Publications by authors named "Fernando de Miguel"

35 Publications

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Cell 2021 01 20;184(1):76-91.e13. Epub 2020 Oct 20.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT 06520, USA; Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA; Yale Cancer Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
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http://dx.doi.org/10.1016/j.cell.2020.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574718PMC
January 2021

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

bioRxiv 2020 Jun 17. Epub 2020 Jun 17.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.
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http://dx.doi.org/10.1101/2020.06.16.155101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457610PMC
June 2020

Consequences of Exon 4 Mutations in Myoblast Function.

Cells 2020 05 21;9(5). Epub 2020 May 21.

Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo km2.2, E-28029 Madrid, Spain.

Laminopathies are causally associated with mutations on the Lamin A/C gene (). To date, more than 400 mutations in have been reported in patients. These mutations are widely distributed throughout the entire gene and are associated with a wide range of phenotypes. Unfortunately, little is known about the mechanisms underlying the effect of the majority of these mutations. This is the case of more than 40 mutations that are located at exon 4. Using CRISPR/Cas9 technology, we generated a collection of exon 4 mutants in mouse C2C12 myoblasts. These cell models included different types of exon 4 deletions and the presence of R249W mutation, one of the human variants associated with a severe type of laminopathy, -associated congenital muscular dystrophy (L-CMD). We characterized these clones by measuring their nuclear circularity, myogenic differentiation capacity in 2D and 3D conditions, DNA damage, and levels of p-ERK and p-AKT (phosphorylated Mitogen-Activated Protein Kinase 1/3 and AKT serine/threonine kinase 1). Our results indicated that exon 4 mutants showed abnormal nuclear morphology. In addition, levels and/or subcellular localization of different members of the lamin and LINC (LInker of Nucleoskeleton and Cytoskeleton) complex were altered in all these mutants. Whereas no significant differences were observed for ERK and AKT activities, the accumulation of DNA damage was associated to the p.R249W mutant myoblasts. Finally, significant myogenic differentiation defects were detected in the exon 4 mutants. These results have key implications in the development of future therapeutic strategies for the treatment of laminopathies.
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http://dx.doi.org/10.3390/cells9051286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291140PMC
May 2020

An Elaborate STING Operation to Take Down NSCLC: Combination of Immunotherapies and Chemotherapies.

J Thorac Oncol 2020 05;15(5):686-688

Yale Cancer Center, Yale School of Medicine, Yale University, New Haven, Connecticut. Electronic address:

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http://dx.doi.org/10.1016/j.jtho.2020.03.018DOI Listing
May 2020

Antiangiogenic Vascular Endothelial Growth Factor-Blocking Peptides Displayed on the Capsid of an Infectious Oncolytic Parvovirus: Assembly and Immune Interactions.

J Virol 2019 10 12;93(19). Epub 2019 Sep 12.

Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain

As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (α-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of α-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an α-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions. Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce α-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.
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http://dx.doi.org/10.1128/JVI.00798-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744233PMC
October 2019

Tumor regression mediated by oncogene withdrawal or erlotinib stimulates infiltration of inflammatory immune cells in EGFR mutant lung tumors.

J Immunother Cancer 2019 07 10;7(1):172. Epub 2019 Jul 10.

Department of Pathology, Yale School of Medicine, 333 Cedar Street, SHM-I 234D, New Haven, CT, 06510, USA.

Background: Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) like erlotinib are effective for treating patients with EGFR mutant lung cancer; however, drug resistance inevitably emerges. Approaches to combine immunotherapies and targeted therapies to overcome or delay drug resistance have been hindered by limited knowledge of the effect of erlotinib on tumor-infiltrating immune cells.

Methods: Using mouse models, we studied the immunological profile of mutant EGFR-driven lung tumors before and after erlotinib treatment.

Results: We found that erlotinib triggered the recruitment of inflammatory T cells into the lungs and increased maturation of alveolar macrophages. Interestingly, this phenotype could be recapitulated by tumor regression mediated by deprivation of the EGFR oncogene indicating that tumor regression alone was sufficient for these immunostimulatory effects. We also found that further efforts to boost the function and abundance of inflammatory cells, by combining erlotinib treatment with anti-PD-1 and/or a CD40 agonist, did not improve survival in an EGFR-driven mouse model.

Conclusions: Our findings lay the foundation for understanding the effects of TKIs on the tumor microenvironment and highlight the importance of investigating targeted and immuno-therapy combination strategies to treat EGFR mutant lung cancer.
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http://dx.doi.org/10.1186/s40425-019-0643-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617639PMC
July 2019

Comparison of RNA-seq and microarray platforms for splice event detection using a cross-platform algorithm.

BMC Genomics 2018 Sep 25;19(1):703. Epub 2018 Sep 25.

CEIT and Tecnun, University of Navarra, Parque Tecnológico de San Sebastián, Paseo Mikeletegi 48, 20009, San Sebastián, Gipuzkoa, Spain.

Background: RNA-seq is a reference technology for determining alternative splicing at genome-wide level. Exon arrays remain widely used for the analysis of gene expression, but show poor validation rate with regard to splicing events. Commercial arrays that include probes within exon junctions have been developed in order to overcome this problem. We compare the performance of RNA-seq (Illumina HiSeq) and junction arrays (Affymetrix Human Transcriptome array) for the analysis of transcript splicing events. Three different breast cancer cell lines were treated with CX-4945, a drug that severely affects splicing. To enable a direct comparison of the two platforms, we adapted EventPointer, an algorithm that detects and labels alternative splicing events using junction arrays, to work also on RNA-seq data. Common results and discrepancies between the technologies were validated and/or resolved by over 200 PCR experiments.

Results: As might be expected, RNA-seq appears superior in cases where the technologies disagree and is able to discover novel splicing events beyond the limitations of physical probe-sets. We observe a high degree of coherence between the two technologies, however, with correlation of EventPointer results over 0.90. Through decimation, the detection power of the junction arrays is equivalent to RNA-seq with up to 60 million reads.

Conclusions: Our results suggest, therefore, that exon-junction arrays are a viable alternative to RNA-seq for detection of alternative splicing events when focusing on well-described transcriptional regions.
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http://dx.doi.org/10.1186/s12864-018-5082-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156849PMC
September 2018

A novel protein-based prognostic signature improves risk stratification to guide clinical management in early-stage lung adenocarcinoma patients.

J Pathol 2018 08 20;245(4):421-432. Epub 2018 Jun 20.

Program in Solid Tumours, CIMA, Pamplona, Spain.

Each of the pathological stages (I-IIIa) of surgically resected non-small-cell lung cancer has hidden biological heterogeneity, manifested as heterogeneous outcomes within each stage. Thus, the finding of robust and precise molecular classifiers with which to assess individual patient risk is an unmet medical need. Here, we identified and validated the clinical utility of a new prognostic signature based on three proteins (BRCA1, QKI, and SLC2A1) to stratify early-stage lung adenocarcinoma patients according to their risk of recurrence or death. Patients were staged according to the new International Association for the Study of Lung Cancer (IASLC) staging criteria (8th edition, 2018). A test cohort (n = 239) was used to assess the value of this new prognostic index (PI) based on the three proteins. The prognostic signature was developed by Cox regression with the use of stringent statistical criteria (TRIPOD: Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis). The model resulted in a highly significant predictor of 5-year outcome for disease-free survival (p < 0.001) and overall survival (p < 0.001). The prognostic ability of the model was externally validated in an independent multi-institutional cohort of patients (n = 114, p = 0.021). We also demonstrated that this molecular classifier adds relevant information to the gold standard TNM-based pathological staging, with a highly significant improvement of the likelihood ratio. We subsequently developed a combined PI including both the molecular and the pathological data that improved the risk stratification in both cohorts (p ≤ 0.001). Moreover, the signature may help to select stage I-IIA patients who might benefit from adjuvant chemotherapy. In summary, this protein-based signature accurately identifies those patients with a high risk of recurrence and death, and adds further prognostic information to the TNM-based clinical staging, even when the new IASLC 8th edition staging criteria are applied. More importantly, it may be a valuable tool for selecting patients for adjuvant therapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563803PMC
August 2018

Endoscopic submucosal injection of adipose-derived mesenchymal stem cells ameliorates TNBS-induced colitis in rats and prevents stenosis.

Stem Cell Res Ther 2018 04 10;9(1):95. Epub 2018 Apr 10.

Cell Therapy Laboratory, La Paz Hospital Institute for Health Research, Madrid, Spain.

Background: Mesenchymal stem cells have potential applications in inflammatory bowel disease due to their immunomodulatory properties. Our aim was to evaluate the feasibility, safety and efficacy of endoscopic administration of adipose-derived mesenchymal stem cells (ASCs) in a colitis model in rats.

Methods: Colitis was induced in rats by rectal trinitrobenzenesulfonic acid (TNBS). After 24 h ASCs (10 cells) or saline vehicle were endoscopically injected into the distal colon. Rats were followed for 11 days. Daily weight, endoscopic score at days 1 and 11, macroscopic appearance at necropsy, colon length and mRNA expression of Foxp3 and IL-10 in mesenteric lymph nodes (MLN) were analyzed.

Results: Endoscopic injection was successful in all the animals. No significant adverse events or mortality due to the procedure occurred. Weight evolution was significantly better in the ASC group, recovering initial weight by day 11 (- 0.8% ± 10.1%, mean ± SD), whereas the vehicle group remained in weight loss (- 6.7% ± 9.2%, p = 0.024). The endoscopic score improved in the ASC group by 47.1% ± 5.3% vs. 21.8% ± 6.6% in the vehicle group (p < 0.01). Stenosis was less frequent in the ASC group (4.8% vs. 41.2%, p < 0.01). Colon length significantly recovered in the ASC group versus the vehicle group (222.6 ± 17.3 mm vs. 193.6 ± 17.9 mm, p < 0.001). The endoscopic score significantly correlated with weight change, macroscopic necropsy score and colon length. Foxp3 and IL-10 mRNA levels in MLN recovered with ASC treatment.

Conclusions: ASC submucosal endoscopic injection is feasible, safe and ameliorates TNBS-induced colitis in rats, especially stenosis.
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http://dx.doi.org/10.1186/s13287-018-0837-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892014PMC
April 2018

Blockade of the Complement C5a/C5aR1 Axis Impairs Lung Cancer Bone Metastasis by CXCL16-mediated Effects.

Am J Respir Crit Care Med 2018 05;197(9):1164-1176

1 Center for Applied Medical Research, Program in Solid Tumors and Biomarkers, Pamplona, Spain.

Rationale: C5aR1 (CD88), a receptor for complement anaphylatoxin C5a, is a potent immune mediator. Its impact on malignant growth and dissemination of non-small cell lung cancer cells is poorly understood.

Objectives: To investigate the contribution of the C5a/C5aR1 axis to the malignant phenotype of non-small cell lung cancer cells, particularly in skeletal colonization, a preferential lung metastasis site.

Methods: Association between C5aR1 expression and clinical outcome was assessed in silico and validated by immunohistochemistry. Functional significance was evaluated by lentiviral gene silencing and ligand l-aptamer inhibition in in vivo models of lung cancer bone metastasis. In vitro functional assays for signaling, migration, invasion, metalloprotease activity, and osteoclastogenesis were also performed.

Measurements And Main Results: High levels of C5aR1 in human lung tumors were significantly associated with shorter recurrence-free survival, overall survival, and bone metastasis. Silencing of C5aR1 in lung cancer cells led to a substantial reduction in skeletal metastatic burden and osteolysis in in vivo models. Furthermore, metalloproteolytic, migratory, and invasive tumor cell activities were modulated in vitro by C5aR1 stimulation or gene silencing. l-Aptamer blockade or C5aR1 silencing significantly reduced the osseous metastatic activity of lung cancer cells in vivo. This effect was associated with decreased osteoclastogenic activity in vitro and was rescued by the exogenous addition of the chemokine CXCL16.

Conclusions: Disruption of C5aR1 signaling in lung cancer cells abrogates their tumor-associated osteoclastogenic activity, impairing osseous colonization. This study unveils the role played by the C5a/C5aR1 axis in lung cancer dissemination and supports its potential use as a novel therapeutic target.
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http://dx.doi.org/10.1164/rccm.201703-0660OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835094PMC
May 2018

A large-scale analysis of alternative splicing reveals a key role of QKI in lung cancer.

Mol Oncol 2016 11 9;10(9):1437-1449. Epub 2016 Aug 9.

Program in Solid Tumors and Biomarkers, CIMA, 31008 Pamplona, Spain; Department of Biochemistry and Genetics, School of Science, University of Navarra, 31008 Pamplona, Spain; Navarra's Health Research Institute (IDISNA), 31008 Pamplona, Spain. Electronic address:

Increasing interest has been devoted in recent years to the understanding of alternative splicing in cancer. In this study, we performed a genome-wide analysis to identify cancer-associated splice variants in non-small cell lung cancer. We discovered and validated novel differences in the splicing of genes known to be relevant to lung cancer biology, such as NFIB, ENAH or SPAG9. Gene enrichment analyses revealed an important contribution of alternative splicing to cancer-related molecular functions, especially those involved in cytoskeletal dynamics. Interestingly, a substantial fraction of the altered genes found in our analysis were targets of the protein quaking (QKI), pointing to this factor as one of the most relevant regulators of alternative splicing in non-small cell lung cancer. We also found that ESYT2, one of the QKI targets, is involved in cytoskeletal organization. ESYT2-short variant inhibition in lung cancer cells resulted in a cortical distribution of actin whereas inhibition of the long variant caused an increase of endocytosis, suggesting that the cancer-associated splicing pattern of ESYT2 has a profound impact in the biology of cancer cells. Finally, we show that low nuclear QKI expression in non-small cell lung cancer is an independent prognostic factor for disease-free survival (HR = 2.47; 95% CI = 1.11-5.46, P = 0.026). In conclusion, we identified several splicing variants with functional relevance in lung cancer largely regulated by the splicing factor QKI, a tumor suppressor associated with prognosis in lung cancer.
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http://dx.doi.org/10.1016/j.molonc.2016.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423218PMC
November 2016

EventPointer: an effective identification of alternative splicing events using junction arrays.

BMC Genomics 2016 06 17;17:467. Epub 2016 Jun 17.

CEIT, Parque Tecnológico de San Sebastián, Paseo Mikeletegi 48, 20009, San Sebastián, Gipuzkoa, Spain.

Background: Alternative splicing (AS) is a major source of variability in the transcriptome of eukaryotes. There is an increasing interest in its role in different pathologies. Before sequencing technology appeared, AS was measured with specific arrays. However, these arrays did not perform well in the detection of AS events and provided very large false discovery rates (FDR). Recently the Human Transcriptome Array 2.0 (HTA 2.0) has been deployed. It includes junction probes. However, the interpretation software provided by its vendor (TAC 3.0) does not fully exploit its potential (does not study jointly the exons and junctions involved in a splicing event) and can only be applied to case-control studies. New statistical algorithms and software must be developed in order to exploit the HTA 2.0 array for event detection.

Results: We have developed EventPointer, an R package (built under the aroma.affymetrix framework) to search and analyze Alternative Splicing events using HTA 2.0 arrays. This software uses a linear model that broadens its application from plain case-control studies to complex experimental designs. Given the CEL files and the design and contrast matrices, the software retrieves a list of all the detected events indicating: 1) the type of event (exon cassette, alternative 3', etc.), 2) its fold change and its statistical significance, and 3) the potential protein domains affected by the AS events and the statistical significance of the possible enrichment. Our tests have shown that EventPointer has an extremely low FDR value (only 1 false positive within the tested top-200 events). This software is publicly available and it has been uploaded to GitHub.

Conclusions: This software empowers the HTA 2.0 arrays for AS event detection as an alternative to RNA-seq: simplifying considerably the required analysis, speeding it up and reducing the required computational power.
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http://dx.doi.org/10.1186/s12864-016-2816-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912780PMC
June 2016

Identification of alternative splicing events regulated by the oncogenic factor SRSF1 in lung cancer.

Cancer Res 2014 Feb 26;74(4):1105-15. Epub 2013 Dec 26.

Authors' Affiliations: Division of Oncology, Center for Applied Medical Research (CIMA); Departments of Histology and Pathology and Biochemistry and Genetics, Schools of Science and Medicine, University of Navarra, Pamplona; and CEIT and TECNUN, University of Navarra, San Sebastian, Spain.

Abnormal alternative splicing has been associated with cancer. Genome-wide microarrays can be used to detect differential splicing events. In this study, we have developed ExonPointer, an algorithm that uses data from exon and junction probes to identify annotated cassette exons. We used the algorithm to profile differential splicing events in lung adenocarcinoma A549 cells after downregulation of the oncogenic serine/arginine-rich splicing factor 1 (SRSF1). Data were generated using two different microarray platforms. The PCR-based validation rate of the top 20 ranked genes was 60% and 100%. Functional enrichment analyses found a substantial number of splicing events in genes related to RNA metabolism. These analyses also identified genes associated with cancer and developmental and hereditary disorders, as well as biologic processes such as cell division, apoptosis, and proliferation. Most of the top 20 ranked genes were validated in other adenocarcinoma and squamous cell lung cancer cells, with validation rates of 80% to 95% and 70% to 75%, respectively. Moreover, the analysis allowed us to identify four genes, ATP11C, IQCB1, TUBD1, and proline-rich coiled-coil 2C (PRRC2C), with a significantly different pattern of alternative splicing in primary non-small cell lung tumors compared with normal lung tissue. In the case of PRRC2C, SRSF1 downregulation led to the skipping of an exon overexpressed in primary lung tumors. Specific siRNA downregulation of the exon-containing variant significantly reduced cell growth. In conclusion, using a novel analytical tool, we have identified new splicing events regulated by the oncogenic splicing factor SRSF1 in lung cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-1481DOI Listing
February 2014

In vitro and in vivo effect of lidocaine on rat muscle-derived cells for treatment of stress urinary incontinence.

Urology 2009 Feb 13;73(2):437-41. Epub 2008 Aug 13.

Department of Urology, Eulji University, Daejeon, Republic of Korea.

Objectives: Lidocaine cytotoxicity has been reported in some cell types, which could affect its use as a local anesthetic in cell-based therapy. We evaluated the in vitro and in vivo effect of lidocaine on rat muscle-derived progenitor cells (MDCs).

Methods: MDCs were isolated from rat skeletal muscle and purified using the preplate technique. For in vitro tests, the MDCs underwent either 2 hours of, or continuous, exposure to lidocaine (50 microM-5 mM). After 72 hours of incubation, cell viability was measured using the methylthiazololetetrazolium assay. For the in vivo tests, periurethral injection of either phosphate-buffered saline, MDCs (1 x 10(6) cells/20 microL), or 2% lidocaine plus MDCs was performed in pudendal nerve-transected rats. The leak point pressure (LPP) was measured at 4 weeks after the injection.

Results: Lidocaine concentrations of < or = 500 microM had no effect on MDCs with continuous exposure. MDCs in 1 mM lidocaine showed decreased survival and no MDCs in 5 mM lidocaine survived. With a 2-hour exposure, only MDCs in the 5-mM lidocaine group showed decreased survival. Rats with nerve transection and phosphate-buffered saline injection showed significantly lower LPPs than the controls. The LPP was restored to a significantly greater level after MDCs only or lidocaine plus MDC injection. No statistically significant difference in LPP restoration was found between the MDC-only and lidocaine plus MDC injections.

Conclusions: Cytotoxicity to lidocaine was minimal at a physiologic concentration in vitro. The functional recovery of LPP by MDC treatment was not affected by lidocaine preinfiltration. Taken together, our data indicate that lidocaine can be applied as a local anesthetic in periurethral MDC injection without decreasing the efficacy of the therapy.
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http://dx.doi.org/10.1016/j.urology.2008.06.019DOI Listing
February 2009

Functional and immunohistochemical characterization of CB1 and CB2 receptors in rat bladder.

Urology 2008 Nov 12;72(5):1174-8. Epub 2008 May 12.

Department of Urology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA.

Objectives: To determined the localization of CB(1) and CB(2) receptors in rat bladder and investigate the effect of a mixed CB(1)/CB(2) receptor agonist, ajulemic acid (AJA), on chemically evoked release of the sensory neuropeptide calcitonin gene-related peptide (CGRP).

Methods: Whole rat bladders were incubated in a series of tissue baths containing physiologic salt solution to measure baseline CGRP release by enzyme immunoassay. Capsaicin (30 nM) and adenosine triphosphate (10 muM) were used to provoke CGRP release in the presence or absence of AJA. Specificity of AJA for CB(1) and CB(2) receptors was determined using antagonists. Localization was determined by immunofluorescence for CB(1) and CB(2) receptors in fixed bladders.

Results: Immunofluorescence showed the localization of CB(1) and CB(2) receptors in the bladder. Mean baseline CGRP release was 605 +/- 62 pg/g of bladder weight, and AJA had no effect on CGRP release. The addition of adenosine triphosphate/capsaicin significantly increased the CGRP release over baseline, by 44% (P < .05), and AJA application significantly decreased CGRP release, by 29% compared with controls (P < .05). The CB(1) and CB(2) antagonists AM 251 and AM 630, respectively, reversed the blunting effect of AJA on evoked CGRP release, resulting in an increase of 40% and 38% over baseline, respectively.

Conclusions: CB(1) and CB(2) receptors are localized in the urothelium of rat bladder, and application of AJA inhibits the evoked release of CGRP by acting on CB(1) and CB(2) receptors. These findings identify a potential new pathway for study in the evaluation and treatment of painful bladder syndrome/interstitial cystitis.
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http://dx.doi.org/10.1016/j.urology.2008.03.044DOI Listing
November 2008

Proteomic investigation on chronic bladder irritation in the rat.

Urology 2008 Mar;71(3):536-40

Department of Urology, University of Pittsburgh School of Medicine; Pittsburgh, Pennsylvania 15213, USA.

Objectives: Interstitial cystitis (IC) is a painful bladder syndrome associated with urinary frequency and urgency. Elusive cause of IC makes its diagnosis only possible by exclusion in many cases. In this study, we used proteomics for identifying disease-associated proteins in a rat model of chronic bladder irritation.

Methods: Chronic irritation of the rat bladder was caused by a brief (90 seconds) intravesical instillation of 0.2 mL of 0.4 N HCl. Whole bladders were collected at different time points after treatment, snap frozen, and nuclear and cytosolic protein extracts were obtained. Samples were resolved in standard 2-dimensional (2D) gels stained with an improved Coomasie stain or by differential gel electrophoresis (DIGE). Differentially expressed spots were excised and identified by MALDI-TOF MS/MS. Histologic and Western blot analyses were also performed.

Results: Bladder morphology and histologic appearance of bladder sections after HCl treatment reflected hemorrhage, edema, epithelial denudation, detrusor mastocytosis, and eosinophilia. Proteomic analysis of irritated rat bladder revealed marked overexpression of 4 nuclear proteins and marked underexpression of 1 nuclear protein compared with normal rat bladders. Among these proteins, inflammation-associated calgranulin A (over) and smooth muscle protein-22/transgelin (under) showed opposed expression patterns after bladder irritation.

Conclusions: Presence of mast cells and eosinophils and overexpression of calgranulin A confirm the inflammatory component of HCl-irritated bladder. Altered expression of nuclear proteins is of particular interest because of their possible role as a prognostic marker in inflammatory bladder disorders. However, more studies are needed before clinical application of these findings can be established.
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http://dx.doi.org/10.1016/j.urology.2007.10.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750729PMC
March 2008

Effects of IP-751, ajulemic acid, on bladder overactivity induced by bladder irritation in rats.

Urology 2007 Jul;70(1):202-8

Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.

Objectives: Ajulemic acid (IP-751) is a synthetic analog of tetrahydrocannabinol, which is a major ingredient of the plant Cannabis. IP-751 reportedly shows potent anti-inflammatory activity and is a powerful analgesic agent. Thus, we examined whether IP-751 can suppress urinary frequency induced by nociceptive stimuli in the bladder.

Methods: Continuous cystometry (infusion rate 0.04 mL/min) under urethane anesthesia was performed to evaluate the effect of intravenous injection of IP-751 with or without a cannabinoid-1 receptor antagonist (AM251) or a cannabinoid-2 receptor antagonist (AM630) on bladder function in normal rats and rats with urinary frequency induced by intravesical infusion with 0.25% acetic acid or cyclophosphamide (CYP) (150 mg/kg intraperitoneally, 48 hours before cystometrography).

Results: When 10 mg/kg of IP-751 was injected in normal rats, the intercontraction interval (ICI) and pressure threshold increased. A 0.25% acetic acid infusion induced urinary frequency, as evidenced by a reduction in ICIs, which were suppressed by injection of IP-751 (10 mg/kg). Urinary frequency, indicated by significant ICI reductions, was also observed in the CYP-treated rats. Administration of IP-751 (10 mg/kg) significantly suppressed CYP-induced urinary frequency, as evidenced by the increase in the ICI. When AM251, but not AM630, was administered before IP-751, the IP-751-induced increases in the ICI and pressure threshold were prevented in all three groups. In addition, administration of AM251 alone decreased the ICIs in CYP-treated rats.

Conclusions: IP-751 can suppress normal bladder activity and urinary frequency induced by bladder nociceptive stimuli, probably by suppression of bladder afferent activity. These inhibitory effects of IP-751 are at least in part mediated by the cannabinoid-1 receptor.
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http://dx.doi.org/10.1016/j.urology.2007.02.069DOI Listing
July 2007

Human muscle-derived cell injection in a rat model of stress urinary incontinence.

Muscle Nerve 2007 Sep;36(3):391-3

Department of Urology, University of Pittsburgh, Suite 700, 3471 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.

We investigated the use of human muscle-derived cells (hMDCs) for the treatment of stress urinary incontinence (SUI) in a nude rat model. hMDCs were isolated from adult skeletal muscle. Three groups of six animals consisting of controls, animals undergoing sciatic nerve transection (SNT) with periurethral sham-injection, and SNT with hMDCs (1 x 10(6) cells/20 microl saline) were utilized. Leak point pressure (LPP) was measured 4 weeks following injection. Bilateral SNT resulted in a significantly lower LPP that was significantly higher following hMDCs than sham injection. The results demonstrate the efficacy of human muscle cell therapy alone in improving physiologic outcomes in an animal model of SUI.
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http://dx.doi.org/10.1002/mus.20827DOI Listing
September 2007

Treatment of overactive bladder: selective use of anticholinergic agents with low drug-drug interaction potential.

Geriatrics 2007 May;62(5):15-24

Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Overactive bladder (OAB) is highly prevalent in the older population and decreases quality of life. Current therapy consists primarily of anticholinergic drugs. Because older individuals typically take multiple medications, clinicians must pay special attention to potential drug-drug interactions that may cause adverse events or alter drug efficacy. The most clinically important drug-drug interactions occur during cytochrome P450 (CYP450) isoenzyme metabolism, resulting in altered metabolism of one or more of the coadministered agents. Of the drugs indicated for OAB, tolterodine, darifenacin, solifenacin, and oxybutynin are extensively metabolized by CYP450, but trospium is not. Trospium is eliminated as unchanged drug, suggesting that it has lower potential for drug-drug interactions and may, therefore, represent a safer treatment option for OAB, particularly in the context of polypharmacy, a significant concern in older adults.
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May 2007

Local drug delivery to bladder using technology innovations.

Urol Clin North Am 2006 Nov;33(4):519-30, x

Department of Urology, University of Pittsburgh School of Medicine, Kaufmann Medical Building, Suite 700, 3471 Fifth Avenue, Pittsburgh, PA 15213-3221, USA.

Local delivery of drugs directly into the bladder by way of a urethral catheter is a clever approach to optimize drug delivery to the disease site while reducing systemic bioavailability. Pharmacotherapy by this route is referred to as intravesical delivery. In recent years, intravesical delivery has been used in combination with and oral regimen of drugs or as second-line treatment for neurogenic bladder and detrusor overactivity. Negligible absorption of instilled drugs into the systemic circulation explains the near-minimal adverse toxicity reported with this form of therapy. The authors discuss shortcomings of the current options available for intravesical delivery and provide a broad overview of the latest advances through technology innovation to overcome these drawbacks.
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http://dx.doi.org/10.1016/j.ucl.2006.06.012DOI Listing
November 2006

Local effects of antimuscarinics.

Urol Clin North Am 2006 Nov;33(4):511-8, ix-x

Department of Urology and Reproductive Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.

Overactive bladder (OAB) syndrome has been estimated to occur in nearly 17% of the population. The most common drug treatments for OAB are antimuscarinic agents that act to increase bladder capacity and decrease the urge to urinate during the storage phase. An increasing number of studies have focused on te role and mechanism of muscarinic acetylcholine receptors for the regulation of afferent activity during urine storage. Interactions between muscarinic receptors in the urothelium, afferent nerves, or myofibroblasts and locally released acetylcholine might be involved in the emergence of detrusor overactivity and OAB. Therefore, antimuscarinic agents may be effective in treating OAB not only by suppression of muscarinic receptor-mediated detrusor muscle contractions but also by modulation of muscarinic receptor-bladder afferent interactions.
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http://dx.doi.org/10.1016/j.ucl.2006.06.011DOI Listing
November 2006

Urinary acidity and pain in interstitial cystitis.

Rev Urol 2005 ;7(2):112

Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1477570PMC
July 2011

Sustained beneficial effects of intraprostatic botulinum toxin type A on lower urinary tract symptoms and quality of life in men with benign prostatic hyperplasia.

BJU Int 2006 Nov 6;98(5):1033-7; discussion 1337. Epub 2006 Sep 6.

Division of Urology, Chang Guang Memorial Hospital Kaohsiung, Chang Gung University Medicine of College, Taiwan.

Objective: To present a comprehensive experience with intraprostatic botulinum toxin-type A (BoNT-A) injection in men with symptomatic benign prostatic hyperplasia (BPH) and to assess the efficacy on lower urinary tract symptoms (LUTS) and quality of life (QoL).

Patients And Methods: In all, 41 men (mean age 69.1 years, sd 7.1 ) with an International Prostate Symptom Score of > or = 8, peak flow rate of < 12 mL/s, and who were refractory to medical treatment were injected with BoNT-A (Botox, Allergan, Inc., CA, USA) at 100 U (21 men, for prostate volume < 30 mL) or 200 U (20, for prostate volume > 30 mL) into the prostate transperineally under transrectal ultrasonography guidance. Study exclusion criteria were confirmed or suspected malignancy, previous pelvic surgery or trauma and previous invasive treatment for BPH. The clinical effects were evaluated at baseline and at 1, 3 and 6 months after treatment.

Results: There were no significant local or systemic side-effects in any men. LUTS and QoL indices improved by > 30% in 31 of the 41 men (76%), and four of five men with urinary retention for > 1 month could void spontaneously at 1 week to 1 month after the BoNT-A injection. In 12 of 41 men (29%) there was no change in prostate volume, yet seven of these men still had a > 30% improvement in maximum flow rate, LUTS and QoL. The efficacy was sustained at 12 months.

Conclusion: BoNT-A injected into the prostate is safe and effective for men with symptomatic BPH. The mechanisms of relief of symptoms might not depend totally on the volume shrinkage; the inhibitory effect on the smooth muscle tone and aberrant sensory function might also be important.
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http://dx.doi.org/10.1111/j.1464-410X.2006.06479.xDOI Listing
November 2006

Qualitative and quantitative expression profile of muscarinic receptors in human urothelium and detrusor.

J Urol 2006 Oct;176(4 Pt 1):1673-8

Department of Urology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.

Purpose: We compared the complete spectrum of receptor subtypes expressed by human detrusor and its primary culture with the expression profile in a human urothelium immortalized cell line, and in fresh urothelium tissue and its primary cell culture.

Materials And Methods: The levels of mRNA expressed for receptor subtypes M1 through M5 were determined with reverse transcriptase-polymerase chain reaction and quantitative polymerase chain reaction in total RNA extracted individually from different human bladder specimens, including fresh tissue of human urothelium and detrusor, and their respective primary cultures, as well as from the UROtsa cell line.

Results: All 5 muscarinic receptors were detected in fresh human bladder tissue by reverse transcriptase-polymerase chain reaction RNA. The same was true in separated urothelium and detrusor tissue except for the lack of the M5 receptor transcript. Receptor subtype mRNA expression in the UROtsa cell line paralleled expression in fresh human bladder. Quantitative polymerase chain reaction data further corroborated these results and showed comparable mRNA expression for M2 and M3 in primary detrusor cultures. Primary cultures also had a decreased copy number of receptor genes than native tissue. The decrease was even more pronounced in primary urothelium culture and the UROtsa cell line in the presence of high calcium. M2 and M3 receptors were also detected in urothelium and detrusor by immunoreactivity.

Conclusions: We identified all 5 existing muscarinic receptor subtypes in detrusor and urothelium, and transcripts levels of M2 and M3 were comparable in detrusor. These results support an alternative site of action in urothelium for anti-muscarinic drugs. Urothelial receptors should be considered in the design of future drugs for overactive bladder.
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http://dx.doi.org/10.1016/j.juro.2006.06.088DOI Listing
October 2006

Periurethral cellular injection: comparison of muscle-derived progenitor cells and fibroblasts with regard to efficacy and tissue contractility in an animal model of stress urinary incontinence.

Urology 2006 Aug;68(2):449-54

Department of Urology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Objectives: To compare muscle-derived cells (MDCs) and fibroblasts with regard to their potential for restoration of urethral function on injection in a previously established animal model of stress urinary incontinence.

Methods: The animals were divided into four (dosage) or five (cell concentration) experimental groups: normal, nontreated controls (normal group) or bilateral sciatic nerve transection with either periurethral injection of saline (saline group), MDCs (MDC group), fibroblasts (fibroblast group), or MDC/fibroblast mixture (mixed group). At 4 weeks after injection, the leak point pressure (LPP) was measured and contractility testing and histologic analysis were performed.

Results: The histologic examination demonstrated muscular atrophy in the saline group and new striated muscle fibers at the sites of MDC injection in the MDC group, but not in the fibroblast group. Denervation of the urethra resulted in a significant decrease of maximal fast-twitch muscle contraction amplitude to only 9% of normal. MDC injection into the denervated urethra significantly improved the fast-twitch muscle contraction amplitude to 73% of normal. The LPP of the normal, saline, MDC, fibroblast, and mixed groups at 4 weeks after treatment was 43.3 +/- 2.5, 25.8 +/- 1.4, 38.2 +/- 4.2, 38.3 +/- 1.2, and 34.5 +/- 3.3 cm H2O, respectively. In the cell dosage experiment, the LPP increased with increases in the injected cell number. Evidence of obstruction was observed in the high-dose (1 x 10(7) cells) fibroblast group.

Conclusions: Although both MDCs and fibroblast injection increased the LPP in a stress urinary incontinence rat model, only MDCs significantly improved urethral muscle strip contractility. Moreover, urinary retention developed with high-dose fibroblast injection, but not with MDC injection.
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http://dx.doi.org/10.1016/j.urology.2006.03.040DOI Listing
August 2006

Green onions: potential mechanism for hepatitis A contamination.

J Food Prot 2006 Jun;69(6):1468-72

Department of Urology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.

The largest documented foodborne hepatitis A outbreak in U.S. history occurred in November 2003. The source of that outbreak was green onions from a farm in Mexico. Two biomarkers were used to determine ways in which hepatitis A virus (HAV) can contaminate onions. Fluorescent microspheres (1.0 to 10 microm) and HAV vaccine were placed on the soil and the surfaces of pot-grown onions and in the liquid medium of hydroponically cultivated onions. Reverse transcription PCR (RT-PCR) was used to identify HAV RNA. Microspheres were found on the outside and inside of the pot-grown onions for up to 60 days. RT-PCR revealed HAV RNA from the vaccine in well-washed green onions. In the hydroponically grown onions, microspheres were found throughout the onion after only 1 day. RT-PCR also revealed HAV RNA inside the hydroponically grown onions. Both biomarkers support the hypothesis that HAV can contaminate the inside of the growing onion and can be taken up intracellularly through the roots. Once inside, the particles are impossible to remove by cleaning.
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http://dx.doi.org/10.4315/0362-028x-69.6.1468DOI Listing
June 2006

Roles of peripheral and central nicotinic receptors in the micturition reflex in rats.

J Urol 2006 Jul;176(1):374-9

Department of Urology, University of Pittsburgh School of Medicine, 3471 Fifth Avenue, Pittsburgh, PA 15213, USA.

Purpose: We investigated the effects of nicotinic acetylcholine receptor activation in the bladder and central nervous system on the micturition reflex in urethane anesthetized rats.

Materials And Methods: The effects of nicotinic acetylcholine receptor activation on bladder activity were examined during continuous infusion cystometrogram. Nicotine with or without the nicotinic acetylcholine receptor antagonist mecamylamine (Sigma Chemical Co., St. Louis, Missouri) was administered intravesically, intrathecally or intracerebroventricularly in normal or capsaicin pretreated rats. We also examined nicotine induced responses in dissociated bladder afferent neurons from L6 to S1 dorsal root ganglia that were sensitive to capsaicin using whole cell patch clamp recordings.

Results: Intravesical nicotine (1 to 10 mM) significantly decreased intercontraction intervals in dose dependent fashion. This excitatory effect was abolished by co-application of mecamylamine (3 mM) as well as by capsaicin pretreatment. On patch clamp recordings 300 muM nicotine evoked rapid inward currents that were antagonized by mecamylamine in capsaicin sensitive bladder afferent neurons. Intrathecal and intracerebroventricular administration of nicotine (10 mug) decreased and increase intercontraction intervals, respectively. Each effect was antagonized by mecamylamine (50 mug) administered intrathecally and intracerebroventricularly. The spinal excitatory effect was significantly inhibited by the N-methyl-D-aspartate receptor antagonist (+)-MK-801 hydrogen maleate (20 mug) given intrathecally or by capsaicin pretreatment, although the effects of capsaicin pretreatment were significantly smaller than those of (+)-MK-801 hydrogen maleate.

Conclusions: These results indicate that nicotinic acetylcholine receptor activation in capsaicin sensitive C-fiber afferents in the bladder can induce detrusor overactivity. In the central nervous system nicotinic acetylcholine receptor activation in the spinal cord and brain has an excitatory and an inhibitory effect on the micturition reflex, respectively. In addition, the nicotine induced spinal excitatory effect may be mediated by the activation of glutamatergic mechanisms.
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http://dx.doi.org/10.1016/S0022-5347(06)00581-7DOI Listing
July 2006

Intravesical instillation of human urine after oral administration of trospium, tolterodine and oxybutynin in a rat model of detrusor overactivity.

BJU Int 2006 Feb;97(2):400-3

Department of Urology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213, USA.

Objective: To study the effects of antimuscarinics excreted into human urine on normal bladder in a rat model of detrusor overactivity.

Materials And Methods: Two 'normal' adult volunteers collected voided urine after taking trospium (20 mg, twice daily), tolterodine LA (4 mg, four times daily), or oxybutynin XL (10 mg, four times daily). The drugs were taken in a random order for 5 days with a 7-day washout period between the drugs. The urine collected from the two volunteers was mixed together and then blindly labelled and used for testing. Control human urine (no oral antimuscarinics) was also used. The effect of intravesical administration of human urine on carbachol-induced bladder overactivity was studied in female Sprague-Dawley rats anaesthetised with urethane. Cystometric variables during continuous infusion (0.04 mL/min) for >1 h each of saline, human urine, then a mixture of carbachol (30 microm) and human urine were compared in the four groups (control and the three different antimuscarinics tested; six rats per group).

Results: Human urine, with or with no intake of antimuscarinic agents, had no effect on normal bladder function. Bladder capacity and intercontraction intervals were significantly decreased after adding carbachol to urine containing vehicle, tolterodine or oxybutynin. However, urine collected from the humans who had taken trospium prevented the carbachol-induced reduction in bladder capacity and intercontraction intervals. Maximum voiding pressure and pressure threshold were not changed in any case.

Conclusion: This is the first report that the urine excreted after oral ingestion of trospium (20 mg, twice daily) has a significant inhibitory effect in a rat model of detrusor overactivity. This suggests that antimuscarinic agents have a local bladder effect during the bladder-storage phase in addition to the smooth muscle-mediated voiding phase.
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http://dx.doi.org/10.1111/j.1464-410X.2005.05913.xDOI Listing
February 2006

Detrusor overactivity induced by intravesical application of adenosine 5'-triphosphate under different delivery conditions in rats.

Urology 2005 Dec;66(6):1332-7

Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.

Objectives: To investigate the effects of intravesical application of adenosine 5'-triphosphate (ATP) on bladder activity to elucidate the role of urothelial barrier function and ecto-ATPase activity in the ATP-mediated mechanism inducing detrusor overactivity.

Methods: Continuous cystometry by an intravesical catheter inserted from the bladder dome was performed in conscious female rats.

Results: ATP solutions adjusted to pH 6.0 did not elicit significant detrusor overactivity at a concentration of 60 mM. However, in bladders pretreated with protamine sulfate (10 mg/mL) to increase urothelial permeability, ATP solution (pH 6.0) induced detrusor overactivity by decreasing the intercontraction intervals. These irritant effects of ATP after protamine treatment were antagonized by P2X receptor antagonists, such as pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (70 micromol/kg) and 2',3'-O-(2,4,6, trinitrophenyl) ATP (30 micromol/kg). These were also suppressed in rats pretreated with systemic capsaicin (125 mg/kg subcutaneously). Alpha,beta-methylene ATP (5 mM, pH 6.0) or ATP (60 mM, pH6) after intravesical infusion of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (5 mM, pH 6.0), an ecto-ATPase inhibitor, induced detrusor overactivity without protamine pretreatment, but the reduction in intercontraction intervals was smaller compared with that with ATP after protamine treatment.

Conclusions: Low permeability of bladder epithelium and ecto-ATPase activity can prevent ATP activation of subepithelial P2X receptors to induce bladder overactivity. Thus, enhanced penetration of endogenous ATP owing to urothelial damage may contribute to urinary frequency and bladder pain in hypersensitive bladder disorders such as interstitial cystitis.
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http://dx.doi.org/10.1016/j.urology.2005.06.099DOI Listing
December 2005