Bertie de Leeuw - Academia.edu (original) (raw)
Papers by Bertie de Leeuw
Molecular Therapy
4602 The limited spread of non-replicating adenoviral vectors after injection into glial tumors m... more 4602 The limited spread of non-replicating adenoviral vectors after injection into glial tumors may contribute to the lack of significant tumor reduction in many human gene therapy trials. To enhance viral spread in the tumor while keeping it restricted to the the brain, we designed conditionally replicating viruses in which the E1A gene is under the control of the promoter of the glia specific intermediate filament Glial Fibrillary Acidic Protein (GFAP). Because of the moderate activity of the normal GFAP promoter (gfa2) compared to the CMV promoter, we also evaluated constructs containing additional glial specific enhancers: gfa2(B)3 and gfa2(ABD)3 promoters. The gfa2 derived promoters followed by a beta galactosidase reporter gene were cloned into the pShuttle plasmid, in an orientation inverse to wildtype E1 expression . Viral vectors were produced according to the Ad-Easy protocol and viruses giving rise to blue plaques upon infection of U251 but not A549 cells were selected and grown to high titers. To check the specificity and effectivity of the promoter constructs, a panel of human cell lines (glial and non-glial) was infected with the gfa2 (triple enhancer) promoter-beta galactosidase vectors. An adenoviral vector containing a CMV-beta galactosidase cassette was included to estimate cell entry. Expression of beta galactosidase was evaluated both in situ using X-gal staining and quantitatively using ONPG staining. Results: Of 11 glial cell lines tested, four were positive for GFAP on a Western blot incubated with anti-GFAP (U251, SNB19, U373 and GOS3). Furthermore, of 12 primary cultures of glial tumors one was positive for GFAP using immunohistochemistry. This positive primary culture was also GFAP positive on Western and was added to the panel of cell lines, as were fourteen non glial cell lines (all GFAP negative on Western). Of the three gfa2 derived promoters, the gfa2 (ABD)3-LacZ construct gives rise to high beta galactosidase expression levels in the GFAP positive cell lines. Beta galactosidase expression levels in some GFAP positive lines are at a level comparable to the CMV promoter construct. The ONPG ratio of GFAP positive cell lines to GFAP negative cell lines (both glial and non-glial) is best for the gfa2-(B)3 promoter containing virus. Preliminary results indicate that conditionally replicating adenovirus in which E1A is regulated by the gfa2-B3 promoter replicate better on the GFAP positive U251 cell line than on the GFAP negative Hep2 cells. Conclusion: The gfa2 promoter constructs are highly active in an adenoviral context and have retained their specificity. Constructs for conditionally replicating viruses using these promoters have been prepared and conditionally replicating virus is being produced.
Synovial sarcomas show a specific translocation involving chromosomes X and 18, t(X;18)(p11.2;q11... more Synovial sarcomas show a specific translocation involving chromosomes X and 18, t(X;18)(p11.2;q11.2). Two distinct X‐chromosomal breakpoints occur in different synovial sarcoma tumour samples. These breakpoints are located within two related genomic regions containing ornithine aminotransferase‐like sequences, termed OATAL1 and OATL2. Preliminary observations indicated the potential correlation of OATL1‐associated breakpoints with biphasic tumours and OATL2‐associated breakpoints with monophasic fibrous tumours. The present study uses interphase cytogenetics to investigate the nature of chromosomal aberrations in frozen synovial sarcoma tissue samples. Two‐colour fluorescence in situ hybridization (FISH) was performed using probes specific for the centromeres of chromosome X or 18, along with yeast artificial chromosome probes corresponding to the distinct breakpoint regions on Xp. One monophasic epithelial and two monophasic fibrous synovial sarcomas showed an OATL2‐associated brea...
The ability to direct transgene expression to astrocytes has become increasingly important as the... more The ability to direct transgene expression to astrocytes has become increasingly important as the roles for these cells continue to expand. Promoters consisting of the 5′‐flanking region of the human or mouse glial fibrillary acidic protein (GFAP) gene have generally proved satisfactory. However, a more powerful promoter would be advantageous for several applications, such as expression of dominant negative RNAs or proteins, or for gene therapy. We investigated the possibility of increasing the transcriptional activity of the human GFAP promoter by inserting into it one or three additional copies of putative GFAP enhancer regions. The promoters enhanced with three additional copies gave 75‐fold higher LacZ expression levels upon plasmid transfection into GFAP‐expressing U251 cells than the parental gfa2 promoter. Surprisingly, in a transgenic mouse model, the enhanced promoters resulted in no or only very low expression of marker genes, probably caused by toxicity. When various cell...
Molecular Therapy
In order to improve the efficacy of first generation adenoviral vectors for glioma treatment, we ... more In order to improve the efficacy of first generation adenoviral vectors for glioma treatment, we designed conditionally replicating viruses in which the E1A gene is under the control of the promoter (gfa2) of the glia specific intermediate filament Glial Fibrillary Acidic Protein (GFAP) and similar constructs containing additional glial specific enhancers.For a control variant of this virus the gfa2 derived promoters (the normal gfa2, gfa2(B)3 and gfa2(ABD)3 promoters resp.) followed by a beta galactosidase reporter gene were cloned into the pShuttle plasmid, in an orientation inverse to wildtype E1 expression. Viral vectors were produced according to the Ad-Easy protocol and viruses giving rise to blue plaques upon infection of U251 but not A549 cells were selected and grown to high titers. To check the specificity and efficacy of the promoter constructs, a panel of human cell lines (glial and non-glial) was infected with the gfa2 (triple enhancer) promoter-beta galactosidase vectors. An adenoviral vector containing a CMV-beta galactosidase cassette was included to determine whether cell entry effects play a role. Expression of beta galactosidase was evaluated both in situ using X-gal staining and quantitatively using ONPG staining. To create the conditionally replicating variant the beta galactosidase gene was replaced by the Ad5 E1 region.Results: A panel of 11 glial cell lines was selected based on GFAP positivity on Western blots and immunohistochemistry. Within this panel GFAP positivity correlated well with beta galactosidase expression upon infection with the beta galactosidase viruses. ONPG values of the Ad5-gfa2-ABD3-LacZ virus in GFAP positive cell lines approached the levels of the Ad5-CMV-LacZ. In 2/14 non-glial cell lines, which were all negative for GFAP protein expression, however, beta galactosidase expression was found with the Ad5-gfa2-LacZ and Ad5-gfa2(ABD)3-LacZ viruses in the same range as for the GFAP positive glial cell lines. After infection with the Ad5-gfa2-B3-LacZ virus, considerable beta galactosidase expression was only seen in one of the 14 non-glial cell lines tested. We discuss possible explanations for and consequences of this GFAP promoter activity in adenoviral context after infection of some non-glial tumor cell lines. Both LacZ viruses containing promoters with extra glial enhancers gave rise to higher ONPG values than the Ad5-gfa2-LacZ virus.Preliminary results indicate that conditionally replicating adenovirus in which E1A is regulated by the gfa2-B3 promoter replicate better on the GFAP positive U251 cell line than on the GFAP negative Hep2 cells.Conclusion: The gfa2 promoter constructs are highly active in an adenoviral context and have retained their specificity, with the exception of some non-glial tumour cell lines. Ad5-gfa2-B3-E1 conditionally replicating viruses have been grown to high titers and are currently being tested for replication in vitro and for effect on xenograft growth.
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2006
The overall median survival of patients with a malignant glioma is <1 y. Because malignant gli... more The overall median survival of patients with a malignant glioma is <1 y. Because malignant gliomas rarely metastasize outside the skull, locoregional treatment strategies, such as gene therapy, are under investigation. Recently, convection-enhanced delivery (CED) has been presented as a method to improve delivery of large molecules. The goal of this study was to evaluate whether CED improves intratumoral delivery of adenoviral vectors and compare it with single injection (SI) and multiple injection (4x, MI). A replication-deficient adenoviral vector encoding the herpes simplex virus thymidine kinase (HSV-tk) and the human somatostatin receptor subtype 2 (sst(2)) was administered into nude mice bearing subcutaneous U87 xenografts. Tumors were injected with 1.5 x 10(9) plaque-forming units of Ad5.tk.sstr by CED, SI, or MI. Three days later, [(99m)Tc-N(4)(0-1),Asp(0),Tyr(3)]octreotate ((99m)Tc-Demotate 2) was injected intravenously to monitor the virus-induced sst(2) expression. gam...
Brain : a journal of neurology, 2003
Paraneoplastic cerebellar degeneration (PCD) is a heterogeneous group of disorders characterized ... more Paraneoplastic cerebellar degeneration (PCD) is a heterogeneous group of disorders characterized by subacute cerebellar ataxia, specific tumour types and (often) associated antineuronal antibodies. Nine specific antineuronal antibodies are associated with PCD. We examined the relative frequency of the antineuronal antibodies associated with PCD and compared the neurological symptoms and signs, associated tumours, disability and survival between groups of PCD with different antibodies. Also, we attempted to identify patient-, tumour- and treatment-related characteristics associated with functional outcome and survival. In a 12-year period, we examined >5000 samples for the presence of antineuronal antibodies. A total of 137 patients were identified with a paraneoplastic neurological syndrome and high titre (> or =400) antineuronal antibodies. Fifty (36%) of these patients had antibody-associated PCD, including 19 anti-Yo, 16 anti-Hu, seven anti-Tr, six anti-Ri and two anti-mGlu...
Nederlands tijdschrift voor geneeskunde, Jan 18, 1998
Paraneoplastic neurological syndromes are believed to result from ectopic expression of onconeura... more Paraneoplastic neurological syndromes are believed to result from ectopic expression of onconeural antigens by tumours. The resulting immune response is not only directed against the tumour but also cross-reacts with the same or similar antigens in the nervous system. The immune response generates high titred autoantibodies that are associated with specific tumours and neurological syndromes. Paraneoplastic autoantibodies help diagnose neurological syndromes and help direct the search for an underlying tumour. In paraneoplastic syndromes, the course of the tumour is relatively mild. Detection of the autoantibodies might lead to early diagnosis and immunomodulation and anti-tumour treatment before irreversible neuronal cell loss and deficits set in.
Oncogene, 1993
In a previous study we localized the synovial sarcoma-associated t(X;18)(p11;q11) breakpoint with... more In a previous study we localized the synovial sarcoma-associated t(X;18)(p11;q11) breakpoint within the ornithine aminotransferase-like 1 (OATL1) cluster on the X chromosome. This localization was delineated from both somatic cell hybrid and fluorescence in situ hybridization (FISH) analysis of patient material, using OAT-specific cDNA and YAC probes. Simultaneously, Knight et al. (1992, Mol. Hum. Genet, in press) mapped this same breakpoint in their patient material adjacent to the more proximal OATL2 region on the X chromosome. Here we report the analysis of two additional tumors and demonstrate that again in these cases the chromosomal break occurs within the OATL1 cluster. In order to further specify the breakpoint, we subcloned the OATL1 YAC (no. 2) into cosmids. At least one of these cosmids (0.38) hybridizes to sequences that bracket the translocation breakpoint, as demonstrated by both Southern blot and FISH analysis. These observations confirm and substantiate our previous ...
Molecular and Cellular Endocrinology, 2008
HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires
Suicide Gene Therapy, 2003
... Hum. Gene Ther. 7, 7987. 29. Roy, S., Shirley, PS, McClelland, A., and Kaleko, M. (1998) Cir... more ... Hum. Gene Ther. 7, 7987. 29. Roy, S., Shirley, PS, McClelland, A., and Kaleko, M. (1998) Circumvention of immu-nity to the adenovirus major coat protein hexon. J. Virol. 72, 68756879. 30. ... Adv. Drug Delivery Rev. 27, 6781. 68. Khuri, FR, Nemunaitis, J., Ganly, I., et al. ...
New England Journal of Medicine, 2000
Neuroscience Research, 1998
We have already reported that non-inflammatory stress induced peripheral IL-6 production is, at l... more We have already reported that non-inflammatory stress induced peripheral IL-6 production is, at least partly, mediated by enteric endotoxin (LPS). In the present study, to clarify the major source of peripheral IL-6 induced by non-inflammatory stress, blood samplings were performed via chronically implanted cannulations into the hepatoportal vein (HV), the vena cava inferior (VCI) and/or the vena cava superior (VCS) in the electrical foot shock (FSS) rat model (ImA, 11 times, each one min). The results were as follows; (1) Plasma IL-6 levels in the HV were higher than those in the VCS (3Omin after FSS). (2) The peak of plasma IL-6 in the HV (30min) were earlier than that in the VCS (60 min). (3) Plasma IL-6 levels in the lower portion of the VCI (below the renal branch) were lower than that in the VCS (0 and 30min). (4) On the contrary, plasma IL-6 levels in the higher portion of the VCI (nearby the hepatic branch) were not differ from those in the VCS. (5)
Neuroscience Letters, 2009
Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC... more Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC) showed high titer immunohistochemical staining of the axon initial segment (AIS) on rat and human brain sections. EM studies showed that the antigen was localized in close proximity of the microtubules in the AIS. Double labeling experiments and absence of staining at the nodes of Ranvier excluded the previously identified betaIV spectrin as autoantigen. Screening a rat hippocampal cDNA library resulted in the isolation of ubiquitin-conjugating enzyme E2E1 (UBE2E1). However, blocking and elution experiments excluded UBE2E1 as the AIS autoantigen.
Molecular Therapy, 2005
targeting adaptors could provide viral retargeting to appropriate receptors. As an example of the... more targeting adaptors could provide viral retargeting to appropriate receptors. As an example of the practical utility of such viral modifications, we investigated if biotinylated fiber could target infection to the EGF receptor (EGFR), overexpressed on cancer cell lines via molecular bridge with EGF-Streptavidin. Our findings indicate that mbAd complexed with EGF-Streptavidin (mbAd/EGF) achieved 20-fold increase of infection efficiency on A431 cells overexpressing EGFR compared to untargeted mbAd. Increase in transduction efficiency of EGFR-retargeted mbAd was also achieved on other cancer cell lines. Enhanced infectivity of fiber-mosaic Ad provided by EGF-related targeting was not abolished by blocking with recombinant Ad5 knob, while wild type infection at the same experimental conditions was blocked. We also investigated in vivo targeting potential of mbAd complexed with EGF-Streptavidin on two different models. First, we utilized mouse model of locoregional EGFR-positive ovarian xenografts. Expression of reporter gene in ovarian tumor nodules was 5 times higher with EGFR-retargeted mbAd compared to the virus without adaptor. Secondly, retargeting capacity of mbAd/EGF was tested on mouse "transient transgenic" model, which is accomplished by preinfection of hCAR transgenic mice with AdfltEGFR and intended to transiently express hEGFR in the mouse lung vasculature. mbAd/EGF achieved 7-fold higher expression in the lung compared to mbAd without adaptor and wt Ad. Moreover, lung/liver ratio of gene expression for retargeted mbAd was also increased in both in vivo models tested. Thus, we demonstrated that additional fiber in fiber-mosaic could be used for biotinylation and this modification in combination with appropriate adaptors can be successfully exploited for adenoviral retargeting strategies.
Molecular Therapy, 2004
Although successful in many rodent models of malignant gliomas, results of clinical adenoviral ge... more Although successful in many rodent models of malignant gliomas, results of clinical adenoviral gene therapy trials using replication deficient vectors have been disappointing. One of the explanations of the discrepancy between results in animal models and clinical trials is the low expression of the Coxsackie Adenovirus receptor (CAR), the main high affinity receptor for binding of adenovirus serotype 5, on primary glioma cells as opposed to established glioma cell lines. To obtain improved adenoviral vectors, we made use of the differential tropism of the naturally occurring 51 human adenoviral serotypes to test the hypothesis that adenoviral vectors, binding to other receptors than CAR, would result in improved infection of primary glioma cells.Design/Methods: Thirty-four of the fifty-one wildtype adenoviral serotypes were collected and grown. Using PCR, sequences coding the adenoviral protein fiber were subcloned into an adenovirus serotype 5 background. All vectors contained the luciferase marker gene. A total of 18 recombinant adenoviral chimeras were constructed and grown on PER.C6 producer cells (Ad5.Fibxx, where xx indicates any of the other serotypes). Fresh glioma tissue from 11 patients was mechanically dissected and treated with trypsin. Early passage glioma cells (6-10) were incubated with Ad5.Fibxx at a multiplicity of infection (m.o.i.) of 1000 and 5000 viral particles (vp) per cell for 2h. Forty-eight hrs after infection, cells were lysed, luciferin was added and relative light units (RLU) were measured and compared to Ad5. CD46 expression on the tumor cells was examined by FACS analysis. In addition, early passage glioma cells were incubated with 34 of the existing 51 wildtype serotypes at m.o.i. 10 and time to CPE was monitored.Results: In 10 out of 11 primary glioma lines, we could identify serotypes that entered the cells more efficiently than Ad5 resulting in higher luciferase expression levels. The Ad5.Fibxx with fibers derived from B-group serotypes (Ad5.Fib11; Ad5.Fib16; Ad5.Fib35; Ad5.Fib50) infected glioma cells 5-100 times better than Ad5.Fib5. Also the F-group Ad5.Fib40s infected glioma cells better than Ad5.Fib5. The results of Ad5Fib35 were confirmed using another batch of the virus. In an additional experiment, infection with Ad5.Fib35 and Ad35.luc resulted in similar luciferase expression levels. CD46 (cell surface receptor for most B-group adenoviruses) was expressed on 6-46% of the low passage glioma tumor cells, but on 75-100% on 5/5 established cell lines.None of the replication competent wildtype serotypes gave significantly quicker CPE than wt Ad5 on five primary glioma cellines.Conclusions: Adenoviral vectors encoding fibers derived from serotypes 11, 16, 35, 50 (B-group) and 40 (F-group) are promising for glioma gene therapy. The Ad5 backbone remains a good choice for the construction of (conditionally) replicating adenoviral vectors for gene therapy of glioma.
Molecular Therapy, 2005
treatment of chronic viral hepatitis. In summary, these mouse model systems offered opportunities... more treatment of chronic viral hepatitis. In summary, these mouse model systems offered opportunities to test in vivo efficacy of HBx-specific siRNAs for control of HBV replication.
Molecular Therapy
4602 The limited spread of non-replicating adenoviral vectors after injection into glial tumors m... more 4602 The limited spread of non-replicating adenoviral vectors after injection into glial tumors may contribute to the lack of significant tumor reduction in many human gene therapy trials. To enhance viral spread in the tumor while keeping it restricted to the the brain, we designed conditionally replicating viruses in which the E1A gene is under the control of the promoter of the glia specific intermediate filament Glial Fibrillary Acidic Protein (GFAP). Because of the moderate activity of the normal GFAP promoter (gfa2) compared to the CMV promoter, we also evaluated constructs containing additional glial specific enhancers: gfa2(B)3 and gfa2(ABD)3 promoters. The gfa2 derived promoters followed by a beta galactosidase reporter gene were cloned into the pShuttle plasmid, in an orientation inverse to wildtype E1 expression . Viral vectors were produced according to the Ad-Easy protocol and viruses giving rise to blue plaques upon infection of U251 but not A549 cells were selected and grown to high titers. To check the specificity and effectivity of the promoter constructs, a panel of human cell lines (glial and non-glial) was infected with the gfa2 (triple enhancer) promoter-beta galactosidase vectors. An adenoviral vector containing a CMV-beta galactosidase cassette was included to estimate cell entry. Expression of beta galactosidase was evaluated both in situ using X-gal staining and quantitatively using ONPG staining. Results: Of 11 glial cell lines tested, four were positive for GFAP on a Western blot incubated with anti-GFAP (U251, SNB19, U373 and GOS3). Furthermore, of 12 primary cultures of glial tumors one was positive for GFAP using immunohistochemistry. This positive primary culture was also GFAP positive on Western and was added to the panel of cell lines, as were fourteen non glial cell lines (all GFAP negative on Western). Of the three gfa2 derived promoters, the gfa2 (ABD)3-LacZ construct gives rise to high beta galactosidase expression levels in the GFAP positive cell lines. Beta galactosidase expression levels in some GFAP positive lines are at a level comparable to the CMV promoter construct. The ONPG ratio of GFAP positive cell lines to GFAP negative cell lines (both glial and non-glial) is best for the gfa2-(B)3 promoter containing virus. Preliminary results indicate that conditionally replicating adenovirus in which E1A is regulated by the gfa2-B3 promoter replicate better on the GFAP positive U251 cell line than on the GFAP negative Hep2 cells. Conclusion: The gfa2 promoter constructs are highly active in an adenoviral context and have retained their specificity. Constructs for conditionally replicating viruses using these promoters have been prepared and conditionally replicating virus is being produced.
Synovial sarcomas show a specific translocation involving chromosomes X and 18, t(X;18)(p11.2;q11... more Synovial sarcomas show a specific translocation involving chromosomes X and 18, t(X;18)(p11.2;q11.2). Two distinct X‐chromosomal breakpoints occur in different synovial sarcoma tumour samples. These breakpoints are located within two related genomic regions containing ornithine aminotransferase‐like sequences, termed OATAL1 and OATL2. Preliminary observations indicated the potential correlation of OATL1‐associated breakpoints with biphasic tumours and OATL2‐associated breakpoints with monophasic fibrous tumours. The present study uses interphase cytogenetics to investigate the nature of chromosomal aberrations in frozen synovial sarcoma tissue samples. Two‐colour fluorescence in situ hybridization (FISH) was performed using probes specific for the centromeres of chromosome X or 18, along with yeast artificial chromosome probes corresponding to the distinct breakpoint regions on Xp. One monophasic epithelial and two monophasic fibrous synovial sarcomas showed an OATL2‐associated brea...
The ability to direct transgene expression to astrocytes has become increasingly important as the... more The ability to direct transgene expression to astrocytes has become increasingly important as the roles for these cells continue to expand. Promoters consisting of the 5′‐flanking region of the human or mouse glial fibrillary acidic protein (GFAP) gene have generally proved satisfactory. However, a more powerful promoter would be advantageous for several applications, such as expression of dominant negative RNAs or proteins, or for gene therapy. We investigated the possibility of increasing the transcriptional activity of the human GFAP promoter by inserting into it one or three additional copies of putative GFAP enhancer regions. The promoters enhanced with three additional copies gave 75‐fold higher LacZ expression levels upon plasmid transfection into GFAP‐expressing U251 cells than the parental gfa2 promoter. Surprisingly, in a transgenic mouse model, the enhanced promoters resulted in no or only very low expression of marker genes, probably caused by toxicity. When various cell...
Molecular Therapy
In order to improve the efficacy of first generation adenoviral vectors for glioma treatment, we ... more In order to improve the efficacy of first generation adenoviral vectors for glioma treatment, we designed conditionally replicating viruses in which the E1A gene is under the control of the promoter (gfa2) of the glia specific intermediate filament Glial Fibrillary Acidic Protein (GFAP) and similar constructs containing additional glial specific enhancers.For a control variant of this virus the gfa2 derived promoters (the normal gfa2, gfa2(B)3 and gfa2(ABD)3 promoters resp.) followed by a beta galactosidase reporter gene were cloned into the pShuttle plasmid, in an orientation inverse to wildtype E1 expression. Viral vectors were produced according to the Ad-Easy protocol and viruses giving rise to blue plaques upon infection of U251 but not A549 cells were selected and grown to high titers. To check the specificity and efficacy of the promoter constructs, a panel of human cell lines (glial and non-glial) was infected with the gfa2 (triple enhancer) promoter-beta galactosidase vectors. An adenoviral vector containing a CMV-beta galactosidase cassette was included to determine whether cell entry effects play a role. Expression of beta galactosidase was evaluated both in situ using X-gal staining and quantitatively using ONPG staining. To create the conditionally replicating variant the beta galactosidase gene was replaced by the Ad5 E1 region.Results: A panel of 11 glial cell lines was selected based on GFAP positivity on Western blots and immunohistochemistry. Within this panel GFAP positivity correlated well with beta galactosidase expression upon infection with the beta galactosidase viruses. ONPG values of the Ad5-gfa2-ABD3-LacZ virus in GFAP positive cell lines approached the levels of the Ad5-CMV-LacZ. In 2/14 non-glial cell lines, which were all negative for GFAP protein expression, however, beta galactosidase expression was found with the Ad5-gfa2-LacZ and Ad5-gfa2(ABD)3-LacZ viruses in the same range as for the GFAP positive glial cell lines. After infection with the Ad5-gfa2-B3-LacZ virus, considerable beta galactosidase expression was only seen in one of the 14 non-glial cell lines tested. We discuss possible explanations for and consequences of this GFAP promoter activity in adenoviral context after infection of some non-glial tumor cell lines. Both LacZ viruses containing promoters with extra glial enhancers gave rise to higher ONPG values than the Ad5-gfa2-LacZ virus.Preliminary results indicate that conditionally replicating adenovirus in which E1A is regulated by the gfa2-B3 promoter replicate better on the GFAP positive U251 cell line than on the GFAP negative Hep2 cells.Conclusion: The gfa2 promoter constructs are highly active in an adenoviral context and have retained their specificity, with the exception of some non-glial tumour cell lines. Ad5-gfa2-B3-E1 conditionally replicating viruses have been grown to high titers and are currently being tested for replication in vitro and for effect on xenograft growth.
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2006
The overall median survival of patients with a malignant glioma is <1 y. Because malignant gli... more The overall median survival of patients with a malignant glioma is <1 y. Because malignant gliomas rarely metastasize outside the skull, locoregional treatment strategies, such as gene therapy, are under investigation. Recently, convection-enhanced delivery (CED) has been presented as a method to improve delivery of large molecules. The goal of this study was to evaluate whether CED improves intratumoral delivery of adenoviral vectors and compare it with single injection (SI) and multiple injection (4x, MI). A replication-deficient adenoviral vector encoding the herpes simplex virus thymidine kinase (HSV-tk) and the human somatostatin receptor subtype 2 (sst(2)) was administered into nude mice bearing subcutaneous U87 xenografts. Tumors were injected with 1.5 x 10(9) plaque-forming units of Ad5.tk.sstr by CED, SI, or MI. Three days later, [(99m)Tc-N(4)(0-1),Asp(0),Tyr(3)]octreotate ((99m)Tc-Demotate 2) was injected intravenously to monitor the virus-induced sst(2) expression. gam...
Brain : a journal of neurology, 2003
Paraneoplastic cerebellar degeneration (PCD) is a heterogeneous group of disorders characterized ... more Paraneoplastic cerebellar degeneration (PCD) is a heterogeneous group of disorders characterized by subacute cerebellar ataxia, specific tumour types and (often) associated antineuronal antibodies. Nine specific antineuronal antibodies are associated with PCD. We examined the relative frequency of the antineuronal antibodies associated with PCD and compared the neurological symptoms and signs, associated tumours, disability and survival between groups of PCD with different antibodies. Also, we attempted to identify patient-, tumour- and treatment-related characteristics associated with functional outcome and survival. In a 12-year period, we examined >5000 samples for the presence of antineuronal antibodies. A total of 137 patients were identified with a paraneoplastic neurological syndrome and high titre (> or =400) antineuronal antibodies. Fifty (36%) of these patients had antibody-associated PCD, including 19 anti-Yo, 16 anti-Hu, seven anti-Tr, six anti-Ri and two anti-mGlu...
Nederlands tijdschrift voor geneeskunde, Jan 18, 1998
Paraneoplastic neurological syndromes are believed to result from ectopic expression of onconeura... more Paraneoplastic neurological syndromes are believed to result from ectopic expression of onconeural antigens by tumours. The resulting immune response is not only directed against the tumour but also cross-reacts with the same or similar antigens in the nervous system. The immune response generates high titred autoantibodies that are associated with specific tumours and neurological syndromes. Paraneoplastic autoantibodies help diagnose neurological syndromes and help direct the search for an underlying tumour. In paraneoplastic syndromes, the course of the tumour is relatively mild. Detection of the autoantibodies might lead to early diagnosis and immunomodulation and anti-tumour treatment before irreversible neuronal cell loss and deficits set in.
Oncogene, 1993
In a previous study we localized the synovial sarcoma-associated t(X;18)(p11;q11) breakpoint with... more In a previous study we localized the synovial sarcoma-associated t(X;18)(p11;q11) breakpoint within the ornithine aminotransferase-like 1 (OATL1) cluster on the X chromosome. This localization was delineated from both somatic cell hybrid and fluorescence in situ hybridization (FISH) analysis of patient material, using OAT-specific cDNA and YAC probes. Simultaneously, Knight et al. (1992, Mol. Hum. Genet, in press) mapped this same breakpoint in their patient material adjacent to the more proximal OATL2 region on the X chromosome. Here we report the analysis of two additional tumors and demonstrate that again in these cases the chromosomal break occurs within the OATL1 cluster. In order to further specify the breakpoint, we subcloned the OATL1 YAC (no. 2) into cosmids. At least one of these cosmids (0.38) hybridizes to sequences that bracket the translocation breakpoint, as demonstrated by both Southern blot and FISH analysis. These observations confirm and substantiate our previous ...
Molecular and Cellular Endocrinology, 2008
HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires
Suicide Gene Therapy, 2003
... Hum. Gene Ther. 7, 7987. 29. Roy, S., Shirley, PS, McClelland, A., and Kaleko, M. (1998) Cir... more ... Hum. Gene Ther. 7, 7987. 29. Roy, S., Shirley, PS, McClelland, A., and Kaleko, M. (1998) Circumvention of immu-nity to the adenovirus major coat protein hexon. J. Virol. 72, 68756879. 30. ... Adv. Drug Delivery Rev. 27, 6781. 68. Khuri, FR, Nemunaitis, J., Ganly, I., et al. ...
New England Journal of Medicine, 2000
Neuroscience Research, 1998
We have already reported that non-inflammatory stress induced peripheral IL-6 production is, at l... more We have already reported that non-inflammatory stress induced peripheral IL-6 production is, at least partly, mediated by enteric endotoxin (LPS). In the present study, to clarify the major source of peripheral IL-6 induced by non-inflammatory stress, blood samplings were performed via chronically implanted cannulations into the hepatoportal vein (HV), the vena cava inferior (VCI) and/or the vena cava superior (VCS) in the electrical foot shock (FSS) rat model (ImA, 11 times, each one min). The results were as follows; (1) Plasma IL-6 levels in the HV were higher than those in the VCS (3Omin after FSS). (2) The peak of plasma IL-6 in the HV (30min) were earlier than that in the VCS (60 min). (3) Plasma IL-6 levels in the lower portion of the VCI (below the renal branch) were lower than that in the VCS (0 and 30min). (4) On the contrary, plasma IL-6 levels in the higher portion of the VCI (nearby the hepatic branch) were not differ from those in the VCS. (5)
Neuroscience Letters, 2009
Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC... more Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC) showed high titer immunohistochemical staining of the axon initial segment (AIS) on rat and human brain sections. EM studies showed that the antigen was localized in close proximity of the microtubules in the AIS. Double labeling experiments and absence of staining at the nodes of Ranvier excluded the previously identified betaIV spectrin as autoantigen. Screening a rat hippocampal cDNA library resulted in the isolation of ubiquitin-conjugating enzyme E2E1 (UBE2E1). However, blocking and elution experiments excluded UBE2E1 as the AIS autoantigen.
Molecular Therapy, 2005
targeting adaptors could provide viral retargeting to appropriate receptors. As an example of the... more targeting adaptors could provide viral retargeting to appropriate receptors. As an example of the practical utility of such viral modifications, we investigated if biotinylated fiber could target infection to the EGF receptor (EGFR), overexpressed on cancer cell lines via molecular bridge with EGF-Streptavidin. Our findings indicate that mbAd complexed with EGF-Streptavidin (mbAd/EGF) achieved 20-fold increase of infection efficiency on A431 cells overexpressing EGFR compared to untargeted mbAd. Increase in transduction efficiency of EGFR-retargeted mbAd was also achieved on other cancer cell lines. Enhanced infectivity of fiber-mosaic Ad provided by EGF-related targeting was not abolished by blocking with recombinant Ad5 knob, while wild type infection at the same experimental conditions was blocked. We also investigated in vivo targeting potential of mbAd complexed with EGF-Streptavidin on two different models. First, we utilized mouse model of locoregional EGFR-positive ovarian xenografts. Expression of reporter gene in ovarian tumor nodules was 5 times higher with EGFR-retargeted mbAd compared to the virus without adaptor. Secondly, retargeting capacity of mbAd/EGF was tested on mouse "transient transgenic" model, which is accomplished by preinfection of hCAR transgenic mice with AdfltEGFR and intended to transiently express hEGFR in the mouse lung vasculature. mbAd/EGF achieved 7-fold higher expression in the lung compared to mbAd without adaptor and wt Ad. Moreover, lung/liver ratio of gene expression for retargeted mbAd was also increased in both in vivo models tested. Thus, we demonstrated that additional fiber in fiber-mosaic could be used for biotinylation and this modification in combination with appropriate adaptors can be successfully exploited for adenoviral retargeting strategies.
Molecular Therapy, 2004
Although successful in many rodent models of malignant gliomas, results of clinical adenoviral ge... more Although successful in many rodent models of malignant gliomas, results of clinical adenoviral gene therapy trials using replication deficient vectors have been disappointing. One of the explanations of the discrepancy between results in animal models and clinical trials is the low expression of the Coxsackie Adenovirus receptor (CAR), the main high affinity receptor for binding of adenovirus serotype 5, on primary glioma cells as opposed to established glioma cell lines. To obtain improved adenoviral vectors, we made use of the differential tropism of the naturally occurring 51 human adenoviral serotypes to test the hypothesis that adenoviral vectors, binding to other receptors than CAR, would result in improved infection of primary glioma cells.Design/Methods: Thirty-four of the fifty-one wildtype adenoviral serotypes were collected and grown. Using PCR, sequences coding the adenoviral protein fiber were subcloned into an adenovirus serotype 5 background. All vectors contained the luciferase marker gene. A total of 18 recombinant adenoviral chimeras were constructed and grown on PER.C6 producer cells (Ad5.Fibxx, where xx indicates any of the other serotypes). Fresh glioma tissue from 11 patients was mechanically dissected and treated with trypsin. Early passage glioma cells (6-10) were incubated with Ad5.Fibxx at a multiplicity of infection (m.o.i.) of 1000 and 5000 viral particles (vp) per cell for 2h. Forty-eight hrs after infection, cells were lysed, luciferin was added and relative light units (RLU) were measured and compared to Ad5. CD46 expression on the tumor cells was examined by FACS analysis. In addition, early passage glioma cells were incubated with 34 of the existing 51 wildtype serotypes at m.o.i. 10 and time to CPE was monitored.Results: In 10 out of 11 primary glioma lines, we could identify serotypes that entered the cells more efficiently than Ad5 resulting in higher luciferase expression levels. The Ad5.Fibxx with fibers derived from B-group serotypes (Ad5.Fib11; Ad5.Fib16; Ad5.Fib35; Ad5.Fib50) infected glioma cells 5-100 times better than Ad5.Fib5. Also the F-group Ad5.Fib40s infected glioma cells better than Ad5.Fib5. The results of Ad5Fib35 were confirmed using another batch of the virus. In an additional experiment, infection with Ad5.Fib35 and Ad35.luc resulted in similar luciferase expression levels. CD46 (cell surface receptor for most B-group adenoviruses) was expressed on 6-46% of the low passage glioma tumor cells, but on 75-100% on 5/5 established cell lines.None of the replication competent wildtype serotypes gave significantly quicker CPE than wt Ad5 on five primary glioma cellines.Conclusions: Adenoviral vectors encoding fibers derived from serotypes 11, 16, 35, 50 (B-group) and 40 (F-group) are promising for glioma gene therapy. The Ad5 backbone remains a good choice for the construction of (conditionally) replicating adenoviral vectors for gene therapy of glioma.
Molecular Therapy, 2005
treatment of chronic viral hepatitis. In summary, these mouse model systems offered opportunities... more treatment of chronic viral hepatitis. In summary, these mouse model systems offered opportunities to test in vivo efficacy of HBx-specific siRNAs for control of HBV replication.