Detecting disease associated biomarkers by luminescence modulating phages.

Assessment of risk for a given disease and the diagnosis of diseases is often based on assays detecting biomarkers. Antibody-based biomarker-assays for diseases such as prostate cancer are often ambiguous and biomarker proteins are frequently also elevated for reasons that are unspecific. We have opted to use luminescence modulating phages for the analysis of known acute inflammatory response biomarker CRP (C-reactive protein) and biomarkers of prostate cancer in urine samples. Firstly, CRP was used to simulate the detection process in a controlled chemical environment. Secondly, we tried to classify more challenging lethal prostate cancer samples from control samples. Our unique method utilizes a special biopanning process in order to create special phages capable of capturing a dye necessary for detection and potential biomarkers. As the biomarker-molecules interfere with the phages, dye is repelled from the phage network resulting in an altered reporter luminescence. These changes can be observed with an absorbance reader and even with the naked eye. The simple method could present an alternative for screening of disease biomarkers. For prostate cancer urine samples, we achieved a sensitivity of 80% and specificity of 75% to detect Grade Group (GG) 4 and 5 prostate cancer.

Analysis of urinary kallikrein-related peptidase 13 for monitoring bladder cancer.

BACKGROUND: Bladder cancer (BC) is one of the 10 most common types of cancer worldwide, with approximately 550,000 new cases each year. Early detection and appropriate diagnosis are important factors in successful treatment of the disease. MATERIAL AND METHODS: We used specific fluorogenic substrate for the quantitative determination of urine kallikrein 13 (KLK13) activity in healthy (n = 15) and BC (n = 54) patients. The proteolytic activity in individual urine samples was determined by fluorescence measurements. Then, immunoenzymatic analyses (ELISA, Western blot) were performed to confirm the presence of KLK13 in the tested samples. RESULTS: Urine samples from patients with G2 and G3 grade BC contained proteolytically active KLK13, as confirmed by kinetic analysis and immunochemical detection. KLK13 was not detected in the urine of patients with G1 grade BC. DISCUSSION: Previous clinical study reveals the KLK13 significance for BC prognosis as increased KLK13 expression was highlighted in bladder tumours compared to normal adjacent tissues. Our findings correlate to the report. KLK13 activity was confirmed in BC patients with G2 and G3 stage of disease development. CONCLUSIONS: Using specific chromogenic/fluorogenic peptides could be useful for the non-invasive disease monitoring of BC progress.

Human urinary kininogenase reduces the endothelial injury by inhibiting Pyk2/MCU pathway.

The injury of endothelial cells is one of the initiating factors in restenosis after endovascular treatment. Human urinary kallidinogenase (HUK) is a tissue kallikrein which is used for ischemia-reperfusion injury treatment. Studies have shown that HUK may be a potential therapeutic agent to prevent stenosis after vascular injury, however, the precise mechanisms have not been fully established. This study is to investigate whether HUK can protect endothelial cells after balloon injury or H2O2-induced endothelial cell damage through the proline-rich tyrosine kinase 2 (Pyk2)/mitochondrial calcium uniporter (MCU) pathway. Intimal hyperplasia, a decrease of pinocytotic vesicles and cell apoptosis were found in the common carotid artery balloon injury and H2O2-induced endothelial cell damage, Pyk2/MCU was also up-regulated in such pathological process. HUK could prevent these injuries partially via the bradykinin B2 receptor by inhibiting Pyk2/MCU pathway, which prevented the mitochondrial damage, maintained calcium balance, and eventually inhibited cell apoptosis. Furthermore, MCU expression was not markedly increased if Pyk2 was suppressed by shRNA technique in the H2O2 treatment group, and cell viability was significantly better than H2O2-treated only. In short, our results indicate that the Pyk2/MCU pathway is involved in endothelial injury induced by balloon injury or H2O2-induced endothelial cell damage. HUK plays an protective role by inhibiting the Pyk2/MCU pathway in the endothelial injury.

N-Butylphthalide vs. Human Urinary Kallidinogenase for the Treatment of Acute Ischemic Stroke: Functional Outcome and Impact on Serum VEGF and TNF-α Expressions.

OBJECTIVE: To compare the e!cacy and functional outcomes of dl-3-n-Butylphthalide (NBP) and human urinary kallidinogenase (HUK) on ischemic stroke patients and to determine their effects on serum tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF). METHODS: A prospective study was conducted on 57 ischemic stroke patients. Functional outcomes were assessed by the National Institute Health Stroke Scale (NIHSS), the modified Rankin Scale (mRS), and the activities of daily living score (ADL), whereas TNF-α and VEGF expressions were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: TNF-α was significantly down-regulated in the NBP group and upregulated in the control group two weeks after treatment (p=0.017 and p=0.047, respectively). A significant difference in VEGF expressions was observed between the two groups (330.25±120.64 vs. 437.15±137.68, p=0.041) two weeks after treatment. Both groups showed significant improvement in NIHSS and ADL scores three months after treatment (p<0.001), with the NBP group exhibiting improvement in NIHSS scores as early as two weeks after treatment (p=0.008). The three-month NIHSS scores of the two groups were significantly lower than those of the control group (p=0.010 and p=0.008, respectively). Both the NBP and HUK groups showed a significant decline in mRS scores two weeks and three months after treatment (p<0.05). CONCLUSIONS: Both treatments are effective and can significantly promote recovery in stroke patients. Additionally, both options have similar effects in promoting long-term recovery, with NBP exerting a greater impact on serum VEGF and TNF-α expressions.

Influence of butyphthalide combined with urinary kallikrein in ACI treatment on neuro-cytokines and vascular endothelial function and its clinical effect.

Objective: To study the influence of butyphthalide combined with urinary kallikrein in acute cerebral infarction (ACI) treatment on neuro-cytokines and indicators of vascular endothelial function, observe the curative effect and adverse effects, and discuss its safety and feasibility.Method: 110 ACI patients were chosen as the objects, and classified into observation group (55 cases) and control group (55 cases) according to the method of random number table. Butyphthalide injection combined with urinary kallikrein was adopted for the observation group based on conventional treatment, while cinepazide maleate injection combined with alprostadil injection was applied for the control group based on conventional treatment. The following indicators of both groups were compared before and after treatment: neurotrophic factor (NTF), nerve growth factor (NGF), neuron specific enolase (NSE); content of CXC chemotactic factor ligand 16 (CXCL16), soluble CD ligand (CD40L), Fibulin-5 and high mobility group box B1 (HMGB1); the content of indicators of vascular endothelial function including plasma endothelin -1 (ET-1) and no therapeutic effects and adverse effects were recorded.Results: NSE of both groups after treatment decreased obviously, and the content of NTF and NGF increased obviously. NSE content of observation group was lower than that of control group. NTF content and NGF content of observation group were higher than those of control group. The differences had statistical significance (p < 0.05). The levels of CXCL16, CD40L, Fibulin-5 and HMGB1 declined obviously, compared with pre-treatment, and the levels of observation groups were significantly lower than those of control grip. The differences had statistical significance (p < 0.05). ET-1 level rose significantly after treatment, and NO level declined obviously after treatment. ET-1 level of observation group was significantly higher than that of control group, and NO level of observation group was significantly lower than that of control group. The difference had statistical significance (p < 0.05). Clinical effect of observation group was significantly higher than that of control group. The difference had statistical significance (p < 0.05). The comparison difference of both groups in the occurrence rate of adverse effects had no statistical significance (p > 0.05).Conclusion: The application of butyphthalide combined with urinary kallikrein in ACI treatment can effectively inhibit secretion and release of neuro-cytokines, and improve patients' vascular endothelial function, with significant treatment effect and high safety. Therefore, it deserves to be promoted clinically.

Targeted Mass Spectrometry of a Clinically Relevant PSA Variant from Post-DRE Urines for Quantitation and Genotype Determination.

PURPOSE: The rs17632542 single nucleotide polymorphism (SNP) results in lower serum prostate specific antigen (PSA) levels which may further mitigate against its clinical utility as a prostate cancer biomarker. Post-digital rectal exam (post-DRE) urine is a minimally invasive fluid that is currently utilized in prostate cancer diagnosis. To detect and quantitate the variant protein in urine. EXPERIMENTAL DESIGN: Fifty-three post-DRE urines from rs17632542 genotyped individuals processed and analyzed by liquid chromatography/mass spectrometry (LC-MS) in a double-blinded randomized study. The ability to distinguish between homozygous wild-type, heterozygous, or homozygous variant is examined before unblinding. RESULTS: Stable-isotope labeled peptides are used in the detection and quantitation of three peptides of interest in each sample using parallel reaction monitoring (PRM). Using these data, groupings are predicted using hierarchical clustering in R. Accuracy of the predictions show 100% concordance across the 53 samples, including individuals homozygous and heterozygous for the SNP. CONCLUSIONS AND CLINICAL RELEVANCE: The study demonstrates that MS based peptide variant quantitation in urine could be useful in determining patient genotype expression. This assay provides a tool to evaluate the utility of PSA variant (rs17632542) in parallel with current and forthcoming urine biomarker panels.

Human urinary kallidinogenase in treating acute ischemic stroke patients: analyses of pooled data from a randomized double-blind placebo-controlled phase IIb and phase III clinical trial.

Objectives: Intravenous thrombolysis and thrombectomy are recommended for patients whose stroke onsets are within first 6 h, and very few options are available for patients whose stroke onset is more than 6 h, which includes most ischemic stroke patients. Human urinary kallidinogenase (HUK) showed potential clinical benefits in acute ischemic stroke patients. This study aims to investigate the safety and clinical benefits of HUK in ischemic stroke patients.Patients and methods: Patients were recruited for a multicenter double-blind, placebo-controlled phase II b and phase III trial. Neurophysiological outcomes were assessed by the European Stroke Scale (ESS) and the functional outcomes were assessed by the activity of daily living scale (ADL). Safety was monitored by recording adverse events.Results: The improvements in ESS scores and ADL scores in the HUK group were significantly greater than that in patients receiving placebo. Furthermore, HUK treatment was also associated with a lower rate of disable, according to ADL. HUK-related adverse events occurred at a low rate, in 1.73% of HUK-treated patients.Conclusion: HUK is safe and provides potential clinical benefits as a treatment for acute ischemic stroke. Further large post-marketing observational studies are needed.

Beyond PSA: The Role of Prostate Health Index (phi).

BACKGROUND: Widespread use of prostate specific antigen (PSA) in screening procedures allowed early identification of an increasing number of prostate cancers (PCas), mainly including indolent cancer. Availability of different therapeutic strategies which have a very different impact on the patient's quality of life suggested a strong need for tools able to identify clinically significant cancer at diagnosis. Multi-parametric magnetic resonance showed very good performance in pre-biopsy diagnosis. However, it is an expensive tool and requires an experienced radiologist. In this context, a simple blood-based test is worth investigating. In this context, researchers focused their attention on the development of a laboratory test able to minimize overdiagnosis without losing the identification of aggressive tumors. RESULTS: Recent literature data on PCa biomarkers revealed a clear tendency towards the use of panels of biomarkers or a combination of biomarkers and clinical variables. Phi, the 4Kscore, and Stockholm3 as circulating biomarkers and the Mi-prostate score, Exo DX Prostate, and Select MD-X as urinary biomarker-based tests have been developed. In this scenario, phi is worthy of attention as a noninvasive test significantly associated with aggressive PCa. CONCLUSIONS: Literature data showed that phi had good diagnostic performance to identify clinically significant (cs) PCa, suggesting that it could be a useful tool for personalized treatment decision-making. In this review, phi potentialities, limitations, and comparisons with other blood- and urinary-based tests were explored.

A critical appraisal of biomarkers in prostate cancer.

PURPOSE: A number of urine and blood-based biomarker tests have been described for prostate cancer, although to date there has only been a limited exploration of the methodology behind the validation studies that underpin these tests. METHODS: In this review, a selection of commercially available urine and blood-based biomarker tests for prostate cancer are described, and the underlying key validation studies for each test are critically appraised using the Standards for Reporting Diagnostic Accuracy (STARD) 2015 statement. RESULTS: The ExoDx Prostate Intelliscore, SelectMDx, Progensa PCA3, Mi-Prostate Score, 4K Score, and Prostate Health Index (PHI) tests were reviewed. Most of the validation studies supporting these tests perform exploratory analyses to determine cut-off values in a post hoc manner, comprise cohorts that are primarily Caucasian, report receiver operating characteristic curves that combine the biomarker's result with established clinical nomograms and are based on a reference standard (prostate biopsy) that lacks central pathology review. Deficiencies in STARD reporting guidelines include frequent failure to provide a published study protocol, prospective study registration in a registry, a flow diagram, justification for sample size determination, a discussion of adverse events with testing, and information on how missing or indeterminate test results should be managed. CONCLUSIONS: Key validation studies that support many commercially available urine and blood-based biomarkers for prostate cancers have deficiencies in transparency based on STARD reporting guidelines, and limitations in methodology must be considered when deciding when these tests should be applied in clinical practice.

"Mix-to-Go" Silver Colloidal Strategy for Prostate Cancer Molecular Profiling and Risk Prediction.

Molecular profiling via analysis of multiple disease biomarkers is a powerful tool for disease diagnosis and risk prediction. Due to simplicity and minimal instrumentation requirement, colloidal-based colorimetric DNA/RNA assays are attractive for driving molecular profiling toward widespread clinical usage. Still, the reliability and speed of current colorimetric assays need to be further improved upon for eventual clinical use. Herein, we propose a "mix-to-go" colloidal strategy that utilizes the electrostatic attraction between negatively charged target sequences and positively charged silver nanoparticles (AgNPs) to induce aggregation of AgNPs to profile a panel of clinically validated urinary prostate cancer (PCa) RNA biomarkers ( TMPRSS2:ERG, T2:ERG; prostate cancer antigen 3, PCA3; and kallikrein-related peptidase 2, KLK2). Our strategy is unique in inducing a rapid (10 s), unambiguous solution color change in the presence of target sequences, without the additional NP aggregation agents that are used in existing electrostatic-mediated aggregation assays. Our strategy is analytically specific and sensitive for the detection of 105 and 104 target copies by the naked eye and UV-vis spectrophotometry, respectively. Analytical accuracies of our strategy in detecting T2:ERG, PCA3, and KLK2 RNA biomarkers were 95.9%, 97.3%, and 100%, respectively, as validated by quantitative reverse transcription-polymerase chain reaction. To further evaluate clinical molecular profiling performance beyond conventional proof-of-concept demonstration, we tested our colloidal strategy for noninvasive PCa risk prediction of 73 patients, using the urinary RNA biomarker panel comprising of T2:ERG, PCA3, and KLK2. We found that elevated T2:ERG and PCA3 levels were positively associated with high-risk PCa and obtained a corresponding area-under-the-curve values of 0.790 and 0.833 for predicting PCa and high-risk PCa on biopsy, respectively. We believe our "mix-to-go" strategy may serve as a reliable and accessible Ag colloidal-based molecular profiling approach for clinical applications.

Genomic Pathology and Cancer Biomarkers: Prostate Cancer.

Our understanding of genomic pathology and biomarkers for prostate cancer is continually growing. Some promising and useful tissue markers are GSTP1, HOXD3, cell cycle proteins, chromatin remodeling proteins, androgen receptor, Stat5a/b, ERG, and PTEN. Serum and urine markers are mostly either prostate-specific antigen or newer tests using one or more other kallikreins or sarcosine. The data and evidence for all of these markers and the commercial tests using them are reviewed here.

Prostate Cancer Patients-Negative Biopsy Controls Discrimination by Untargeted Metabolomics Analysis of Urine by LC-QTOF: Upstream Information on Other Omics.

The existing clinical biomarkers for prostate cancer (PCa) diagnosis are far from ideal (e.g., the prostate specific antigen (PSA) serum level suffers from lack of specificity, providing frequent false positives leading to over-diagnosis). A key step in the search for minimum invasive tests to complement or replace PSA should be supported on the changes experienced by the biochemical pathways in PCa patients as compared to negative biopsy control individuals. In this research a comprehensive global analysis by LC-QTOF was applied to urine from 62 patients with a clinically significant PCa and 42 healthy individuals, both groups confirmed by biopsy. An unpaired t-test (p-value < 0.05) provided 28 significant metabolites tentatively identified in urine, used to develop a partial least squares discriminant analysis (PLS-DA) model characterized by 88.4 and 92.9% of sensitivity and specificity, respectively. Among the 28 significant metabolites 27 were present at lower concentrations in PCa patients than in control individuals, while only one reported higher concentrations in PCa patients. The connection among the biochemical pathways in which they are involved (DNA methylation, epigenetic marks on histones and RNA cap methylation) could explain the concentration changes with PCa and supports, once again, the role of metabolomics in upstream processes.

Effect of Urinary Kallidinogenase on Transforming Growth Factor-ß1 and High-Sensitivity C-Reactive Protein Expression in Rat Focal Cerebral Ischemic Injury.

BACKGROUND In this study we investigated the effect of urinary kallidinogenase (UK) on transforming growth factor beta 1 (TGF-ß1) expression in brain tissue. We also explored the neuroprotective mechanism of UK against ischemic injury by measuring serum high-sensitivity C-reactive protein (hs-CRP) level changes after rat cerebral ischemic injury. MATERIAL AND METHODS The rat middle cerebral artery ischemia/reperfusion model was established using the suture method. Sprague-Dawley rats were randomly divided into 3 groups: treatment, Gegen control, and blank control. Each group was subsequently divided into 5 subgroups according to time (6, 12, 24, 48, and 72 h). Rats in the treatment group were administered UK as treatment. TGF-ß1 expression was observed at each time point using SABC and immunohistochemical staining methods to estimate cerebral infarct volume percentage. Serum hs-CRP levels were also measured. RESULTS TGF-ß1 protein expression in ischemic brain tissues of the treatment group significantly increased at each time point (P<0.01) compared with both control groups. Treatment group serum hs-CRP levels significantly decreased at each time point (P<0.05) compared with both control groups. CONCLUSIONS UK exerts a neuroprotective effect by upregulating TGF-ß1 expression and inhibiting excessive inflammatory responses.

Analysis of Urinary Prostate-Specific Antigen Glycoforms in Samples of Prostate Cancer and Benign Prostate Hyperplasia.

Glycans of prostate-specific antigen (PSA) in prostate cancer were found to be different from that in benign disease. It is difficult to analyze heterogeneous PSA glycoforms in each individual specimen because of low protein abundance and the limitation of detection sensitivity. We developed a method for prostate cancer diagnosis based on PSA glycoforms. Specific glycoforms were screened in each clinical sample based on liquid chromatography-tandem mass spectrometry with ion accumulation. To look for potential biomarkers, normalized abundance of each glycoform in benign prostate hyperplasia (BPH) and in prostate cancer was evaluated. The PSA glycoform, Hex5HexNAc4NeuAc1dHex1, and monosialylated, sialylated, and unfucosylated glycoforms differed significantly between the prostate cancer and BPH samples. The detection sensitivity (87.5%) and specificity (60%) for prostate cancer identification are higher than those of the serum PSA marker. As low as 100 amol PSA could be detected with the ion accumulation method which has not been reported before. The improved detection specificity can help reduce unnecessary examinations.

Exogenous human urinary kallidinogenase increases cerebral blood flow in patients with acute ischemic stroke.

OBJECTIVE: To study the effects of human urinary kallidinogenase (HUK) treatment on acute cerebral ischemia (ACI) using magnetic resonance perfusion weighted imaging (MRP) methods. METHODS: In a non-randomized controlled clinical trial, 30 patients diagnosed with ACI were enrolled and divided manually into 2 groups. The experimental group, consisting of 18 participants, was treated with HUK (0.15 Perinatal Assessment Unit/day) for 7 consecutive days. The control group was treated with routine medication. The participants underwent MRP examination on the first and fourteenth day after onset. The National Institutes of Health Stroke Scale (NIHSS) score, cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), and time to peak (TTP) were compared between the groups. RESULTS: After undergoing therapy, the experimental HUK-treated group had lower NIHSS scores than the control group (p<0.05). The CBF improved more in the HUK-treated group than in the control group (p<0.05). Additionally, MTT and TTP were shorter in the HUK-treated group than in the control group (p<0.05). CONCLUSION: Human urinary kallidinogenase improves CBF and ameliorates neurological deficits. Human urinary kallidinogenase is a safe and effective treatment approach for treating patients with ACI.

Human urine kininogenase attenuates balloon-induced intimal hyperplasia in rabbit carotid artery through transforming growth factor ß1/Smad2/3 signaling pathway.

OBJECTIVE: Effective treatments against restenosis after percutaneous transluminal angioplasty and stenting are largely lacking. Human tissue kallikrein gene transfer has been shown to be able to attenuate neointima formation induced by balloon catheter. As a tissue kallikrein in vivo, human urinary kininogenase (HUK) is widely used to prevent ischemia-reperfusion injury. However, the effects of HUK on neointima formation have not been explored. We therefore investigated whether HUK could alleviate balloon catheter-induced intimal hyperplasia in rabbits fed with high-fat diets. METHODS: The effects of HUK on neointima and atherosclerosis formation were analyzed by hematoxylin-eosin staining and immunohistochemical staining in balloon-injured carotid arteries of rabbits. Local inflammatory response was evaluated by detecting the gene expression of tumor necrosis factor α and interleukin 1ß with real-time quantitative polymerase chain reaction plus the invasion of macrophages with immunohistochemical staining. Western blotting was employed to investigate the effects of HUK on activities of endothelial nitric oxide synthase (eNOS), transforming growth factor ß1 (TGF-ß1), and Smad signaling pathway. The long-term effect of HUK on intimal hyperplasia of the injured carotid artery was assessed by angiography. RESULTS: Quantitative image analysis showed that intravenous administration of HUK for 14 days significantly decreased the intimal areas and intima area/media area ratios (day 14, 54% decrease in intimal area and 58% decrease in intima area/media area ratios; day 28, 63% and 85%). Significant decreases were also noted in macrophage foam cell-positive area after 7-day or 14-day administration of HUK (day 7, 69% decrease in intimal area and 78% decrease in media area; day 14, 79% and 60%; day 28, 68% and 44%). Actin staining for smooth muscle cells in neointima at 2 months showed similar results (vascular smooth muscle cell-positive area of neointima, 28.21% ± 5.58% vs 43.78% ± 8.36%; P < .05). Real-time quantitative polymerase chain reaction or Western blot analysis showed that HUK reduced expression of tumor necrosis factor α, interleukin 1ß, TGF-ß1, and p-Smad2/3 but increased the expression of p-eNOS. Angiography analysis showed that 14-day administration of HUK significantly decreased the degree of stenosis (26.8% ± 7.1% vs 47.9% ± 5.7%; P < .01) at 2 months after balloon injury. CONCLUSIONS: Our results indicate that HUK is able to attenuate atherosclerosis formation and to inhibit intimal hyperplasia by downregulating TGF-ß1 expression and Smad2/3 phosphorylation, upregulating eNOS activity. HUK may be a potential therapeutic agent to prevent stenosis after vascular injury.

Increased Klk9 Urinary Excretion Is Associated to Hypertension-Induced Cardiovascular Damage and Renal Alterations.

Early detection of hypertensive end-organ damage and secondary diseases are key determinants of cardiovascular prognosis in patients suffering from arterial hypertension. Presently, there are no biomarkers for the detection of hypertensive target organ damage, most outstandingly including blood vessels, the heart, and the kidneys.We aimed to validate the usefulness of the urinary excretion of the serine protease kallikrein-related peptidase 9 (KLK9) as a biomarker of hypertension-induced target organ damage.Urinary, plasma, and renal tissue levels of KLK9 were measured by the Western blot in different rat models of hypertension, including angiotensin-II infusion, DOCA-salt, L-NAME administration, and spontaneous hypertension. Urinary levels were associated to cardiovascular and renal injury, assessed by histopathology. The origin of urinary KLK9 was investigated through in situ renal perfusion experiments.The urinary excretion of KLK9 is increased in different experimental models of hypertension in rats. The ACE inhibitor trandolapril significantly reduced arterial pressure and the urinary level of KLK9. Hypertension did not increase kidney, heart, liver, lung, or plasma KLK9 levels. Hypertension-induced increased urinary excretion of KLK9 results from specific alterations in its tubular reabsorption, even in the absence of overt nephropathy. KLK9 urinary excretion strongly correlates with cardiac hypertrophy and aortic wall thickening.KLK9 appears in the urine in the presence of hypertension as a result of subtle renal handling alterations. Urinary KLK9 might be potentially used as an indicator of hypertensive cardiac and vascular damage.

Improved sensitivity and specificity for prostate cancer diagnosis based on the urine PCA3/PSA ratio acquired by sequence­specific RNA capture.

Prostate cancer antigen 3 (PCA3) is a non-coding RNA fragment that is overexpressed in prostate cancer cells. However, the clinical applications of PCA3 are highly limited due to the instability of RNA and the lack of reliable and efficient RNA extraction and purification methods. Thus, in the present study, we compared three different methods of RNA extraction to further confirm the higher yield of commercial magnetic beads with poly-T functionalization and a capturer strand. The current protocols for RNA extraction of i) the phenol-chloroform method, ii) the affinity column method and iii) magnetic beads with poly-T functionalization and a capturer strand were applied separately for RNA extraction in urine samples. Reverse transcription­quantitative polymerase chain reaction was performed to evaluate the yield of the three methods of RNA extraction. Furthermore, 52 urine samples after prostate massage from patients suspected of a diagnosis of prostate cancer were collected. The Mag-Cap method and RT-PCR were applied to obtain the PCA3 score. The clinical value of the PCA3 score was investigated by comparison with the pathology of the prostate biopsy. The yield of the Mag-Cap method was higher than that of the phenol­chloroform method and commercial kits. Thirty­four patients were pathologically diagnosed with prostate cancer and 18 with benign prostatic hyperplasia (BPH). It was confirmed that the median PCA3 score was higher among the prostate cancer patients than those with benign disease (53.5 vs. 17, p=0.000). A sensitivity of 82.4% and a specificity of 77.8% were obtained when the cut-off value for the PCA3 score was 28.5. The Mag-Cap method was found to be more efficient for RNA extraction. The urinary PCA3 score is a promising method for prostate cancer screening, detection and diagnosis, and has the potential to reduce unnecessary prostate biopsies.

Human Urinary Kallidinogenase Promotes Angiogenesis and Cerebral Perfusion in Experimental Stroke.

Angiogenesisis a key restorative mechanism in response to ischemia, and pro-angiogenic therapy could be beneficial in stroke. Accumulating experimental and clinical evidence suggest that human urinary kallidinogenase (HUK) improves stroke outcome, but the underlying mechanisms are not clear. The aim of current study was to verify roles of HUK in post-ischemic angiogenesis and identify relevant mediators. In rat middle cerebral artery occlusion (MCAO) model, we confirmed that HUK treatment could improve stroke outcome, indicated by reduced infarct size and improved neurological function. Notably, the 18F-FDG micro-PET scan indicated that HUK enhanced cerebral perfusion in rats after MCAO treatment. In addition, HUK promotespost-ischemic angiogenesis, with increased vessel density as well as up-regulated VEGF andapelin/APJ expression in HUK-treated MCAO mice. In endothelial cell cultures, induction of VEGF and apelin/APJ expression, and ERK1/2 phosphorylation by HUK was further confirmed. These changes were abrogated by U0126, a selective ERK1/2 inhibitor. Moreover, F13A, a competitive antagonist of APJ receptor, significantly suppressed HUK-induced VEGF expression. Furthermore, angiogenic functions of HUK were inhibited in the presence of selective bradykinin B1 or B2 receptor antagonist both in vitro and in vivo. Our findings indicate that HUK treatment promotes post-ischemic angiogenesis and cerebral perfusion via activation of bradykinin B1 and B2 receptors, which is potentially due to enhancement expression of VEGF and apelin/APJ in ERK1/2 dependent way.

Study on the effect of urinary kallidinogenase after thrombolytic treatment for acute cerebral infarction.

OBJECTIVE: To evaluate the safety and efficacy of urinary kallidinogenase for recombinant tissue-type plasminogen activator (rt-PA) intravenous thrombolytic treatment in patients with acute cerebral infarction. PATIENTS AND METHODS: All 200 patients with acute cerebral infarction were randomized 1:1 into an experimental group (100 cases) and a control group (100 cases). Patients in the control group were administrated rt-PA (0/9 mg/kg) while patients in the experimental group were given urinary kallidinogenase by intravenous drip (0.15 PNAU/d, for 7 days) after rt-PA intravenous thrombolytic treatment (0.9 mg/kg). The main evaluation index was NIHSS and BI. RESULTS: Compared to the control group, the NIHSS scores were significantly lower 7 and 90 days after thrombolytic therapy (t = 2.391, 2.714; p < 0.05). BI scores were obviously higher at 90 days after thrombolytic therapy in the experimental group (t = 2.675, p < 0.05). CONCLUSIONS: Urinary kallidinogenase may improve the treatment effect for rt-PA intravenous thrombolytic treatment in patients with acute cerebral infraction.