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1.  Bladder Cancer Diagnosis and Identification of Clinically Significant Disease by Combined Urinary Detection of Mcm5 and Nuclear Matrix Protein 22 
PLoS ONE  2012;7(7):e40305.
Urinary biomarkers for bladder cancer detection are constrained by inadequate sensitivity or specificity. Here we evaluate the diagnostic accuracy of Mcm5, a novel cell cycle biomarker of aberrant growth, alone and in combination with NMP22.
1677 consecutive patients under investigation for urinary tract malignancy were recruited to a prospective blinded observational study. All patients underwent ultrasound, intravenous urography, cystoscopy, urine culture and cytologic analysis. An immunofluorometric assay was used to measure Mcm5 levels in urine cell sediments. NMP22 urinary levels were determined with the FDA-approved NMP22® Test Kit.
Genito-urinary tract cancers were identified in 210/1564 (13%) patients with an Mcm5 result and in 195/1396 (14%) patients with an NMP22 result. At the assay cut-point where sensitivity and specificity were equal, the Mcm5 test detected primary and recurrent bladder cancers with 69% sensitivity (95% confidence interval = 62–75%) and 93% negative predictive value (95% CI = 92–95%). The area under the receiver operating characteristic curve for Mcm5 was 0.75 (95% CI = 0.71–0.79) and 0.72 (95% CI = 0.67–0.77) for NMP22. Importantly, Mcm5 combined with NMP22 identified 95% (79/83; 95% CI = 88–99%) of potentially life threatening diagnoses (i.e. grade 3 or carcinoma in situ or stage ≥pT1) with high specificity (72%, 95% CI = 69–74%).
The Mcm5 immunoassay is a non-invasive test for identifying patients with urothelial cancers with similar accuracy to the FDA-approved NMP22 ELISA Test Kit. The combination of Mcm5 plus NMP22 improves the detection of UCC and identifies 95% of clinically significant disease. Trials of a commercially developed Mcm5 assay suitable for an end-user laboratory alongside NMP22 are required to assess their potential clinical utility in improving diagnostic and surveillance care pathways.
PMCID: PMC3392249  PMID: 22792272
2.  Usefulness of the NMP22BladderChek Test for Screening and Follow-up of Bladder Cancer 
Korean Journal of Urology  2010;51(2):88-93.
We evaluated the usefulness of the nuclear matrix protein 22 BladderChek (NMP22BC) test for the screening and follow-up of bladder cancer.
Materials and Methods
From February 2006 to September 2009, we enrolled 1,070 patients who had hematuria or who were being followed up for bladder cancer. We compared the sensitivity and specificity of the NMP22BC test with those of urine cytology.
The sensitivity of the NMP22BC test (77.5%) was significantly higher than that of urine cytology (46.3%). The specificity of the NMP22BC test was 88.8%, compared with 97.9% for urine cytology. The sensitivity of the NMP22BC test (81.8%) in non-muscle-invasive bladder cancer was higher than that of cytology (36.4%). However, the sensitivity of the NMP22BC test and of urine cytology in invasive bladder cancer were 57.1% and 92.9%, respectively. The sensitivity of the NMP22BC test was higher for low-grade bladder cancer (83.9%) than for high-grade (62.5%), and the sensitivity of cytology was higher for high-grade bladder cancer (66.7%) than for low-grade (37.5%). Follow-up bladder cancer was detected in 262 patients. The sensitivity of the NMP22BC test in that group (72.7%) was decreased and the specificity (91.7%) was increased. The sensitivity of cytology (54.5%) in the follow-up group was increased and the specificity (95.6%) was decreased. The presence of pyuria was significantly associated with the lower specificity of the NMP22BC test.
The greater sensitivity of the NMP22BC test may be more useful for the diagnosis of non-muscle-invasive bladder cancer and low-grade bladder cancer than for the diagnosis of invasive or high-grade bladder cancer. If the NMP22BC test is performed in the absence of pyuria, it may play a compensatory role for urine cytology.
PMCID: PMC2855476  PMID: 20414419
Nuclear matrix protein 22; Cytology; Urinary bladder neoplasms
3.  Galactosyl Human Serum Albumin-NMP1 Conjugate: A Near Infrared (NIR)-Activatable Fluorescence Imaging Agent to Detect Peritoneal Ovarian Cancer Metastases 
Bioconjugate chemistry  2012;23(8):1671-1679.
Patient survival depends on the completeness of resection of peritoneal ovarian cancer metastases (POCM) and therefore, it is important to develop methods to enhance detection. Previous probe designs based on activatable galactosyl human serum albumin (hGSA)-fluorophore pairs, which target lectin receptors expressed on POCM, have used only visible range dyes conjugated to hGSA. However, imaging probes emitting fluorescence in the NIR range are advantageous because NIR photons have deeper in vivo tissue penetration and result in lower background autofluorescence than those emitting in the visible range. A NIR-activatable hGSA fluorophore was synthesized using a bacteriochlorin-based dye, NMP1. NMP1 has two unique absorption peaks, one in the green range and the other in the NIR range, but emits at a NIR peak of 780 nm. NMP1, thus, has two different Stokes shifts that have the potential to allow imaging of POCM both at the peritoneal surface and just below it.
hGSA was conjugated with 2 NMP1 molecules to create a self-quenching complex (hGSA-NMP1). The activation ratio of hGSA-NMP1 was measured by the fluorescence intensity before and after exposure to 10% SDS. The activation ratio of hGSA-NMP1 was ~100-fold in vitro. Flow cytometry, fluorescence microscopy, and in vivo spectral fluorescence imaging were carried out to compare hGSA-NMP1 with hGSA-IR800 and hGSA-ICG (two always-on control agents with similar emission to NMP1) in terms of comparative fluorescence signal and the ability to detect POCM in mice models. The sensitivity and specificity of hGSA-NMP1 for POCM implant detection were determined by co-localizing NMP1 emission spectra with red fluorescent protein (RFP) expressed constitutively in SHIN3 tumor implants at different depths below the peritoneal surface. In vitro, SHIN3 cells were easily detectable after 3 hours of incubation with hGSA-NMP1. In vivo submillimeter POCM foci were clearly detectable with spectral fluorescence imaging using hGSA-NMP1. Among 555 peritoneal lesions, hGSA-NMP, using NIR and green excitation light, respectively, detect 75% of all lesions and 91% of lesions ~0.8 mm or greater in diameter. Few false positives were encountered. Nodules located at a depth below the small bowel surface were only depicted with hGSA-NMP1.
We conclude that hGSA-NMP1 is useful in imaging peritoneal ovarian cancer metastases, located both superficially and deep in the abdominal cavity.
PMCID: PMC3432315  PMID: 22799539
fluorescence imaging; activatable; near infrared; multiple excitations
4.  The Clinical Usefulness of Nuclear Matrix Protein-22 in Patients with Atypical Urine Cytology 
Korean Journal of Urology  2011;52(9):603-606.
Difficulty exists in interpreting the significance of atypical urine cytology. This study was performed to assess the diagnostic utility of nuclear matrix protein-22 (NMP-22) testing when atypical cells are detected during urine cytology.
Materials and Methods
Among patients whose urine cytology was reported as atypical between January 2004 and December 2009, a total of 275 who also underwent NMP-22 testing were enrolled in the present study. These patients were further divided into the screening group (143 patients examined as outpatients for hematuria) and the follow-up group (132 patients followed up for previously diagnosed bladder cancer). The sensitivity, specificity, positive and negative predictive values, and accuracy were assessed for atypical cytology alone and in conjunction with NMP-22.
Of the 275 patients exhibiting atypical urine cytology, cancer was confirmed in 85, yielding a positive predictive value of 30.9% (85/275). Of the 96 patients testing positive for NMP-22, 58 were diagnosed with bladder cancer. The positive predictive value in conjunction with NMP-22 was 60.4% (58/96). The sensitivity, specificity, negative predictive value, and accuracy were 68.2% (58/85), 80.0% (152/190), 84.9% (152/179), and 76.2% (210/275), respectively. Testing for NMP-22 in the screening and follow-up groups increased the positive predictive value from 30.0% (43/143) to 64.0% (32/50) and from 31.3% (42/132) to 56.5% (26/46), respectively; there was no significant difference between the screening and follow-up groups (p=0.106).
When only cases with atypical urine cytology were examined, NMP-22 testing increased the detection rate of bladder cancer regardless of whether the test was used in screening hematuria or in following up patients.
PMCID: PMC3198232  PMID: 22025954
Cytology; Nuclear matrix; Urinary bladder neoplasms
5.  Assessing the clinical benefit of NMP22 in the surveillance of patients with non–muscle-invasive bladder cancer and negative cytology: a decision-curve analysis 
Cancer  2011;117(13):2892-2897.
Several studies have shown that abnormal levels of nuclear matrix protein 22 (NMP22) are associated with bladder cancer, leading to NMP22 being approved as a urinary biomarker by the FDA. Nonetheless, the clinical significance of NMP22 remains unclear.
To use decision analysis to determine whether NMP22 improves medical decision-making.
Design, Setting, and Participants
The study included 2,222 patients with a history of non–muscle-invasive bladder cancer and current negative cytology. We developed models to predict cancer recurrence or progression to muscle-invasive disease using NMP22 levels, age, and gender.
Voided NMP22 and cystoscopy.
Clinical net benefit was calculated by summing the benefits (true positives) and subtracting the harms (false positives) and weighting these by the threshold probability at which a patient or clinician would opt for cytoscopy.
Results and limitations
After cystoscopy, 581 (26%) patients were found to have cancer. NMP22 level was significantly associated with bladder cancer recurrence and progression (p<0.001 for both). Using NMP22 in a model with age and gender was associated with better patient outcomes than performing cystoscopy on everyone for threshold probabilities above 8% for recurrence and above 3% for progression. Only offering cystoscopy to those with a 15% or greater risk would reduce the number of cystoscopies by 229, while missing only 25 cancer recurrences per 1000 men with a negative cytology. The study was limited by its multicenter design.
For clinicians who would perform a cystoscopy at a threshold of 5% for recurrence or 1% for progression, NMP22 will not aid clinical decision-making. For less risk-averse clinicians who would only perform a cystoscopy at a threshold probability >8% for recurrence or >3% for progression, NMP22 can help determine which patients require cystoscopy and which can be spared this procedure.
PMCID: PMC3334293  PMID: 21692050
nuclear matrix protein 22; bladder cancer; urothelial carcinoma; detection; surveillance
6.  Biomarkers for detection and surveillance of bladder cancer 
Bladder cancer is the fourth most common cancer in men and the ninth most common cancer in women in Canada. Early detection of tumours is essential for improved prognosis and long-term survival. The standard method for detection and surveillance is cystoscopy together with urine cytology. Cystoscopy is relatively sensitive but is expensive and invasive. Urinary cytology is a noninvasive method that has poor sensitivity but high specificity; it is relied on for the detection of carcinoma in situ. Currently, several urinary-based bladder tumour biomarkers with USFDA/Health Canada approval are available commercially, but none have been widely adopted by urologists despite their offering high sensitivity and/or specificity. We present here a review of recent studies evaluating 7 commercial biomarker assays for the detection and/or surveillance of bladder cancer.
Sensitivity and specificity ranges, respectively, for each marker were reported as follows: BTA Stat (Polymedco), 52.5%–78.0% and 69.0%–87.1%; BTA Trak (Polymedco), 51%–100% and 73%–92.5%; cytology, 12.1%–84.6% and 78.0%–100%; hematuria dipstick, 47.0%–92.6% and 51.0%–84.0%; NMP22 Bladder Cancer Test (Matritech), 34.6%–100% and 60.0%–95.0%; NMP22 BladderChek (Matritech), 49.5%–65.0% and 40.0%–89.8%; ImmunoCyt/uCyt+ (DiagnoCure), 63.3%–84.9% and 62.0%–78.1%; ImmunoCyt/uCyt+ and cytology, 81.0%–89.3% and 61.0%–77.7%; and UroVysion (Abbott Molecular)/florescence in situ hybridization, 68.6%–100% and 65.0%–96.0%.
We find that no currently available bladder cancer urinary marker is sensitive enough to eliminate the need for cystoscopy. In addition, cytology remains integral to the detection of occult cancer. However, owing to their relatively high sensitivities, these markers may be used to extend the period between cystoscopies in the surveillance of patients with transitional cell carcinoma. Further study is required to determine which markers, alone or in panel, would best accomplish this.
PMCID: PMC2494897  PMID: 18682775
7.  Prognostic relevance of positive urine markers in patients with negative cystoscopy during surveillance of bladder cancer 
BMC Cancer  2015;15:155.
The role of urine markers in the surveillance of patients with non-muscle invasive bladder cancer (NMIBC) is discussed extensively. In case of negative cystoscopy the additional prognostic value of these markers has not been clearly defined yet. The present study is the first systematic approach to directly compare the ability of a urine marker panel to predict the risk of recurrence and progression in bladder cancer (BC) patients with no evidence of relapse during surveillance for NMIBC.
One hundred fourteen patients who underwent urine marker testing during surveillance for NMIBC and who had no evidence of BC recurrence were included. For all patients cytology, Fluorescence-in-situ-hybridization (FISH), immunocytology (uCyt+) and Nuclear matrix protein 22 enzyme-linked immunosorbent assay (NMP22) were performed. All patients completed at least 24 months of endoscopic and clinical follow-up of after inclusion.
Within 24 months of follow-up, 38 (33.0%) patients experienced disease recurrence and 11 (9.8%) progression. Recurrence rates in patients with positive vs. negative cytology, FISH, uCyt+ and NMP22 were 52.6% vs. 21.9% (HR = 3.9; 95% CI 1.75-9.2; p < 0.001), 47.6% vs. 25.0% (HR 2.7; 1.2-6.2; p = 0.01), 43.8% vs. 22.4% (HR 3.3; 1.5-7.6; p = 0.003) and 43.8% vs. 16.7% (HR 4.2; 1.7-10.8; p = 0.001). In patients with negative cytology, a positive NMP22 test was associated with a shorter time to recurrence (p = 0.01), whereas FISH or uCyt+ were not predictive of recurrence in these patients. In the group of patients with negative cytology and negative NMP22, only 13.5% and 5.4% developed recurrence and progression after 24 months.
Patients with positive urine markers at time of negative cystoscopy are at increased risk of recurrence and progression. In patients with negative cytology, only NMP22 is predictive for recurrence. Patients with negative marker combinations including NMP22 harbour a low risk of recurrence. Therefore, the endoscopic follow-up regimen may be attenuated in this group of patients.
PMCID: PMC4374530  PMID: 25884545
Urine markers; Prediction; Recurrence; Risk; Surveillance; Anticipatory positive
8.  Urinary Tumor Markers Could Predict Survival in Bladder Carcinoma 
The early diagnosis of bladder cancer is important for effective treatment of the disease. This study aimed to evaluate the nuclear matrix protein 22 (NMP 22), soluble epithelial cadherin (E-cadherin), cathepthin-D and total protein with clinico-pathological features of bladder cancer, and to determine the relation between each marker and tumor progression after treatment. The study includes 65 patients with bladder cancer, 14 benign urinary diseases and 11 healthy volunteers. Patients were categorized according to bilharzial infestation, T stage, tumor grade, size and the presence of lymph node metastasis. Forty patients were followed for disease progression after surgery. There was a significant increase of NMP22, E-cadherin, cathepthin-D and total protein detected in cancer group compared to healthy and benign groups. It was found that NMP 22 and E-cadherin had highest sensitivity (84.4, 76.9 %, respectively) while, total ddedprotein showed highest specificity (77.4 %). Tumor size correlated with urinary NMP22 (r = 0.3, p = 0.02), although, E-cadherin, cathepsin-D and total protein correlated with tumor size (r = 0.3, 0.28, 0.2; p = 0.01, p = 0.01, 0.04, respectively) and lymph node metastasis (r = 0.32, 0.34, 0.2; p = 0.003, 0.005, 0.04, respectively). Elevated pretreatment urinary NMP22, E-cadherin and total protein levels was associated significantly with bladder cancer recurrence (p = 0.02, 0.001, 0.005, respectively). In conclusion, determination of urinary NMP22, E-cadherin and total protein in bladder cancer patients or persons at risk of developing bladder cancer will help in early detection of the disease and prediction of recurrence. The use of a combination of tumor markers is markedly useful than the assessment of single one.
PMCID: PMC3689337  PMID: 24426222
NMP22; Cadherin; Cathepsin D; Total protein; Bladder carcinoma
9.  NMP22 is predictive of recurrence in high-risk superficial bladder cancer patients 
The nuclear matrix protein 22 (NMP22) assay has been shown to have greater sensitivity for the diagnosis and detection of recurrent urothelial carcinoma of the bladder (UCB) over that of traditional urine cytology. We assessed the use of NMP22 to predict which high-risk superficial UCB patients will have recurrence, progression or disease-related death; we compared these results to standard urine cytology.
One hundred consecutive patients with high-risk superficial UCB were enrolled. During surveillance, urine was collected for cytology and NMP22 testing. Patients were followed for at least 6 months. Retrospective chart review was undertaken to collect data on previous tumour history, tumour characteristics, disease recurrences, progression and death. Kaplan-Meier analyses were performed to determine the significance between NMP22-positive and -negative patients in terms of recurrence-free, progression-free and overall survival. Similar analyses were performed for urine cytology.
From 94 eligible patients, 15 and 79 were NMP22 positive and negative, respectively. The baseline characteristics between the 2 groups were not significantly different in terms of patient characteristics, prior tumour history or intravesical therapies received. Mean recurrence-free survival time was significantly lower in the NMP22 positive group (p = 0.038); however, mean progression-free and overall survival were not significantly different between the 2 groups (p = 0.297 and 0.519, respectively). Urine cytology demonstrated no significant predictive power for disease recurrence, progression or survival.
The nuclear matrix protein 22 assay appears to have predictive value for future tumour recurrences, but not progression or overall survival in patients with high-risk superficial UCB.
PMCID: PMC2792415  PMID: 20019971
10.  Experimental exposure of male volunteers to N-methyl-2-pyrrolidone (NMP): acute effects and pharmacokinetics of NMP in plasma and urine. 
OBJECTIVES: To study the acute effects of exposure to the increasingly used solvent, N-methyl-2-pyrrolidone (NMP) in male volunteers. Further, to determine the NMP concentration in plasma and urine during and after the exposure. METHODS: Six male volunteers were exposed for eight hours on four different days to 0, 10, 25, and 50 mg/m3 NMP. Plasma was collected and urine was sampled during and after the exposure. Changes in nasal volume were measured by acoustic rhinometry and in airway resistance by spirometry. RESULTS: The eight-hour experimental exposure to 10, 25, and 50 mg/m3 did not induce discomfort to eyes or upper airways. Acute changes in nasal volume were not found, and no changes in the spirometric data could be registered. The elimination curves suggested a non-linear pattern and at the end of exposure showed mean (range) half lifes of NMP in plasma of about 4.0 (2.9-5.8) hours and in urine 4.5 (3.5-6.6) hours. The unmetabolised NMP found in urine samples collected during exposure and at the subsequent 44 hours corresponded to about 2% of the calculated absorbed dose. At the end of the exposure there was a close correlation between exposures and the plasma concentration and urinary excretion of NMP. CONCLUSIONS: NMP was absorbed through the respiratory tract and readily eliminated from the body, mainly by biotransformation to other compounds. Exposure to 10, 25, or 50 mg/m3 NMP did not cause nose, eye, or airway irritation. Thus, NMP is a mild irritant.
PMCID: PMC1128696  PMID: 9166128
11.  Prospective evaluation of fluorescence-guided cystoscopy to detect bladder cancer in a high-risk population: results from the UroScreen-Study 
SpringerPlus  2014;3:24.
To prospectively evaluate the role of fluorescence-guided cystoscopy in a high-risk bladder cancer population undergoing screening based on a multi-marker panel of urine-tests (UroScreen-study).
Patients and methods
UroScreen was conducted as a validation study for tumor markers within the frame of a health surveillance program of workers with occupational exposure to aromatic amines. Voluntary annual screens were done in 1,609 men. Cytology, quantitative NMP22® assay, and UroVysion (FISH) were applied to 7091 urine samples. Subjects with at least one positive urine-based tumor marker and/or persisting microscopic hematuria were offered fluorescence-guided (PDD) instead of white light cystoscopy. In case of suspicious findings histopathological evaluation by transurethral biopsy was performed. Data were statistically summarized and compared to tumors found by the standard algorithm of the screening study.
Twenty-two subjects with a mean age of 58 years (39–72) underwent PDD cystoscopy. Of those 3 had positive NMP22 tests, 14 positive FISH tests and 9 suspicious cytologies. Two had persisting microscopic hematuria only. PDD cystoscopy revealed enhanced unifocal fluorescence in 14. All had subsequent transurethral biopsy or resection. In total, 1 urothelial carcinoma (pTaG1, low grade) was diagnosed. In the other participants urothelial cancer of the bladder was ruled out. Chronic cystitis was revealed in 8 of 14 biopsies. No higher detection rate was found using PDD than with the standard algorithm of the UroScreen study in which 17 tumors were detected by white light cystoscopy.
The use of PDD does not lead to a higher cancer detection rate in a high-risk screening population. Larger sample sizes may be needed to ultimately asses the value of PDD for bladder cancer screening.
PMCID: PMC3905106  PMID: 24478941
Urothelial cancer of the bladder; Urine based tumor marker; Bladder cancer screening; NMP22; UroVysion; UroScreen; Cytology; Photodynamic diagnostics; Cystoscopy
12.  Activatable Organic Near-Infrared Fluorescent Probes Based on a Bacteriochlorin Platform: Synthesis and Multicolor in Vivo Imaging with a Single Excitation 
Bioconjugate Chemistry  2014;25(2):362-369.
Near infrared (NIR) fluorescent probes are ideal for in vivo imaging because they offer deeper tissue penetration and lower background autofluorescence. Although most fluorophores in this range are cyanine-based dyes, several new classes of fluorescent NIR probes have been developed. In this study, we developed organic bacteriochlorin derivatives, NMP4 and NMP5, which are excited with a single green light and emit different narrow, well-resolved bands in the NIR (peak of 739 and 770 nm for NMP4 and NMP5, respectively). When conjugated to galactosyl-human serum albumin (hGSA) or glucosyl-human serum albumin (glu-HSA), both targeting H-type lectins, including the β-d-galactose receptor expressing on ovarian cancer, these agents become targeted, activatable, single excitation, multicolor NIR fluorescence probes. After conjugation to either glu-HSA or hGSA, substantial quenching of fluorescence occurs that is reversed after cell binding and internalization. In vitro studies showed higher cancer cell uptake with NMP4 or NMP5 conjugated to hGSA compared to the same conjugates with glu-HSA. In vivo single excitation two-color imaging was performed after intraperitoneal injection of these agents into mice with disseminated ovarian cancer. Excited with a single green light, distinct NIR emission spectra from each fluorophore were detected and could be distinguished with spectral unmixing. In vivo results using a red fluorescence protein (RFP) labeled tumor model of disseminated ovarian cancer demonstrated high sensitivity and specificity for all probes. The success of single excitation, 2-color NIR fluorescence imaging with a new class of bacteriochlorin-based activatable fluorophores, NMP4 and NMP5, paves the way for further exploration of noncyanine dye-based NIR fluorophores.
PMCID: PMC3983136  PMID: 24450401
13.  Medical follow-up for workers exposed to bladder carcinogens: the French evidence-based and pragmatic statement 
BMC Public Health  2014;14:1155.
The aim of this work was to establish recommendations for the medical follow-up of workers currently or previously exposed to carcinogenic substances for the bladder.
A critical synthesis of the literature was conducted. Sectors of activity where workers are or were exposed to carcinogenic substances for the bladder were listed and classified according to the level of bladder cancer risk. Performances of techniques available for the targeted screening of bladder cancer were analysed, including a simulation of results among high-risk populations in France.
The risk level for the professional group and the latency period between the start of exposure and the natural history of the disease were selected to define a targeted screening protocol. The NMP22BC test, exclusive haematuria testing, and combinations of urine cytology with, respectively, the NMP22BC test and haematuria test, generated an extremely high proportion of false positive results.
Urine cytology is the test that offers the best specificity. Although poor for all bladder cancer stages and grades combined, its sensitivity is better for high grades, which require early diagnosis since late-stage cancers are of very poor prognosis. These results suggest that urine cytology is currently the only technique suitable for proposal within the context of a first line targeted screening strategy for occupational bladder cancer. An algorithm summarising the recommended medical follow-up for workers currently or previously exposed to carcinogenic substances for the bladder is proposed, based on the level of risk of bladder cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2458-14-1155) contains supplementary material, which is available to authorized users.
PMCID: PMC4230399  PMID: 25377503
Bladder cancer; Occupational exposure; Medico-professional follow-up; Recommendations
14.  Stepwise Application of Urine Markers to Detect Tumor Recurrence in Patients Undergoing Surveillance for Non-Muscle-Invasive Bladder Cancer 
Disease Markers  2014;2014:973406.
Background. The optimal use of urine markers in the surveillance of non-muscle-invasive bladder cancer (NMIBC) remains unclear. Aim of the present study was to investigate the combined and stepwise use of the four most broadly available urine markers to detect tumor recurrence in patients undergoing surveillance of NMIBC. Patients and Methods. 483 patients with history of NMIBC were included. Cytology, UroVysion, fluorescence in situ hybridization (FISH), immunocytology (uCyt+), and NMP22 ELISA were performed before surveillance cystoscopy. Characteristics of single tests and combinations were assessed by contingency analysis. Results. 128 (26.5%) patients had evidence of tumor recurrence. Sensitivities and negative predictive values (NPVs) of the single tests ranged between 66.4–74.3 and 82.3–88.2%. Two-marker combinations showed sensitivities and NPVs of 80.5–89.8 and 89.5–91.2%. A stepwise application of the two-test combinations with highest accuracy (cytology and FISH; cytology and uCyt+; uCyt+ and FISH) showed NPVs for high-risk recurrences (G3/Cis/pT1) of 98.8, 98.8, and 99.1%, respectively. Conclusions. Combinations of cytology, FISH, immunocytology, and NMP22 show remarkable detection rates for recurrent NMIBC. Stepwise two-test combinations of cytology, FISH, and immunocytology have a low probability of missing a high-risk tumor. The high sensitivities may justify the use of these combinations in prospective studies assessing the use of urine markers to individualize intervals between cystoscopies during follow-up.
PMCID: PMC4284969  PMID: 25587206
15.  Investigation of CCL18 and A1AT as potential urinary biomarkers for bladder cancer detection 
BMC Urology  2013;13:42.
In this study, we further investigated the association of two biomarkers, CCL18 and A1AT, with bladder cancer (BCa) and evaluated the influence of potentially confounding factors in an experimental model.
In a cohort of 308 subjects (102 with BCa), urinary concentrations of CCL18 and A1AT were assessed by enzyme-linked immunosorbent assay (ELISA). In an experimental model, benign or cancerous cells, in addition to blood, were added to urines from healthy controls and analyzed by ELISA. Lastly, immunohistochemical staining for CCL18 and A1AT in human bladder tumors was performed.
Median urinary protein concentrations of CCL18 (52.84 pg/ml vs. 11.13 pg/ml, p < 0.0001) and A1AT (606.4 ng/ml vs. 120.0 ng/ml, p < 0.0001) were significantly elevated in BCa subjects compared to controls. Furthermore, the addition of whole blood to pooled normal urine resulted in a significant increase in both CCL18 and A1AT. IHC staining of bladder tumors revealed CCL18 immunoreactivity in inflammatory cells only, and there was no significant increase in these immunoreactive cells within benign and cancerous tissue and no association with BCa grade nor stage was noted. A1AT immunoreactivity was observed in the cytoplasm of epithelia cells and intensity of immunostaining increased with tumor grade, but not tumor stage.
Further development of A1AT as a diagnostic biomarker for BCa is warranted.
PMCID: PMC3846766  PMID: 24011266
Biomarkers; Bladder cancer; Specificity; Urine
Drug and alcohol dependence  2012;131(0):71-77.
Use of prescription stimulants used to treat Attention Deficit/Hyperactivity Disorder (ADHD) for reasons other than prescribed, known as non-medical use, is a growing problem among undergraduates. Previous studies show that non-medical prescription stimulant (NMPS) users consume more alcohol than individuals who do not use NMPS. However, research on simultaneous use of NMPS and alcohol is limited. The objectives of this study were to: (1) determine the prevalence of simultaneous use of alcohol and NMPS; (2) examine predictors and consequences of simultaneous NMPS and alcohol use among undergraduates.
In fall 2009, 4,090 students from eight North Carolina universities completed a web-based survey.
Past year prevalence of NMPS use among this sample was 10.6% and simultaneous use of NMPS with alcohol was 4.9%. Among NMPS users, 46.4% used NMPS simultaneously with alcohol within the past year. Multivariable analysis revealed that simultaneous NMPS and alcohol use was associated with low grade point averages, use of other substances, and increased alcohol-related consequences. Simultaneous NMPS and alcohol users reported experiencing significantly more negative consequences than either past year drinkers who did not use prescription stimulants and concurrent NMPS and alcohol users (use over the past year but not at the same time).
Simultaneous use of NMPS and alcohol is high among NMPS users in our sample of undergraduate students. Simultaneous users are at increased risk of experiencing negative consequences. Thus, prevention and intervention efforts should include a focus on simultaneous NMPS and alcohol use.
PMCID: PMC3644523  PMID: 23274057
polydrug; alcohol; prescription stimulants; nonmedical use; college students
17.  Ribonucleotide incorporation by yeast DNA polymerase ζ 
DNA repair  2014;18:63-67.
During replication in yeast, the three B family DNA replicases frequently incorporate ribonucleotides (rNMPs) into DNA, and their presence in the nuclear genome can affect genome stability. This prompted us to examine ribonucleotide incorporation by the fourth B family member, Pol ζ the enzyme responsible for the majority of damage-induced mutagenesis in eukaryotes. We first show that Pol ζ inserts rNMPs into DNA and can extend primer termini containing 3’-ribonucleotides. We then measure rNMP incorporation by Pol ζ in the presence of its cofactors, RPA, RFC and PCNA and at normal cellular dNTP and rNTP concentrations that exist under unstressed conditions. Under these conditions, Pol ζ stably incorporates one rNMP for every 200-300 dNMPs incorporated, a frequency that is slightly higher than for the high fidelity replicative DNA polymerases. Under damage-induced conditions wherein cellular dNTP concentrations are elevated 5-fold, Pol ζ only incorporates one rNMP per 1,300 dNMPs. Functional interaction of Pol ζ with the mutasome assembly factor Rev1 gives comparable rNMP incorporation frequencies. These results suggest that ribonucleotide incorporation into DNA during Pol ζ-mediated mutagenesis in vivo may be rare.
PMCID: PMC4402711  PMID: 24674899
Ribonucleotides; Mutagenesis; DNA polymerase; Translesion synthesis
18.  TNF-α Promotes Caspase Activation and Apoptosis in Human Fetal Membranes 
Purpose: Increased amniotic fluid tumor necrosis factor (TNF) is a marker of infection when associated with preterm labor and preterm premature rupture of the amniochorionic membranes (PROM). We have noted increased apoptosis in membranes derived from women with PROM. This study examines the role of TNF in promoting fetal membrane apoptosis. Methods: Amniochorion (n = 8), collected at the time of elective repeat cesarean section prior to labor from normal term gestation, were placed in an organ explant system. After 48 h in culture, the membranes were stimulated with recombinant TNF-α (20 ng/mL) for 24 h. Tissue frozen after stimulation was subjected to RT-PCR to study the expression of TNF-induced caspase genes. ELISA assayed the levels of proapoptotic p53 in tissues and cell death related nuclear matrix protein (NMP) in tissue culture supernatants. The activity of caspases in tissue homogenates was measured using substrates specific for caspase 2, 3, 6, 8, and 9. Results were analyzed by using the Wilcoxon nonparametric test for paired samples. A p < 0.05 was considered significant. Results: RT-PCR showed induction of caspases 2, 8, and 9 (caspase cascade initiators) in human fetal membranes after TNF stimulation. Caspases 3 and 6 (effector caspases) expression was constitutive in both TNF stimulated- and control membranes. Caspases, 2, 3, 8, and 9 activity was significantly higher in TNF-stimulated tissues compared with control, whereas, no significant change in caspase 6 activity was noticed. TNF-stimulated tissues released increased levels of NMP (24.03 U/mL) compared with control (13.5U/mL) (p = 0.03). TNF also increased p53 levels in the tissues (0.05 ng/mL) compared with control cultures (0.03 ng/mL; p = 0.02). Conclusions: TNF increases proapoptotic p53 levels and caspase activities in fetal membranes. Increased NMP reflects cell death.
PMCID: PMC3455651  PMID: 12036088
amniochorion; caspases; preterm labor; p53; PROM; TNF
19.  Nmp4/CIZ inhibits mechanically-induced β-catenin signaling activity in osteoblasts 
Journal of cellular physiology  2010;223(2):435-441.
Cellular mechanotransduction, the process of converting mechanical signals into biochemical responses within cells, is a critical aspect of bone health. While the effects of mechanical loading on bone are well recognized, elucidating the specific molecular pathways involved in the processing of mechanical signals by bone cells represents a challenge and an opportunity to identify therapeutic strategies to combat bone loss. In this study we have for the first time examined the relationship between the nucleocytoplasmic shuttling transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ) and β-catenin signaling in response to a physiologic mechanical stimulation (oscillatory fluid shear stress, OFSS) in osteoblasts. Using calvaria-derived osteoblasts from Nmp4-deficient and wild-type mice, we found that the normal translocation of β-catenin to the nucleus in osteoblasts that is induced by OFSS is enhanced when Nmp4/CIZ is absent. Furthermore, we found that other aspects of OFSS-induced mechanotransduction generally associated with the β-catenin signaling pathway, including ERK, Akt and GSK3β activity, as well as expression of the β-catenin-responsive protein cyclin D1 are also enhanced in cells lacking Nmp4/CIZ. Finally, we found that in the absence of Nmp4/CIZ, OFSS-induced cytoskeletal reorganization and the formation of focal adhesions between osteoblasts and the extracellular substrate is qualitatively enhanced, suggesting that Nmp4/CIZ may reduce the sensitivity of bone cells to mechanical stimuli. Together these results provide experimental support for the concept that Nmp4/CIZ plays an inhibitory role in the response of bone cells to mechanical stimulation induced by OFSS.
PMCID: PMC2872931  PMID: 20112285
Akt; bone; ERK; fluid shear mechanosome; mechanotransduction
20.  Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist 
Molecular Pain  2013;9:32.
Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181.
NMP-181 action on CB1 and CB2 receptors was characterized in radioligand binding and in vitro GTPγ[35S] functional assays, and block of transiently expressed human Cav3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak CaV3.2 currents with IC50 values in the low micromolar range and acted as a CB2 agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mechanical hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in CaV3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB2 antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB1 antagonist).
Our work shows that both T-type channels as well as CB2 receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands.
PMCID: PMC3703287  PMID: 23815854
T-type Channels; Cannabinoid Receptors; Pain; Patch-Clamp; Mice
21.  Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT) 
Journal of Cellular Physiology  2012;227(5):1873-1882.
Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, ALK3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism.
PMCID: PMC3209493  PMID: 21732358
adipogenesis; BMP2; differentiation; osteoporosis; PTH; proliferation
22.  Point-of-Care Tests for Bladder Cancer: The Influencing Role of Hematuria 
Advances in Urology  2011;2011:937561.
Introduction. Several point-of-care tests (POCT) are available for the diagnosis of bladder cancer (BC). We evaluate the impact of HU (hematuria) on performance of POCTs. Materials and Methods. Urine from 10 donors was diluted with blood from 0.5 to 0.00625%. BladderCheckR, BTAstatR, BCMR, and BTAR tests were applied. Tests were additionally conducted in 54 patients with HU. HU was stratified according to the amount of erythrocytes (RBC)/μL using two systems: (1) no HU; mild microscopic HU; severe microscopic HU; gross HU; (2) I <25 RBCs; <250 II; ≥250 III. Results were compared to HU status and histopathology. Results. Gross HU became evident between 2090 RBCs/μL and 1065/μL. Addition of blood led to default tests in all 4: BladderCheckR 0.25%; BCM 0.025%, BioNexia 0.00625%, and BTAstat <0.00625%. Rates of false positives for BladderCheck, BTAstat, BCM, and BioNexia were 5.9, 11.8, 0, and 1.8% without HU and 0, 66.7, 44.4, and 66.7% with HU. BTAstat, BCM, and BioNexia were independently influenced by HU (P < 0.0002). Conclusions. NMP22-BladderCheck was most resistant to blood. The diagnostic yield of all others was significantly influenced by HU. A well-defined HU grading helps to define limits of HU for a reliable interpretation of BC-POCTs.
PMCID: PMC3227231  PMID: 22162681
23.  Urinary levels of Hepatocarcinoma-intestine-pancreas/Pancreatitis-associated protein as a diagnostic biomarker in patients with bladder cancer 
BMC Urology  2012;12:24.
To assess the possibility of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) as a biological marker for detecting Bladder cancer (BCa), we examined the expression of HIP/PAP in both BCa specimens and BCa cell lines and measured HIP/PAP levels in urine from patients with BCa.
HIP/PAP expression in BCa samples was evaluated by western blot analysis, and urinary levels of HIP/PAP in patients with BCa were measured by enzyme-linked immunosorbent assay. Urine samples were collected from 10 healthy volunteers and 109 with benign urological disorders as controls, and from 101 patients who were diagnosed with BCa.
HIP/PAP was highly expressed in BCa samples as compared with control bladder. Urinary HIP/PAP concentrations were significantly higher in BCa patients than in controls (median value; 3.184 pg/mL vs. 55.200 pg/mL, P <0.0001, by Mann–Whitney U test). Urinary HIP/PAP levels in BCa patients correlated positively with pathological T stages and progression-risk groups among non-muscle invasive BCa (P = 0.0008, by Kruskal-Wallis test). Regarding the recurrence-risk classifications of non-muscle invasive BCa, the urinary levels of HIP/PAP were significantly higher in the intermediate than in the low risk group (P = 0.0002, by Mann–Whitney U test). Based on a cut-off of 8.5 pg/mL, the ability of urinary HIP/PAP levels to detect BCa had a sensitivity of 80.2%, specificity of 78.2%, positive predictive value (PPV) of 75.7%, and negative predictive value (NPV) of 82.3%.
HIP/PAP was abundantly expressed in BCa, and the urinary levels of HIP/PAP could be a novel and potent biomarker for detection of BCa, and also for predicting the risks of recurrence- and progression-risk of non-muscle invasive BCa. A large scale study will be needed to establish the usefulness of this biomarker.
PMCID: PMC3487857  PMID: 22943287
Bladder cancer; Urinary marker; HIP/PAP; ELISA; ROC
24.  Identification of Second Messenger Mediating Signal Transduction in the Olfactory Receptor Cell 
The Journal of General Physiology  2003;122(5):557-567.
One of the biggest controversial issues in the research of olfaction has been the mechanism underlying response generation to odorants that have been shown to fail to produce cAMP when tested by biochemical assays with olfactory ciliary preparations. Such observations are actually the original source proposing a possibility for the presence of multiple and parallel transduction pathways. In this study the activity of transduction channels in the olfactory cilia was recorded in cells that retained their abilities of responding to odorants that have been reported to produce InsP3 (instead of producing cAMP, and therefore tentatively termed “InsP3 odorants”). At the same time, the cytoplasmic cNMP concentration ([cNMP]i) was manipulated through the photolysis of caged compounds to examine their real-time interactions with odorant responses. Properties of responses induced by both InsP3 odorants and cytoplasmic cNMP resembled each other in their unique characteristics. Reversal potentials of currents were 2 mV for InsP3 odorant responses and 3 mV for responses induced by cNMP. Current and voltage (I-V) relations showed slight outward rectification. Both responses showed voltage-dependent adaptation when examined with double pulse protocols. When brief pulses of the InsP3 odorant and cytoplasmic cNMP were applied alternatively, responses expressed cross-adaptation with each other. Furthermore, both responses were additive in a manner as predicted quantitatively by the theory that signal transduction is mediated by the increase in cytoplasmic cAMP. With InsP3 odorants, actually, remarkable responses could be detected in a small fraction of cells (∼2%), explaining the observation for a small production of cAMP in ciliary preparations obtained from the entire epithelium. The data will provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.
PMCID: PMC2229575  PMID: 14581582
olfactory receptor cell; signal transduction; second messenger; cAMP; caged compound
25.  Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands 
Molecular Pain  2011;7:89.
Both T-type calcium channels and cannabinoid receptors modulate signalling in the primary afferent pain pathway. Here, we investigate the analgesics activities of a series of novel cannabinoid receptor ligands with T-type calcium channel blocking activity.
Novel compounds were characterized in radioligand binding assays and in vitro functional assays at human and rat CB1 and CB2 receptors. The inhibitory effects of these compounds on transient expressed human T-type calcium channels were examined in tsA-201 cells using standard whole-cell voltage clamp techniques, and their analgesic effects in response to various administration routes (intrathecally, intraplantarly, intraperitoneally) assessed in the formalin model. A series of compounds were synthesized and evaluated for channel and receptor activity. Compound NMP-7 acted as non-selective CB1/CB2 agonist while NMP4 was found to be a CB1 partial agonist and CB2 inverse agonist. Furthermore, NMP-144 behaved as a selective CB2 inverse agonist. All of these three compounds completely inhibited peak Cav3.2 currents with IC50 values in the low micromolar range. All compounds mediated analgesic effects in the formalin model, but depending on the route of administration, could differentially affect phase 1 and phase 2 of the formalin response.
Our results reveal that a set of novel cannabinioid receptor ligands potently inhibit T-type calcium channels and show analgesic effects in vivo. Our findings suggest possible novel means of mediating pain relief through mixed T-type/cannabinoid receptor ligands.
PMCID: PMC3250956  PMID: 22093952

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