseline and 1, 3, 6 and 12 months later. Specimens from 20 controls were obtained twice at a one month interval. The concentration of 14 different markers was determined at each time point and the data were analyzed by statistical methodology., Results: The markers could be divided by the method of principal components analysis into five clusters of related markers: inflammation markers (C-reactive protein, tumor necrosis receptor type I and tumor necrosis receptor type II, interleukin 6, eosinophilic cationic protein), bone markers (bone sialoprotein, hydroxylysyl pyridinoline, lysyl pyridinoline), putative markers of cartilage anabolism (carboxypropeptide of type II procollagen, hyaluronan, epitope 846) and catabolism (keratan sulfate, cartilage oligomeric matrix protein), and transforming growth factor beta. Three markers (tumor necrosis factor receptor II, cartilage oligomeric matrix protein and epitope 846) from independent clusters discriminated osteoarthritis patients from controls. Inflammation was not a confounding factor in measurement, but a recognizable distinguishing factor in osteoarthritis., Conclusions: The markers separated into rational groups on the basis of their covariance, a finding with independent biochemical support. The covariance of markers from the same cluster suggests the use of a representative marker from the cluster to reflect changes in osteoarthritis. If multiple markers are being measured within a single cluster, then the use of a weighted cluster 'factor' may be preferable to the separate use of individual markers.

Thrombin activatable fibrinolysis inhibitor (TAFI, or procarboxypeptidase B) is the precursor of a recently described carboxypeptidase that potently attenuates fibrinolysis. Therefore, we hypothesized that elevated plasma TAFI levels induce a hypofibrinolytic state associated with an increased risk for venous thrombosis. To evaluate this hypothesis, we developed an electroimmunoassay for TAFI antigen and used this assay to measure TAFI levels in the Leiden Thrombophilia Study, a case-control study of venous thrombosis in 474 patients with a first deep vein thrombosis and 474 age- and sex-matched control subjects. In 474 healthy control subjects, an increase of TAFI with age was observed in women but not in men. Oral contraceptive use also increased the TAFI concentration. TAFI levels above the 90th percentile of the controls (> 122 U/dL) increased the risk for thrombosis nearly 2-fold compared with TAFI levels below the 90th percentile (odds ratio, 1.7; 95% confidence interval, 1.1-2.5). Adjustment for various possible confounders did not materially affect this estimate. These results indicate that elevated TAFI levels form a mild risk factor for venous thrombosis. Such levels were found in 9% of healthy controls and in 14% of patients with a first deep vein thrombosis. Elevated TAFI levels did not enhance the thrombotic risk associated with factor V Leiden but may interact with high factor VIII levels. (Blood. 2000;95:2855-2859)