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Biomedical subjects

B J Roessler

Publications and source records attributed to B J Roessler.

At least 37 records · Page 2Linked to original sources
PubMed1997

Perifollicular transgenic expression of human interleukin-1 receptor antagonist protein following topical application of novel liposome-plasmid DNA formulations in vivo.

Expression plasmid DNA for the human interleukin-1 receptor antagonist (IL-1ra) protein was formulated with nonionic:cationic (NC) liposomes or phosphatidylcholine:cationic (PC) liposomes and applied to the auricular skin of hamsters in single- and multiple-dose protocols. Confocal microscopy identified delivery of plasmid DNA proximal to perifollicular cells, and successful transfection of perifollicular cells was identified by immunohistochemistry and ELISA. Skin treated for 3 days with the NC liposomes had statistically significant levels of transgenic IL-1ra present for 5 days post-treatment. Expression of transgenic IL-1ra was specific to areas of skin treated with NC liposomes but not PC liposomes. The results indicate that the NC liposomes can deliver expression plasmid DNA to perifollicular cells and mediate transient transfection in vivo.

Administration, Topical↗
PubMed1997

The absence of accessible vitronectin receptors in differentiated tissue hinders adenoviral-mediated gene transfer to the intestinal epithelium in vitro.

PURPOSE: Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished. METHODS: Caco-2 cell monolayers were chosen as an in vitro model for the differentiated intestinal epithelium. Fluorescence-labeled adenoviral particles were used for binding studies to cell surfaces. Internalization receptors for adenoviral uptake were detected by a fluorescence-labeled vitronectin antibody. Gene expression was studied by using the beta-galactosidase reporter gene. All experiments were done on undifferentiated and differentiated Caco-2 cells. Furthermore, adenoviral particles were allowed to bind to differentiated Caco-2 monolayers followed by a trypsinization step that disintegrates the monolayers and result in a cell suspension. Gene expression was tested after reseeding the cells into dishes. RESULTS: The results from adenoviral binding studies, vitronectin immunofluorescence detection and gene expression are in good agreement and indicate that virion binding as well as the expression of internalization receptors almost disappear in fully differentiated cells. Nonetheless, adenoviral binding to differentiated monolayers seems to be sufficient to cause up to 53% gene expression, but only if internalization of the vector can be induced by disintegrating the monolayers and releasing free vitronectin receptors. CONCLUSIONS: These findings indicate that gene transfer to the intestinal epithelium utilizing adenoviral vectors is poor and ineffective, because of the lack of sufficient internalization receptors. If these receptors can be exposed in differentiated epithelium, transduction can be made more efficient. Alternatively, a viral vector must be developed whose uptake mechanism is independent of integrin receptor expression like the enteral virus Ad40, or Ad5 could be conjugated to ligands that trigger viral internalization by receptor-mediated endocytosis.

Adenoviridae↗
PubMed1996

Systemic delivery of the interleukin-1 receptor antagonist protein using a new strategy of direct adenoviral-mediated gene transfer to skeletal muscle capillary endothelium in the isolated rat hindlimb.

Current gene therapy strategies using adenoviral vectors to target the lung or liver have been complicated by an acute inflammatory response that can result in loss of transgene expression as well as tissue injury and necrosis. Skeletal muscle comprises 40% of total body weight; it possesses a high density, accessible capillary network that is resistant to injury and thus may be a logical target for adenoviral vectors. We hypothesized that adenoviral transduction of the rat skeletal muscle capillary bed during vascular isolation would achieve efficient gene transfer sufficient to achieve systemic serum levels of a recombinant protein without significant tissue injury. During vascular isolation of the hindleg, a replication-incompetent adenovirus (Ad) encoding for either the marker gene, human placental alkaline phosphatase (hpAP), or interleukin-1 receptor antagonist (IL-1ra) was infused and subsequently flushed from the circulation after a 30-min dwell period. Gene transfer over a 10(9)-10(12) particle/ml range to the gastrocnemius capillary endothelium and muscle fibers was highly efficient and titer-dependent, reaching maximum transduction rates of 71 +/- 7% and 25 +/- 5%, respectively, 5 days after gene transfer (n = 3-8 rats/group, p < 0.05). hpAP transgene expression was barely detectable at 14 days. No significant tissue injury or necrosis of the skeletal muscle was observed at 5 and 14 days, and distant organ gene transfer was minimal or absent. Gastrocnemius muscle from rats (n = 4) given Ad-IL-1ra had 241 +/- 66 pg IL-1ra/mg protein at 5 days, while those given Ad-hpAP, negative control (n = 3) had 35 +/- 14 pg IL-1ra/mg protein (p < 0.05). Ad-IL-1ra rats (n = 4) had serum levels of 185 +/- 20 pg/ml IL-1ra at 5 days whereas Ad-hpAP control rats (n = 5) had no IL-1ra detectable (p < 0.0001). Athymic rats given Ad-IL-1ra (n = 6) had serum levels of 493 +/- 62 pg/ml IL-1ra 14 days after transduction, and IL-1ra was detected for up to 98 days. Sera from Ad-IL-1ra athymic rats significantly inhibited IL-1 beta-induced (1 ng/ml) prostaglandin E2 (PGE2) production from cultured endothelial cells by 82 +/- 2% (p < 0.001). Thus, this gene transfer strategy is the first to result in substantial transduction of both skeletal muscle capillary endothelium and fibers, sufficient to achieve pharmacologic levels of IL-1ra. Although no acute tissue injury or necrosis was observed, persistence of transgene expression in athymic rats suggests that loss of expression in normal rats was by an immune-mediated mechanism.

Adenoviridae↗
PubMed1996

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression in the central nervous system of HPRT-deficient mice following adenoviral-mediated gene transfer.

In this study we show that recombinant adenovirus can augment hypoxanthine-guanine phosphoribosyltransferase (HPRT) levels in the central nervous system (CNS) of HPRT-deficient mice. Recombinant adenovirus containing the cDNA for rat HPRT (rHPRT) expressed from the Rous sarcoma virus LTR (RSV LTR) was constructed (AdRSVrHPRT). AdRSVrHPRT was injected into the right caudate nucleus of 7-week-old HPRT-deficient mice. Brains were analyzed for gene transfer, transgene expression and function by DNA PCR, in situ RNA hybridization, and enzyme bioactivity. The results show that rHPRT cDNA delivered by an adenoviral vector can augment HPRT levels in brain tissue and documents the utility of gene transfer to restore HPRT activity in an HPRT-deficient CNS.

Adenoviridae↗
PubMed1996

Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.

Degradable matrices containing expression plasmid DNA [gene-activated matrices (GAMs)] were implanted into segmental gaps created in the adult rat femur. Implantation of GAMs containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells (granulation tissue) growing into the gap. Implantation of a GAM containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulted in a biological response of new bone filling the gap. Finally, implantation of a two-plasmid GAM encoding bone morphogenetic protein-4 and the parathyroid hormone fragment, which act synergistically in vitro, caused new bone to form faster than with either factor alone. These studies demonstrate for the first time that repair cells (fibroblasts) in bone can be genetically manipulated in vivo. While serving as a useful tool to study the biology of repair fibroblasts and the wound healing response, the GAM technology may also have wide therapeutic utility.

Animals↗
PubMed1996

Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.

Loss of ganglion cells is a common and irreversible complication of hair cell loss in the cochlea. Gene transfer could potentially be used to prevent this neuronal degeneration and other pathologies in the cochlea. Human adenoviruses should provide a feasible gene transfer vehicle for transducing the quiescent cochlear neurons and organ of Corti epithelium. We now describe in vivo experiments in which a replication-deficient adenoviral vector, Ad.RSVntlacZ was injected into the perilymphatic fluid of six normal guinea pigs. Postoperative recovery of animals was complete. Inner ear tissues were assessed for histology and for presence of lacZ-positive cells 1 or 2 weeks after the injection. A large number of blue (lacZ-positive) cells were observed in the neural, epithelial and connective tissues of the cochlea. In four ears spiral ganglion cell infection exceeded 50%, throughout the length of the cochlear spiral. No major pathology was detected in the organ of Corti and other cochlear tissues, and no infection was present in the vestibular tissues or the contralateral cochlea. Immunocytochemical assessment of T cells revealed an increased in the number of lymphocytes in the connective tissue lining the perilymphatic spaces. We conclude that efficient gene transfer into multiple types of cochlear cells in vivo can be achieved without major morphological signs of pathology or toxicity.

Adenoviruses, Human↗
PubMed1996

HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: in vitro-in vivo correlation with permeability data from rats and humans.

The diverse secretory and absorptive functions of the intestinal epithelium are conducted by a mixed population of absorptive cells and mucus-producing goblet cells as the major cell types. In order to approach the main characteristics in an in vitro model, a coculture system of absorptive Caco-2 cells and mucus-secreting HT29-MTX cells was developed and the permeability of a range of different drugs was tested. Variable goblet cell frequency can be achieved, preserving a significant barrier to drug transport and maintaining the differentiated features of both cell types. Absorption rates for actively transported drugs are rather underestimated in the cell culture model when compared to in vivo data. However, a good correlation with fraction absorbed in humans was attained separating the range of passively transported drugs into two groups of well-absorbable compounds with Peff > or = 10 x 10(-6) cm/s and drugs that are absorbed 40-70% with Peff = 0.1-1 x 10(-5) cm/s. A permeability of Peff < 0.1 x 10(-5) cm/s is suggested for low absorbable drugs.

Animals↗
PubMed1996

Adenovirus-mediated transfer of human factor IX gene in immunodeficient and normal mice: evidence for prolonged stability and activity of the transgene in liver.

Recombinant adenovirus has recently become a promising gene delivery vehicle that may be used therapeutically for various medical disorders. However, in vivo expression of transgenes delivered by E1 region-deleted adenoviral vectors is transient in immunocompetent animals. It has been proposed that destruction of adenovirally transduced cells by the host immune mechanisms, particularly cytotoxic T-lymphocytes, may play a major role in limiting the duration of transgene expression in vivo. In the present study, Southern blot analysis of genomic DNA prepared from transduced liver tissues showed the persistent presence of the viral genome in both immunocompetent and immunodeficient animals, indicating the survival of the adenovirally transduced liver cells. Furthermore, active expression of the surviving factor IX transgenes was shown by the presence of recombinant human factor IX as well as specific human factor IX mRNA and protein in the transduced liver tissues. The transient appearance of human factor IX in the circulation of normal as well as partially immunodeficient mice is primarily due to the generation of mouse antihuman factor IX antibodies in these mice rather than host immune destruction of transduced cells. These results suggest that liver cells transduced with recombinant adenoviral vectors can escape from being destroyed by the host immune mechanism in normal animals. The present study thus provides a new rationale for further engineering of adenoviral vectors into a durable expression system for gene therapy of various diseases including congenital disorders such as hemophilia B.

Adenoviruses, Human↗
PubMed1995

Pulmonary inflammation induced by incomplete or inactivated adenoviral particles.

One of the major obstacles to pulmonary-directed gene therapy using adenoviral vectors is the induction of inflammation. We investigated whether the adenoviral particles that constitute the initial inoculum can serve as an inflammatory stimulus, independent of their ability to express genes that they contain. Viral particles were prepared that are defective in gene expression by (i) isolating particles that have incomplete genomes by selecting those that have buoyant densities on CsCl density gradients lighter than complete viruses; and (ii) cross-linking viral DNA by exposure to ultraviolet light in the presence of 8-methoxypsoralen. The defective particles retained their icosahedral appearance when viewed by electron microscopy but lost their plaque-forming ability on 293 cells. High doses of intact, incomplete, or inactivated viral particles were instilled intratracheally into CBA/J mice, and after 6 days the amount of inflammation was quantified by counting inflammatory cells contained within lung tissue. We found that the inflammatory responses induced by the incomplete or inactivated viral vectors were quantitatively similar to those caused by intact, competent viral vectors. We conclude that high doses of adenoviral vectors that are used for gene therapy can induce pulmonary inflammation, independent of expressing the genes they contain.

Adenoviridae↗
PubMed1995

Inhibition of interleukin-1-induced effects in synoviocytes transduced with the human IL-1 receptor antagonist cDNA using an adenoviral vector.

In this report, we present data showing that a recombinant adenoviral vector (Ad.RSVIL-1ra) containing the cDNA for human interleukin-1 receptor antagonist protein (IL-1ra) can genetically modify synoviocytes both in vitro and in vivo. Human synoviocytes infected with Ad.RSVIL-1ra in vitro expressed and secreted high levels of human IL-1ra that were detected by ELISA of tissue culture supernatants. New Zealand White rabbits that received intra-articular injections of Ad.RSVIL-1ra expressed transgenic IL-1ra in synoviocytes, and secretion was detected for at least 4 weeks post-infection. Further, biological activity of the transgenic IL-1ra was demonstrated by its ability to inhibit IL-1-induced prostaglandin E2 (PGE2) synthesis in vitro and IL-1-induced glycosaminoglycan (GAG) degradation in vivo. These data demonstrate that recombinant adenoviral vectors can mediate the intra-articular expression of anti-inflammatory proteins and may be a reasonable method to deliver therapeutically relevant proteins for the regional treatment of synovial inflammation.

Adenoviridae↗
PubMed1995

Transplantation of transduced chondrocytes protects articular cartilage from interleukin 1-induced extracellular matrix degradation.

Gene therapy used in the context of delivering a therapeutic gene(s) to chondrocytes offers a new approach for treating chondrocyte-mediated cartilage degradation associated with various human arthropathies including osteoarthritis. In this study, gene delivery to human osteoarthritis chondrocytes in monolayer culture was demonstrated using two adenoviral vectors (Ad.CMVlacZ and Ad.RSVntlacZ) carrying the Escherichia coli beta-galactosidase marker gene, and a third vector (Ad.RSV hIL-1ra) containing the cDNA for human interleukin-1 receptor antagonist. At an moi of 10(3) plaque-forming units/chondrocyte, > 90% of the infected cells stained positive for E. coli beta-galactosidase activity, indicating a high efficiency of transduction. Genetically modified chondrocytes were then transplanted onto the articular surface of osteoarthritic cartilage organ cultures with and without the underlying subchondral bone. Both in situ staining of the cartilage organ cultures for E. coli beta-galactosidase activity and examination by scanning electron microscopy indicated that the transplanted chondrocytes adhered and integrated into the articular surface and continued to express transgenic protein. Chondrocytes transduced with Ad.RSV hIL-1ra and seeded onto the surface of osteoarthritic cartilage secreted high levels of biologically active IL-1 receptor antagonist. The Ad.RSV hIL-1ra-treated cartilage samples were resistant to IL1-induced proteoglycan degradation over 10 d of sustained organ culture. These data demonstrate that transplantation of transduced chondrocytes onto the articular surface protects cartilage from IL-1-induced extracellular matrix degradation.

Adenoviridae↗
PubMed1995

Gene expression from recombinant viral vectors in the central nervous system after blood-brain barrier disruption.

Direct intracerebral injection of recombinant adenoviral vectors within the brain parenchyma or the ventricular system results in a limited volume of distribution of virus, as demonstrated by transgene expression. Global delivery to the central nervous system may increase the use of these vectors but only if the viral vectors can cross the blood-brain barrier and result in transduction of the underlying cells. This short-term study examines whether osmotic disruption with mannitol can result in sufficient opening of the vascular endothelium to allow for passage of replication-defective adenovirus containing the Escherichia coli beta-galactosidase gene (lacZ). Virus was injected into the carotid artery of rats after blood-brain barrier disruption with intracarotid hypertonic mannitol, and the animals were killed and analyzed after 4 days. Histochemical analysis and electron microscopy confirmed expression of the E. coli lacZ gene in the pericapillary astrocytes of the ipsilateral cerebral cortex and deep grey matter. Furthermore, the extent of gene transfer and expression correlated with the degree of barrier opening, as measured by Evans blue staining. Transgene expression was not seen in control animals that received intracarotid saline before recombinant virus injection. These data demonstrate, for the first time, that blood-brain barrier disruption can allow for the delivery of functional viral vectors to the central nervous system.

Adenoviridae↗
PubMed1995

Expression of human interleukin-1 receptor antagonist in mouse lungs using a recombinant adenovirus: effects on vector-induced inflammation.

Pulmonary inflammation is a major obstacle to using adenovirus-based vectors for gene transfer to the lung. Since the pro-inflammatory cytokine, interleukin-1 (IL-1), is expressed early following adenovirus infection, we hypothesized that inhibition of IL-1 might block the inflammation caused by adenoviral vectors. To inhibit IL-1 activity at the site of infection continuously, we employed a recombinant adenovirus that contained the cDNA for human IL-1 receptor antagonist protein (IL-1ra) designated as Ad.RSVIL-1ra. When Ad.RSVIL-1ra was instilled intratracheally into CBA/J mice, human IL-1ra was recovered in lung tissue and bronchoalveolar lavage fluid for up to 30 days. Human IL-1ra is known to bind to murine IL-1 receptors and inhibit IL-1-mediated responses. To measure pulmonary inflammation, the number of inflammatory cells contained within suspensions of protease-digested lung tissue were counted 6 days after virus administration. Ad.RSVIL-1ra failed to reduce the number of inflammatory cells below that induced by a control vector that lacked an expression cassette (Ad.BgIII). Light microscopy showed that the lung tissue from Ad.RSVIL-1ra and Ad.BgIII-treated mice contained qualitatively similar amounts of inflammatory infiltrate. We conclude that adenovirus-based vectors can be used to induce high levels of IL-1ra expression within the lung, but such expression was unable to prevent adenoviral vector-induced inflammation.

Adenoviridae↗
PubMed1995

A recombinant adenoviral vector expressing a soluble form of VCAM-1 inhibits VCAM-1/VLA-4 adhesion in transduced synoviocytes.

Intra-articular injection of recombinant adenovirus has been shown to be a feasible approach to the introduction of genetic reagents into synovial tissues in vivo. Rheumatoid arthritis (RA) is an autoimmune disorder characterized by the infiltration of lymphocytes and monocytes into inflamed synovium. It has been hypothesized that the recruitment of T lymphocytes/monocytes into sites of chronic inflammation is mediated by enhanced binding of very late antigen-4 (VLA-4) to vascular cell adhesion molecule-1 (VCAM-1) expressed on microvascular endothelial cells. Additional evidence suggests that VLA-4 binding continues to be important within the inflamed synovial membrane, where it appears to play a role in T cell retention and activation. A feasible therapeutic strategy for RA could be to utilize a soluble congener of the VCAM-1 molecule to block VLA-4 binding. In order to test this concept, a recombinant serotype Ad5 human adenovirus encoding a secreted form of VCAM-1 (Ad.CBsVCAM) was constructed. Human synoviocytes were readily infected in vitro with Ad.CBsVCAM, and sVCAM-1 expression and processing were analyzed by immunoprecipitation studies. Secretion of transgenic sVCAM was identified by ELISA of tissue culture supernatants, and biological activity was demonstrated with cell adhesion assays. In vivo, transgenic sVCAM-1 expression was determined by immunohistochemical analysis and in situ hybridization of synovial tissue, and secretion of transgenic sVCAM-1 was demonstrated by ELISA of tidal knee lavage fluid. The results showed that recombinant adenovirus can mediate the expression of a biologically active sVCAM-1 by synoviocytes in vivo and suggest that this strategy may be useful for inhibiting T lymphocyte retention and activation within rheumatoid synovium.

Adenoviridae↗
PubMed1994

Direct plasmid mediated transfection of adult murine brain cells in vivo using cationic liposomes.

Gene transfer to the central nervous system (CNS) is complicated by the anatomic and physiologic isolation of the brain. Direct injection techniques circumvent this, and allow delivery of transgenes to specific areas of the CNS. Previously, direct transfection of cellular components of the CNS has been achieved using plasmid DNA. We report the use of cationic liposomes as a means of transfecting plasmids into adult mammalian brain. Using the gene for E. coli beta-galactosidase or the cDNA or human beta-glucuronidase as reporters, we demonstrate plasmid mediated gene transfer into the caudate putamen of adult mice with expression of the transgene for at least 21 days post-transfection.

Animals↗
PubMed1994

Expression of Escherichia coli beta-galactosidase and rat HPRT in the CNS of Macaca mulatta following adenoviral mediated gene transfer.

Adenoviral-mediated gene transfer to the caudate nucleus of Macaca mulatta was accomplished using stereotactic injection of two distinct recombinant Ad5 vectors containing the gene for Escherichia coli beta-galactosidase and the cDNA for rat hypoxanthine-guanine phosphoribosylphosphotransferase (HPRT), respectively. Multiple analyses (including immunohistochemistry, histochemistry, transmission electron microscopy, RNA in situ hybridization, nucleotide pool analysis, and enzyme assay) confirmed efficient expression of beta-galactosidase and rat HPRT. Transgene expression was evident in both neurons and glia. Clinically, no evidence of meningitis or cerebritis was observed and no focal neurological deficits were detected in the animal. These preliminary studies indicate that recombinant adenovirus is capable of mediating high level transgene expression to the brains of higher order mammals.

Adenoviridae↗
PubMed1994

A 13 base pair deletion in exon 1 of HPRTIllinois forms a functional GUG initiation codon.

More than 50 mutations in the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus have been described, yet only 2 alter the AUG initiation codon. One, variant HPRT1151, results in Lesch-Nyhan syndrome (LNS), and the other, HPRTIllinois, results in partial HPRT deficiency. Although previously undetectable, we used a sensitive gel assay to demonstrate that HPRTIllinois is not only active, but has a native Mr indistinguishable from normal. Confirmatory evidence of activity and native Mr is demonstrated following transfection of HPRT cells with expression plasmids containing cDNA sequences representing HPRTIllinois. These data provide support for the hypothesis that patient RT, or variant HPRTIllinois, is spared manifestations of the LNS as a result of translation at the newly formed GUG initiation codon.

Base Composition↗