Pub Date : 2001-12-01DOI: 10.1089/10915360152745795
C. Niederberger
Neural computation is a field in which mathematical models are derived from algorithms based loosely on the physiological function of the biological neuron. This paper serves as an introduction to those that follow in this issue of Molecular Urology, which describe actual applications of neural computational modeling in the urologic domain. In this introductory paper, the history of computer technology and the foundations of neural computation are discussed. Methods of determining the accuracy of computation models are reviewed, and a statistical method of evaluating the significance of individual input features to the model's output, a process known as "feature extraction," is presented. Resources that provide free and commercial neural computational programs are cited for those readers interested in applying this technology to their own datasets, and a brief description of the author's neural computational programming environment is included. Finally, deployment of computational models via the Internet and various computer platforms is discussed.
{"title":"Neural computation in urology: an orientation.","authors":"C. Niederberger","doi":"10.1089/10915360152745795","DOIUrl":"https://doi.org/10.1089/10915360152745795","url":null,"abstract":"Neural computation is a field in which mathematical models are derived from algorithms based loosely on the physiological function of the biological neuron. This paper serves as an introduction to those that follow in this issue of Molecular Urology, which describe actual applications of neural computational modeling in the urologic domain. In this introductory paper, the history of computer technology and the foundations of neural computation are discussed. Methods of determining the accuracy of computation models are reviewed, and a statistical method of evaluating the significance of individual input features to the model's output, a process known as \"feature extraction,\" is presented. Resources that provide free and commercial neural computational programs are cited for those readers interested in applying this technology to their own datasets, and a brief description of the author's neural computational programming environment is included. Finally, deployment of computational models via the Internet and various computer platforms is discussed.","PeriodicalId":80296,"journal":{"name":"Molecular urology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2001-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/10915360152745795","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60627255","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-12-01DOI: 10.1089/10915360152745786
H. Tazaki, W. Fair
{"title":"Farewell and Thank You","authors":"H. Tazaki, W. Fair","doi":"10.1089/10915360152745786","DOIUrl":"https://doi.org/10.1089/10915360152745786","url":null,"abstract":"","PeriodicalId":80296,"journal":{"name":"Molecular urology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2001-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60627142","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-12-01DOI: 10.1089/10915360152745849
Ashutosh Tewari, Mutta Issa, R. El-Galley, H. Stricker, J. Peabody, Julio M. Pow-Sang, Asim Shukla, Zev Wajsman, Mark Rubin, John T. Wei, James Montie, Raymond Demers, Christine C. Johnson, Lois Lamerato, George W. Divine, E. David Crawford, E. Gamito, Riad Farah, Perinchery Narayan, Grant Carlson, M. Menon
BACKGROUND AND PURPOSE Despite many new procedures, radical prostatectomy remains one of the commonest methods of treating clinically localized prostate cancer. Both from the physician's and the patient's point of view, it is important to have objective estimation of the likelihood of recurrence, which forms the foundation for treatment selection for an individual patient. Currently, it is difficult to predict the probability of biochemical recurrence (rising serum prostate specific antigen [PSA] concentration) in an individual patient, and approximately 30% of the patients do experience recurrence. Tools predicting the recurrence will be of immense practical utility in the treatment selection and planning follow up. We have utilized preoperative parameters through a computer based genetic adaptive neural network model to predict recurrence in such patients, which can help primary care physicians and urologists in making management recommendations. PATIENTS AND METHODS Fourteen hundred patients who underwent radical prostatectomy at participating institutions form the subjects of this study. Demographic data such as age, race, preoperative PSA, systemic biopsy based staging and Gleason scores were used to construct a neural network model. This model simulated the functioning of a trained human mind and learned from the database. Once trained, it was used to predict the outcomes in new patients. RESULTS The patients in this comprehensive database were representative of the average prostate cancer patients as seen in USA. Their mean age was 68.4 years, the mean PSA concentration before surgery was 11.6 ng/mL, and 67% patients had a Gleason sum of 5 to 7. The mean length of follow-up was 41.5 months. Eighty percent of the cancers were clinical stage T2 and 5% T3. In our series, 64% of patients had pathologically organ-confined cancer, 33% positive margins, and 14% had seminal vesicle invasion. Lymph node positive patients were not included in this series. Progression as judged by serum PSA was noted in 30.6%. With entry of a few routinely used parameters, the model could correctly predict recurrence in 76% of the patients in the validation set. The area under the curve was 0.831. The sensitivity was 85%, the specificity 74%, the positive predictive value 77%, and the negative predictive value of 83%. CONCLUSION It was possible to predict PSA recurrence with a high accuracy (76%). Physicians desiring objective treatment counseling can use this model, and significant cost savings are anticipated because of appropriate treatment selection and patient-specific follow-up protocols. This technology can be extended to other treatments such as watchful waiting, external-beam radiation, and brachytherapy.
{"title":"Genetic adaptive neural network to predict biochemical failure after radical prostatectomy: a multi-institutional study.","authors":"Ashutosh Tewari, Mutta Issa, R. El-Galley, H. Stricker, J. Peabody, Julio M. Pow-Sang, Asim Shukla, Zev Wajsman, Mark Rubin, John T. Wei, James Montie, Raymond Demers, Christine C. Johnson, Lois Lamerato, George W. Divine, E. David Crawford, E. Gamito, Riad Farah, Perinchery Narayan, Grant Carlson, M. Menon","doi":"10.1089/10915360152745849","DOIUrl":"https://doi.org/10.1089/10915360152745849","url":null,"abstract":"BACKGROUND AND PURPOSE\u0000Despite many new procedures, radical prostatectomy remains one of the commonest methods of treating clinically localized prostate cancer. Both from the physician's and the patient's point of view, it is important to have objective estimation of the likelihood of recurrence, which forms the foundation for treatment selection for an individual patient. Currently, it is difficult to predict the probability of biochemical recurrence (rising serum prostate specific antigen [PSA] concentration) in an individual patient, and approximately 30% of the patients do experience recurrence. Tools predicting the recurrence will be of immense practical utility in the treatment selection and planning follow up. We have utilized preoperative parameters through a computer based genetic adaptive neural network model to predict recurrence in such patients, which can help primary care physicians and urologists in making management recommendations.\u0000\u0000\u0000PATIENTS AND METHODS\u0000Fourteen hundred patients who underwent radical prostatectomy at participating institutions form the subjects of this study. Demographic data such as age, race, preoperative PSA, systemic biopsy based staging and Gleason scores were used to construct a neural network model. This model simulated the functioning of a trained human mind and learned from the database. Once trained, it was used to predict the outcomes in new patients.\u0000\u0000\u0000RESULTS\u0000The patients in this comprehensive database were representative of the average prostate cancer patients as seen in USA. Their mean age was 68.4 years, the mean PSA concentration before surgery was 11.6 ng/mL, and 67% patients had a Gleason sum of 5 to 7. The mean length of follow-up was 41.5 months. Eighty percent of the cancers were clinical stage T2 and 5% T3. In our series, 64% of patients had pathologically organ-confined cancer, 33% positive margins, and 14% had seminal vesicle invasion. Lymph node positive patients were not included in this series. Progression as judged by serum PSA was noted in 30.6%. With entry of a few routinely used parameters, the model could correctly predict recurrence in 76% of the patients in the validation set. The area under the curve was 0.831. The sensitivity was 85%, the specificity 74%, the positive predictive value 77%, and the negative predictive value of 83%.\u0000\u0000\u0000CONCLUSION\u0000It was possible to predict PSA recurrence with a high accuracy (76%). Physicians desiring objective treatment counseling can use this model, and significant cost savings are anticipated because of appropriate treatment selection and patient-specific follow-up protocols. This technology can be extended to other treatments such as watchful waiting, external-beam radiation, and brachytherapy.","PeriodicalId":80296,"journal":{"name":"Molecular urology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2001-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/10915360152745849","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60627345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-12-01DOI: 10.1089/10915360152745812
Ashutosh Tewari, Christopher Porter, J. Peabody, E. David Crawford, Raymond Demers, Christine C. Johnson, John T. Wei, George W. Divine, Colin O'Donnell, E. Gamito, Mani Menon
A number of new predictive modeling techniques have emerged in the past several years. These methods can be used independently or in combination with traditional modeling techniques to produce useful tools for the management of prostate cancer. Investigators should be aware of these techniques and avail themselves of their potentially useful properties. This review outlines selected predictive methods that can be used to develop models that may be useful to patients and clinicians for prostate cancer management.
{"title":"Predictive modeling techniques in prostate cancer.","authors":"Ashutosh Tewari, Christopher Porter, J. Peabody, E. David Crawford, Raymond Demers, Christine C. Johnson, John T. Wei, George W. Divine, Colin O'Donnell, E. Gamito, Mani Menon","doi":"10.1089/10915360152745812","DOIUrl":"https://doi.org/10.1089/10915360152745812","url":null,"abstract":"A number of new predictive modeling techniques have emerged in the past several years. These methods can be used independently or in combination with traditional modeling techniques to produce useful tools for the management of prostate cancer. Investigators should be aware of these techniques and avail themselves of their potentially useful properties. This review outlines selected predictive methods that can be used to develop models that may be useful to patients and clinicians for prostate cancer management.","PeriodicalId":80296,"journal":{"name":"Molecular urology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2001-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/10915360152745812","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60627464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/109153601300177600
I. Yoshimura, S. Suzuki, M. Hayakawa
The bacteriophage P1-derived Cre-loxP system is a powerful and versatile tool for in vivo DNA recombination. It is widely used in gene targeting research. The combination of the Cre-loxP system and a specific promoter enables conditional "knockout" of a target gene in a particular tissue or cell type. It has also made it possible to delete genes in adult animals that are essential for embryogenesis. This system is also used to enhance tissue- or tumor-specific promoter activities that are useful for gene therapy of certain types of cancer. It is expected that this simple and effective system will be further utilized in various research applications.
{"title":"Application of Cre-loxP system to the urinary tract and cancer gene therapy.","authors":"I. Yoshimura, S. Suzuki, M. Hayakawa","doi":"10.1089/109153601300177600","DOIUrl":"https://doi.org/10.1089/109153601300177600","url":null,"abstract":"The bacteriophage P1-derived Cre-loxP system is a powerful and versatile tool for in vivo DNA recombination. It is widely used in gene targeting research. The combination of the Cre-loxP system and a specific promoter enables conditional \"knockout\" of a target gene in a particular tissue or cell type. It has also made it possible to delete genes in adult animals that are essential for embryogenesis. This system is also used to enhance tissue- or tumor-specific promoter activities that are useful for gene therapy of certain types of cancer. It is expected that this simple and effective system will be further utilized in various research applications.","PeriodicalId":80296,"journal":{"name":"Molecular urology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/109153601300177600","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60626660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/109153601300177619
Y. Kaneda
We have combined hemagglutinating virus of Japan (HVJ; Sendai virus) with liposomes for efficient in vitro and in vivo fusion-mediated gene delivery. The HVJ-liposome was a highly efficient vehicle for the introduction of oligonucleotides into cells in vivo as well as for the transfer of genes <100 kbp without damaging cells. By coupling the Epstein-Barr (EB) virus replicon apparatus with HVJ-liposomes (virosomes), transgene expression was sustained in vitro and in vivo. When we added cationic lipids, the HVJ-cationic liposomes increased gene delivery 100 to 800 times in vitro compared with the conventional anionic virosomes and were also more useful for gene expression in restricted areas of organs and for gene therapy of disseminated cancers. We further discovered that the use of anionic virosomes with a virus-mimicking lipid composition (artificial viral envelope; AVE type) increased transfection efficiency approximately 10 fold in vivo, especially in the heart, liver, kidney, and muscle. Most animal organs were found to be suitable targets for the fusigenic virosomes, and numerous gene therapy strategies using this system were successful in animals. The combination of suicide gene therapy with radiation was very effective for killing hepatomas in a mouse model. Arteriosclerosis obliterans in animal models was cured by the transfer of hepatocyte growth factor.
{"title":"Improvements in gene therapy technologies.","authors":"Y. Kaneda","doi":"10.1089/109153601300177619","DOIUrl":"https://doi.org/10.1089/109153601300177619","url":null,"abstract":"We have combined hemagglutinating virus of Japan (HVJ; Sendai virus) with liposomes for efficient in vitro and in vivo fusion-mediated gene delivery. The HVJ-liposome was a highly efficient vehicle for the introduction of oligonucleotides into cells in vivo as well as for the transfer of genes <100 kbp without damaging cells. By coupling the Epstein-Barr (EB) virus replicon apparatus with HVJ-liposomes (virosomes), transgene expression was sustained in vitro and in vivo. When we added cationic lipids, the HVJ-cationic liposomes increased gene delivery 100 to 800 times in vitro compared with the conventional anionic virosomes and were also more useful for gene expression in restricted areas of organs and for gene therapy of disseminated cancers. We further discovered that the use of anionic virosomes with a virus-mimicking lipid composition (artificial viral envelope; AVE type) increased transfection efficiency approximately 10 fold in vivo, especially in the heart, liver, kidney, and muscle. Most animal organs were found to be suitable targets for the fusigenic virosomes, and numerous gene therapy strategies using this system were successful in animals. The combination of suicide gene therapy with radiation was very effective for killing hepatomas in a mouse model. Arteriosclerosis obliterans in animal models was cured by the transfer of hepatocyte growth factor.","PeriodicalId":80296,"journal":{"name":"Molecular urology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/109153601300177619","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60626712","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/10915360152559576
Zhao Wang, Xingyao Wu, Robert M. Levin, Alan P. Hudson
When the rabbit bladder outlet is partially obstructed, the relative amount of mitochondrial (mt) DNA per cell in bladder smooth muscle falls rapidly. In order to assess whether this loss of organellar genome results from attenuation of mt DNA replication, we cloned portions of rabbit genes specifying the single-strand binding (SSB) protein required for initiation of mt DNA replication, and the catalytic subunit of DNA polymerase gamma (pol gamma), the replication enzyme itself. We then designed primer-probe systems for real-time RT-PCR (TaqMan) analyses for each gene. These were used to assess mRNA in preparations from bladder smooth muscle and mucosa from rabbits subjected to surgical obstruction of the bladder outlet for up to 14 days. mRNA from the pol gamma gene remained essentially at control level in smooth muscle and mucosa in all samples. In mucosa, mRNA from the SSB protein gene remained virtually at control levels in all samples, as did mt genome copy number. In smooth muscle, however, levels of this mRNA declined by >95% within 3 days of obstruction and remained at that level through 14 days; this attenuation of SSB protein mRNA paralleled the loss of mt DNA in the same smooth muscle samples. Thus, lack of mt SSB protein, and consequently attenuated mt DNA replication, is a primary factor in the loss of mt genome copies in bladder smooth muscle after outlet obstruction in the rabbit model of benign bladder dysfunction.
{"title":"Loss of mitochondrial DNA in rabbit bladder smooth muscle following partial outlet obstruction results from lack of organellar DNA replication.","authors":"Zhao Wang, Xingyao Wu, Robert M. Levin, Alan P. Hudson","doi":"10.1089/10915360152559576","DOIUrl":"https://doi.org/10.1089/10915360152559576","url":null,"abstract":"When the rabbit bladder outlet is partially obstructed, the relative amount of mitochondrial (mt) DNA per cell in bladder smooth muscle falls rapidly. In order to assess whether this loss of organellar genome results from attenuation of mt DNA replication, we cloned portions of rabbit genes specifying the single-strand binding (SSB) protein required for initiation of mt DNA replication, and the catalytic subunit of DNA polymerase gamma (pol gamma), the replication enzyme itself. We then designed primer-probe systems for real-time RT-PCR (TaqMan) analyses for each gene. These were used to assess mRNA in preparations from bladder smooth muscle and mucosa from rabbits subjected to surgical obstruction of the bladder outlet for up to 14 days. mRNA from the pol gamma gene remained essentially at control level in smooth muscle and mucosa in all samples. In mucosa, mRNA from the SSB protein gene remained virtually at control levels in all samples, as did mt genome copy number. In smooth muscle, however, levels of this mRNA declined by >95% within 3 days of obstruction and remained at that level through 14 days; this attenuation of SSB protein mRNA paralleled the loss of mt DNA in the same smooth muscle samples. Thus, lack of mt SSB protein, and consequently attenuated mt DNA replication, is a primary factor in the loss of mt genome copies in bladder smooth muscle after outlet obstruction in the rabbit model of benign bladder dysfunction.","PeriodicalId":80296,"journal":{"name":"Molecular urology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/10915360152559576","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60626863","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/10915360152745867
R. Babaian, Z. Zhang
Artificial neural networks (ANNs) have only recently been applied to solve problems in the diagnosis, staging, and prediction of treatment outcome in prostate cancer. A literature search provided information on 10 published journal articles that were selected for review and analysis. In all but one of the studies that compared the ANN output with logistic regression modeling, the ANN performed better. Specific training issues for neural networks are discussed and examples provided. We conclude that the continued development and refinement of computer-assisted diagnostic methodology are warranted to enhance conventional statistical approaches to the classification and pattern recognition found in data sets from men either suspected of having or known to have prostate cancer.
{"title":"Computer-assisted diagnostics: application to prostate cancer.","authors":"R. Babaian, Z. Zhang","doi":"10.1089/10915360152745867","DOIUrl":"https://doi.org/10.1089/10915360152745867","url":null,"abstract":"Artificial neural networks (ANNs) have only recently been applied to solve problems in the diagnosis, staging, and prediction of treatment outcome in prostate cancer. A literature search provided information on 10 published journal articles that were selected for review and analysis. In all but one of the studies that compared the ANN output with logistic regression modeling, the ANN performed better. Specific training issues for neural networks are discussed and examples provided. We conclude that the continued development and refinement of computer-assisted diagnostic methodology are warranted to enhance conventional statistical approaches to the classification and pattern recognition found in data sets from men either suspected of having or known to have prostate cancer.","PeriodicalId":80296,"journal":{"name":"Molecular urology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/10915360152745867","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60627789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/109153601300177565
N. Okui, Y. Kitamura, N. Kobayashi, R. Sakuma, T. Ishikawa, T. Kitamura
BACKGROUND Acquired immune deficiency syndrome (AIDS) is resistant to all current therapy. Gene therapy is an attractive alternative or additive to current, unsatisfactory AIDS therapy. MATERIALS AND METHODS To develop an antiviral molecule targeting viral integrase (HIV IN), we generated a single-chain antibody, termed scAb, which interacted with human immunodeficiency virus type 1 (HIV-1) IN and inhibited virus replication at the integration step when expressed intracellularly. To reduce infectivity from within the virus particles, we made expression plasmids (pC-scAbE-Vpr, pC-scAbE-CA, and pC-scAbE-WXXF), which expressed the anti-HIV IN scAb fused to the N-terminus of HIV-1-associated accessory protein R (Vpr), capsid protein (CA), and specific binding motif to Vpr (WXXF), respectively. All fusion proteins were tagged with a nine-amino acid peptide derived from influenza virus hemagglutinin (HA) at the C terminus. RESULTS The fusion molecules, termed scAbE-Vpr, scAbE-CA, and scAbE-WXXF, interacted specifically with HIV IN immobilized on a nitrocellulose membrane. Immunoblot analysis showed that scAbE-Vpr, scAbE-CA, and scAbE-WXXF were incorporated into the virions produced by cotransfection of 293T cells with HIV-1 infectious clone DNA (pLAI) and pC-scAbE-Vpr, pC-scAbE-WXXF. A multinuclear activation galactosidase indicator (MAGI) assay revealed that the virions released from 293T cells cotransfected with pLAI and pC-scAbE-Vpr, pC-scAbE-WXXF had as little 1000-fold of the infectivity of the control wild-type virions, which were produced from the 293T cells transfected with pLAI alone. Furthermore, the virions produced from the 293T cells cotransfected with pLAI and an scAb expression vector (pC-scAb) showed only 1% of the infectivity of the control HIV-1 in a MAGI assay, although scAb was not incorporated into the virions. In either instance, the total quantity of the progeny virions released from the transfected 293T cells and the patterns of the virion proteins were hardly affected by the presence of scAb, scAbE-Vpr, or scAbE-WXXF, as determined by virion-associated reverse transcriptase assay and by immunoblot analysis, respectively. Because G418-selected HeLa clones carrying the expression plasmid for scAbE-WXXF were obtained much more frequently than those for scAbE-Vpr, scAbE-WXXF was inferred to be less toxic to cells than scAbE-Vpr. The result that scAbE-WXXF with viral incorporation achieved more than a 10-fold reduction in infectivity of the progeny virions than scAb without incorporation suggests that scAbE-WXXF is a potential antiviral molecule, inhibiting replication by neutralization of HIV IN activity both within cells and within virions. Moreover, it is nontoxic to human cells. We termed this gene therapy "virion-"targeted-viral inactivation" and these molecules "packageable antiviral therapeutics." CONCLUSION This new gene therapy has the potential for wide application in many viral infectious diseases.
{"title":"Virion-targeted viral inactivation: new therapy against viral infection.","authors":"N. Okui, Y. Kitamura, N. Kobayashi, R. Sakuma, T. Ishikawa, T. Kitamura","doi":"10.1089/109153601300177565","DOIUrl":"https://doi.org/10.1089/109153601300177565","url":null,"abstract":"BACKGROUND Acquired immune deficiency syndrome (AIDS) is resistant to all current therapy. Gene therapy is an attractive alternative or additive to current, unsatisfactory AIDS therapy. MATERIALS AND METHODS To develop an antiviral molecule targeting viral integrase (HIV IN), we generated a single-chain antibody, termed scAb, which interacted with human immunodeficiency virus type 1 (HIV-1) IN and inhibited virus replication at the integration step when expressed intracellularly. To reduce infectivity from within the virus particles, we made expression plasmids (pC-scAbE-Vpr, pC-scAbE-CA, and pC-scAbE-WXXF), which expressed the anti-HIV IN scAb fused to the N-terminus of HIV-1-associated accessory protein R (Vpr), capsid protein (CA), and specific binding motif to Vpr (WXXF), respectively. All fusion proteins were tagged with a nine-amino acid peptide derived from influenza virus hemagglutinin (HA) at the C terminus. RESULTS The fusion molecules, termed scAbE-Vpr, scAbE-CA, and scAbE-WXXF, interacted specifically with HIV IN immobilized on a nitrocellulose membrane. Immunoblot analysis showed that scAbE-Vpr, scAbE-CA, and scAbE-WXXF were incorporated into the virions produced by cotransfection of 293T cells with HIV-1 infectious clone DNA (pLAI) and pC-scAbE-Vpr, pC-scAbE-WXXF. A multinuclear activation galactosidase indicator (MAGI) assay revealed that the virions released from 293T cells cotransfected with pLAI and pC-scAbE-Vpr, pC-scAbE-WXXF had as little 1000-fold of the infectivity of the control wild-type virions, which were produced from the 293T cells transfected with pLAI alone. Furthermore, the virions produced from the 293T cells cotransfected with pLAI and an scAb expression vector (pC-scAb) showed only 1% of the infectivity of the control HIV-1 in a MAGI assay, although scAb was not incorporated into the virions. In either instance, the total quantity of the progeny virions released from the transfected 293T cells and the patterns of the virion proteins were hardly affected by the presence of scAb, scAbE-Vpr, or scAbE-WXXF, as determined by virion-associated reverse transcriptase assay and by immunoblot analysis, respectively. Because G418-selected HeLa clones carrying the expression plasmid for scAbE-WXXF were obtained much more frequently than those for scAbE-Vpr, scAbE-WXXF was inferred to be less toxic to cells than scAbE-Vpr. The result that scAbE-WXXF with viral incorporation achieved more than a 10-fold reduction in infectivity of the progeny virions than scAb without incorporation suggests that scAbE-WXXF is a potential antiviral molecule, inhibiting replication by neutralization of HIV IN activity both within cells and within virions. Moreover, it is nontoxic to human cells. We termed this gene therapy \"virion-\"targeted-viral inactivation\" and these molecules \"packageable antiviral therapeutics.\" CONCLUSION This new gene therapy has the potential for wide application in many viral infectious diseases.","PeriodicalId":80296,"journal":{"name":"Molecular urology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/109153601300177565","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60626895","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/10915360152559594
Y. Murakami, K. Kanda, K. Yokota, H. Kanayama, S. Kagawa
Our purpose was to elucidate the clinical roles of the "immuno-proteosome," which is involved in the accelerated pathway of the major histocompatibility complex (MHC) class I-restricted antigen presentation system, in renal cell carcinoma (RCC). The relative expression of six proteosome subunits (existing subunits X, Y, and Z and immunoproteosome subunits LMP7, LMP2, and MECL1) in 54 RCCs was investigated using RT-PCR analysis and was compared with clinicopathological measures, including patient outcome. Expression of the LMP7 and LMP2 genes was significantly low in high-grade tumors, and that of the LMP7 and MECL1 genes was significantly low in high-stage tumors. Low levels of LMP7, LMP2, and MECL1 expression were strongly associated with shortened survival (LMP7: P = 0.0002, LMP2: P < 0.0001, MECL1: P < 0.0047). The levels of subunits X, Y, and Z had no significant correlation with those measures. These findings suggest that RCCs with low level of immuno-proteosome subunit expression have a disorder in their antigen-presentation system. As a consequence, they may escape from immune surveillance and worsen patient outcome.
我们的目的是阐明“免疫蛋白体”在肾细胞癌(RCC)中的临床作用，它参与了主要组织相容性复合体(MHC) i类限制性抗原呈递系统的加速途径。使用RT-PCR分析研究了54例rcc中6个蛋白体亚基(现有的X、Y和Z亚基以及免疫蛋白体亚基LMP7、LMP2和MECL1)的相对表达，并与包括患者预后在内的临床病理指标进行了比较。LMP7和LMP2基因在高级别肿瘤中表达明显低，LMP7和MECL1基因在高级别肿瘤中表达明显低。低水平的LMP7、LMP2和MECL1表达与缩短生存期密切相关(LMP7: P = 0.0002, LMP2: P < 0.0001, MECL1: P < 0.0047)。亚单位X、Y和Z的水平与这些测量无显著相关。这些发现表明，低水平的免疫蛋白体亚基表达的rcc在其抗原呈递系统中存在障碍。因此，它们可能会逃避免疫监视并使患者预后恶化。
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