Papers by Pedro Lowenstein
Background: High-grade gliomas are fatal with universally poor prognosis. Initiation of effective... more Background: High-grade gliomas are fatal with universally poor prognosis. Initiation of effective cancer immune responses requires functional immune cells, particularly afferent antigen-presenting cells, which are typically absent from the brain parenchyma. To overcome this limitation, two adenoviral vectors expressing HSV1-TK and Flt3L were combined to target human gliomas. This first-in-human trial assessed safety, cytotoxicity, and recruitment of immune cells to the brain, in support of a future phase 1b/2 clinical trial. Methods: Treatment-naive high-grade glioma adult patients received injections of adenoviral vectors expressing HSV1-TK and Flt3L to the tumor bed, following maximal safe resection, at six escalating doses ranging from a total of 1.1x1010 to a maximum of 2x1011 viral particles. This was followed by two 14-day courses of valacyclovir and standard upfront chemoradiation. Key inclusion criteria were age between 18 to 75, KPS≥70, and treatment-naive possible high-gra...
Neoplasia, 2011
We have demonstrated that modifying the tumor microenvironment through intratumoral administratio... more We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6 −/− mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6 −/− mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in
A Companion to Genethics
ABSTRACT
Apoptosis Genes, 1998
... 3.1 Ischaemia Various types of brain ischaemia have been shown to trigger apoptosis (Choi, 19... more ... 3.1 Ischaemia Various types of brain ischaemia have been shown to trigger apoptosis (Choi, 1996 ... expressed through an adenoviral vector was also useful in a neonatal rat model of excitotoxicdamage, another putative mechanism underlying ischemic damage (Hagan et ...
Trends in Immunology, 2002
Opinion 'Continuous forms are the perfect ones. Usually, a distinction is created between support... more Opinion 'Continuous forms are the perfect ones. Usually, a distinction is created between supporting and supported elements, which is obviously wrong; there are those that both support and are supported. This distinction creates a degree of imperfection.' (Antoni Gaudi, 1852-1926). 'Constraints upon evolutionary change may be ordered into at least two categories. All evolutionists are familiar with phyletic constraints... Developmental constraints, a subcategory of phyletic restrictions, may hold the most powerful rein of all over possible evolutionary pathways. In complex organisms, early stages of ontogeny are remarkably refractory to evolutionary change, presumably because the differentiation of organ systems and their integration into a functioning body is such a delicate process, so easily derailed by early errors with accumulating effects...' [1]
PLoS ONE, 2011
Background: Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purp... more Background: Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD). Methodology/Principal Finding: A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replicationcompetent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35-45% of splenic T cells were transduced by Ad-RGD. Conclusions: Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.
Molecular Therapy, 2004
We tested the activity of the dopaminergic neuron differentiation factor sonic hedgehog, its down... more We tested the activity of the dopaminergic neuron differentiation factor sonic hedgehog, its downstream transcription factor target Gli-1, and an orphan nuclear receptor, Nurr-1, necessary for the induction of the dopaminergic phenotype of nigrostriatal neurons, in an in vivo model of nigrostriatal neurodegeneration. Our preliminary experiments demonstrated that all three constructs expressed the proper molecules and that these had the predicted biological activities in vitro. We expressed the N-terminal of sonic hedgehog (ShhN) and the Gli-1 and Nurr-1 entire coding regions from highly purified, and quality controlled, replication-defective adenoviral vectors injected into the brains of rats and used the dopaminergic growth factor GDNF as a positive control. The neurotoxin 6-hydroxydopamine was used to lesion the nigrostriatal dopaminergic innervation; RAd-ShhN and RAd-Gli-1 protected dopaminergic neuronal cell bodies in the substantia nigra, but not axonal terminals in the striatum, from 6-OHDA-induced cell death, while RAd-Nurr-1 was ineffective in protecting either cell bodies or axons. RAd-GDNF was able to protect both the dopaminergic cell bodies and the striatal axon terminals. Our results establish for the first time, to the best of our knowledge, that gene transfer of ShhN and one of its target transcription factors can selectively protect dopaminergic nigrostriatal neuronal cell bodies from a specific neurotoxic insult. Selective protection of nigrostriatal dopaminergic cell bodies by the differentiation factor ShhN and the transcription factor Gli-1 was achieved in a neurotoxic model that eliminates more than 70% of the nigral neurons under consideration. Differentiation and transcription factors can thus be used for the treatment of neurodegeneration by gene therapy.
Molecular Therapy, 2005
Jump to main content; Jump to navigation; nature.com homepage; Publications AZ index; Browse by s... more Jump to main content; Jump to navigation; nature.com homepage; Publications AZ index; Browse by subject. My account; Submit manuscript; Register; Subscribe; ASGCT. Login. Molecular Therapy homepage Search This journal Advanced search. ...
Journal of Endocrinology, 2002
Gene therapy for pituitary disease requires evaluation for safety as well as efficacy. We have re... more Gene therapy for pituitary disease requires evaluation for safety as well as efficacy. We have reported results of adenovirus-mediated gene transfer using the sheep as a large animal model that allows longitudinal evaluation of hormone secretion and have confirmed high levels of transgene expression up to 7 days after direct stereotaxic injection into the pituitary gland. Here we report the results of detailed histological examination of the pituitary glands from animals injected with two recombinant adenoviruses expressing the beta-galactosidase marker gene, or with saline vehicle to control for the potential tissue-disruptive effect of the injection volume itself. Pituitaries injected with saline showed no evidence of inflammatory response apart from occasional minor foci of apoptosis. In all other respects they were indistinguishable from normal uninjected control pituitary glands. Glands injected with recombinant adenoviruses containing either the hCMV-beta-gal or the hPRL-beta-...
Current Opinion in Pharmacology, 2004
The choice of vectors, transgenes, regulatory elements, delivery approaches and the capacity to t... more The choice of vectors, transgenes, regulatory elements, delivery approaches and the capacity to transduce the appropriate target cell type all influence the effectiveness of gene therapy for neurological diseases. Furthermore, even if many strategies are sound and effective in experimental animals, issues relating to side effects of gene therapy, longevity of therapeutic transgene expression and diffusion throughout the brain can limit the clinical potential of gene therapy. During the past 12-18 months, there have been significant advances in the following areas: the capacity to target vectors to predetermined cells types; the development of gene therapy approaches for the treatment of dominant inherited and neurodegenerative diseases; the capacity to achieve systemic delivery of viral vectors to the brain; and the development of viral vectors to model neurological diseases.
Nature Communications
Intra-tumoral heterogeneity is a hallmark of glioblastoma that challenges treatment efficacy. How... more Intra-tumoral heterogeneity is a hallmark of glioblastoma that challenges treatment efficacy. However, the mechanisms that set up tumor heterogeneity and tumor cell migration remain poorly understood. Herein, we present a comprehensive spatiotemporal study that aligns distinctive intra-tumoral histopathological structures, oncostreams, with dynamic properties and a specific, actionable, spatial transcriptomic signature. Oncostreams are dynamic multicellular fascicles of spindle-like and aligned cells with mesenchymal properties, detected using ex vivo explants and in vivo intravital imaging. Their density correlates with tumor aggressiveness in genetically engineered mouse glioma models, and high grade human gliomas. Oncostreams facilitate the intra-tumoral distribution of tumoral and non-tumoral cells, and potentially the collective invasion of the normal brain. These fascicles are defined by a specific molecular signature that regulates their organization and function. Oncostreams...
Molecular and Cellular Endocrinology, 1998
Procorticotrophin-releasing hormone (proCRH) is expressed mainly in the hypothalamus and in the p... more Procorticotrophin-releasing hormone (proCRH) is expressed mainly in the hypothalamus and in the placenta, where it undergoes tissue-specific endoproteolysis. Our results show that within stably transfected AtT20/D16V cells proCRH is cleaved to generate two fragments of approximately 8 and 3 kDa which could account for proCRH(125-194) and proCRH(125-151), respectively, and a 4.5 kDa product which could account for mature IR-CRH(1-41). The immunofluorescence staining patterns for IR-CRH and IR-ACTH and their response of secretagogues indicate targeting of proCRH and POMC to the secretory pathway in transfected AtT20 cells. In this work, we have used a unique set of specific RIAs and IRMAs to the full length POMC and proCRH molecules and several products of endoproteolytic processing to assess if they could be released differentially in response to stimulation. Although the release of both IR-ACTH and IR-CRH peptides from transfected AtT20 cells is stimulated in response to exposure to high potassium stimulation (51 mM KCl/SmM CaCl2), the sorting index (SI) suggests that mature ACTH is sorted to the regulated secretory pathway 2.1-fold more efficiently than mature CRH(1-41). Mature ACTH is also sorted to the regulated secretory pathway 9-fold more efficiently than IR-proCRH(125-151). Also, mature CRH(1-41) is sorted to the regulated secretory pathway 3-fold more efficiently than IR-proCRH(125-151). These results therefore indicate that the intracellular mechanisms for the storage and release of POMC, proCRH and their endoproteolytic products differ and would sustain the hypothesis that within mammalian peptidergic cells, different biologically active peptides originating from the same or different precursor molecules, could be differentially released in response to specific stimuli. This would give these cells the capacity to finely regulate neurotransmitter release in response to environmental and physiological demands.
Molecular and Cellular Endocrinology, 1998
In this paper we demonstrate the use of recombinant viral vectors derived from herpes simplex vir... more In this paper we demonstrate the use of recombinant viral vectors derived from herpes simplex virus type 1 (HSV1) to transfer reporter genes in vitro into rat anterior pituitary cells grown in primary cultures and the anterior pituitary tumour cell lines GH3 and AtT20. The three vectors used were, tsK/beta-galactosidase (beta-gal), tsK/CRH and tsK/TIMP, the corresponding transgene products respectively being E. coli beta-gal, pre-procorticotropin releasing hormone (ppCRH), and the chimeric protein TIMP/Thy1 (tissue inhibitor of metalloproteinases (TIMP)/linked to the carboxy terminus of Thy1 which confers the addition of a glycolipid glycosyl-phosphatidylinositol anchor in the ER). Double labelling immunofluorescence experiments to detect reporter proteins and transduced cell types indicated that the three vectors could transfer and express the reporter genes in normal and tumour anterior pituitary cells. Virus infection of pituitary cells was characterised, and it was shown that infection with tsK/beta-gal at multiplicities of infection (MOI)=10, 100% of tumour and non-endocrine anterior pituitary cells expressed beta-gal, whereas 75% endocrine anterior pituitary cells expressed the transgene. Long-term expression studies after infection with tsK/beta-gal indicated that anterior pituitary cells in primary cultures expressed the transgene for significant longer periods than tumour anterior pituitary cells. Growth arrest by serum starvation markedly decreased the frequency of transgene expression in anterior pituitary cells following infection with tsK/beta-gal. Transgenic products expressed from tsK were targeted to their correct intracellular domain in both anterior pituitary cells in primary cultures and in pituitary tumour cell lines. We conclude that transgenes can be delivered into anterior pituitary cells in primary culture and pituitary tumour cell lines using tsK derived HSV1 vectors. The prospect of employing viral vectors to transfer genes into endocrine cells opens up the potential exploration of various molecular aspects of pituitary cell function both in vitro and in vivo, as well as the use of gene transfer into the pituitary for potentially therapeutic applications, such as the treatment of pituitary tumours.
Molecular and Cellular Endocrinology, 1995
To investigate the intracellular localisation and biological activity of procorticotrophin-releas... more To investigate the intracellular localisation and biological activity of procorticotrophin-releasing hormone (proCRH), we have established stably transfected CHO-Kl cells expressing the rat pre-proCRH cDNA. Using immunoblot analysis of cell lysates of transfected CHO-Kl cells, we detected a major CRH immunoreactive band with an apparent molecular weight of approximately 19 kDa. This 19 kDa band could account for full length proCRH molecule which has not undergone post-translational modifications. Metabolic labelling followed by immunoprecipitation, SDS-PAGE and autoradiography indicated that no endoproteolytic processing of proCRH takes place within the transfected CHO-KI cells. Immunofluorescence staining localises the CRH precursor to both the cytoplasm and to the nucleus in transfected CHO-Kl cells. This result was confirmed using subcellular fractionation techniques on radiolabelled CHO-Kl cells expressing immunoreactive CRH. A major CRH-immunoreactive band of 19 kDa was detected both in the microsomal and secreted fractions, indicating the presence of proCRH within the secretory pathway of these cells. This was also evident in the nuclear fraction, therefore confirming the nuclear localisation of proCRH. Analysis of DNA concentration, ceil number and DNA synthesis showed that stably transfected CHO-Kl cells expressing proCRH have a higher proliferation and DNA synthesis rate than wildtype CHO-Kl cells or CHO-Kl cells transfected with pEE14 alone. Our results therefore suggest a mitogenic role for the intact proCRH molecule within CHO-Kl cells. Furthermore, treatment of mouse corticotrophic tumour cells (AtT20/D16-16) with conditioned medium from transfected CHO-Kl cells expressing proCRH, stimulated both DNA synthesis and cell proliferation above basal levels. Our results constitute the first reported direct evidence of a mitogenic role for proCRH acting on a corticotrophic cell population.
Journal of Virology, 2001
The effect of combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inh... more The effect of combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H) on the infectivity of foot-and-mouth disease virus (FMDV) in cell culture has been investigated. Related FMDV clones differing up to 106-fold in relative fitness in BHK-21 cells have been compared. Systematic extinction of intermediate fitness virus was attained with a combination of FU and G but not with the mutagen or the inhibitor alone. Systematic extinction of high-fitness FMDV required the combination of FU, G, and H. FMDV showing high relative fitness in BHK-21 cells but decreased replicative ability in CHO cells behaved as a low-fitness virus with regard to extinction mutagenesis in CHO cells. This confirms that relative fitness, rather than a specific genomic sequence, determines the FMDV response to enhanced mutagenesis. Mutant spectrum analysis of several genomic regions from a preextinction population showed a statistically ...
Journal of Virology, 2005
Enhanced mutagenesis may result in RNA virus extinction, but the molecular events underlying this... more Enhanced mutagenesis may result in RNA virus extinction, but the molecular events underlying this process are not well understood. Here we show that 5-fluorouracil (FU)-induced mutagenesis of the arenavirus lymphocytic choriomeningitis virus (LCMV) resulted in preextinction populations whose consensus genomic nucleotide sequence remained unaltered. Furthermore, fitness recovery passages in the absence of FU, or alternate virus passages in the presence and absence of FU, led to profound differences in the capacity of LCMV to produce progeny, without modification of the consensus genomic sequence. Molecular genetic analysis failed to produce evidence of hypermutated LCMV genomes. The results suggest that low-level mutagenesis to enrich the viral population with defector, interfering genomes harboring limited numbers of mutations may mediate the loss of infectivity that accompanies viral extinction.
Journal of Virology, 2002
Intracranial administration of adenovirus vectors elicits rapid, capsid-mediated dose-dependent b... more Intracranial administration of adenovirus vectors elicits rapid, capsid-mediated dose-dependent brain inflammation. The mechanisms through which adenovirus capsids trigger inflammation in the brain remain unknown. We determined whether adenovirus interaction with the primary and secondary cell surface receptors for infection (CAR and αv integrins) was necessary to trigger acute adenovirus-mediated brain inflammation, and, furthermore, whether capsid mutations that abrogated CAR and integrin binding altered vector tropism in the brain. Vectors ablated for CAR binding, but retaining integrin binding function, transduced equivalent areas of brain compared to vectors with wild-type capsids; however, vector tropsim was dramatically altered. Vectors with wild-type capsids predominantly transduced oligodendrocytes, whereas mutation of the fiber protein to ablate CAR binding resulted in a loss of oligodendrocyte transduction and a consequent redirection of transduction to neurons and other ...
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Papers by Pedro Lowenstein