DNA sequence analysis of spontaneous mutations at the aprt locus of hamster cells.

To determine the nature of spontaneous mutational events in cellular genes in hamster cells, mutant adenine phosphoribosyltransferase (aprt) genes were cloned and the regions to which we mapped alterations were sequenced. A variety of nucleotide changes were found to occur in the 12 mutant genes analyzed. Most mutations were simple base-pair substitutions-transitions (both G X C----A X T and A X T----G X C) and transversions. The only multiple mutation was a simple transition next to a single-base-pair insertion. Of the 12 mutations, 4 were more complex, involving small deletions or duplications. Two of these were similar to previously described deletions in that they occurred between short direct sequence repeats. No hot spots were detected. Three independent mutations were characterized at one restriction endonuclease site, although no other mutations were detected in the nucleotides surrounding this site in other mutant strains. At a functional level, sequence changes were either in exons (resulting in missense and, in one instance, nonsense mutations) or at splicing sites.

The principles of gene therapy for the nervous system.

Research pertaining to gene transfer into cells of the nervous system is one of the fastest growing fields in neuroscience. An important application of gene transfer is gene therapy, which is based on introducing therapeutic genes into cells of the nervous system by ex vivo or in vivo techniques. With the eventual development of efficient and safe vectors, therapeutic genes, under the control of a suitable promoter, can be targeted to the appropriate neurons or glial cells. Gene therapy is not only applicable to the treatment of genetic diseases of the nervous system and the control of malignant neoplasia, but it also has therapeutic potential for acquired degenerative encephalopathies (Alzheimer's disease, Parkinson's disease), as well as for promoting neuronal survival and regeneration in various pathological states.

p53 gene mutation and mdm2 gene amplification are uncommon in medulloblastoma.

Loss of heterozygosity is common for the short arm of chromosome 17 in medulloblastomas, and putative medulloblastoma suppressor loci have been localized to 17p13. The colocalization of the p53 tumor suppressor gene to 17p13 raises the possibility that its mutant alleles may play a role in the malignant transformation of "medulloblasts." Mutations and deletions of the p53 gene have been described in many tumor types and in the germline of some individuals with the Li-Fraumeni syndrome, but reports on the status of the p53 and mdm2 (a gene coding for a p53-associated protein reportedly amplified in human sarcomas) genes in medulloblastomas are few and an indication of their roles, if any, in the etiology of this important childhood tumor has yet to emerge. Here we have analyzed polymerase chain reaction-amplified products of exons 4-9 (95% of reported p53 mutations occur within this region) of the p53 gene in 9 medulloblastomas for potential mutations using the technique of single strand conformation polymorphism analysis and DNA sequencing. We found only one mutation, an A-T to T-A transversion involving the second base of codon 285 and resulting in the substitution of valine for glutamic acid, amplification of the mdm2 gene could be detected in zero of eight of these tumors. These findings suggest that genetic events associated with the inactivation of p53 gene occur in only a minor subset of medulloblastomas.

Vesicular stomatitis virus G pseudotyped retrovector mediates effective in vivo suicide gene delivery in experimental brain cancer.

Direct in vivo tumor-targeting with "suicide" viral vectors is limited by either inefficient gene transfer (i.e., retroviral vectors) or indiscriminate transfer of a conditionally toxic gene to surrounding nonmalignant tissue (i.e., adenoviral vectors). Retrovectors pseudotyped with the vesicular stomatitis virus G protein (VSVG) may serve as a remedy to this conundrum. These retroviral particles differ from standard murine retroviruses by their very broad tropism and the capacity to be concentrated by ultracentrifugation without loss of activity. We propose that a VSVG-typed retrovector can be used for efficient and tumor-specific herpes simplex virus thymidine kinase (TK) gene delivery in vivo. To test this hypothesis, we developed a bicistronic retroviral vector that expresses TK and green fluorescence protein (pTKiGFP). The 293GPG packaging cell line was used to generate vTKiGFP retroparticles. In cytotoxicity assays, vTKiGFP-transduced human glioma cell lines were sensitized to the cytotoxic effects of gangciclovir (GCV) 10,000-fold. Subsequently, virus was concentrated by ultracentrifugation to a titer of 2.3 x 10(10) cfu/ml. We tested the antitumor activity of vTKiGFP retroparticles in a rat C6 glioma model of brain cancer. Concentrated retrovector stock (9 microl volume) was injected stereotactically in preestablished intracerebral tumor. Subsequently, rats were treated with GCV for 10 days. Control rats (no GCV) had a mean survival of 38 days (range, 20-52 days). Sections performed on postmortem brain tissue revealed large tumors with evidence of high efficiency retrovector transfer and expression (as assessed by GFP fluorescence). Fluorescence was restricted to malignant tissue. In the experimental group (GCV treated), 8 of 12 remain alive and well >120 days after glioma implantation. In conclusion, vTKiGFP is very efficient at transducing human glioma cell lines in vitro and leads to significant GCV sensitization. Recombinant retroviral particles can be concentrated to titers that allow in vivo intratumoral delivery of large viral doses. The therapeutic efficiency of this reagent has been demonstrated in a preclinical model of brain cancer.

Localization of coxsackie virus and adenovirus receptor (CAR) in normal and regenerating human muscle.

The primary receptor for Adenovirus and Coxsackie virus (CAR) serves as main port of entry of the adenovirus vector mediating gene transfer into skeletal muscle. Information about CAR expression in normal and diseased human skeletal muscle is lacking. C'- or N'-terminally directed polyclonal antibodies against CAR were generated and immunohistochemical analysis of CAR on morphologically normal and regenerating human skeletal muscle of children and adults was performed. In morphologically normal human muscle fibers, CAR immunoreactivity was limited to the neuromuscular junction. In regenerating muscle fibers, CAR was abundantly co-expressed with markers of regeneration. The function of CAR at the neuromuscular junction is currently unknown. Co-expression of CAR with markers of regeneration suggests that CAR is developmentally regulated, and may serve as a marker of skeletal muscle fiber regeneration.

Insertion of unique and repetitive DNA fragments into the aprt locus of hamster cells.

Two insertion mutations occurring at the aprt locus of Chinese hamster ovary cells were analyzed at the nucleotide level by cloning and sequencing the mutant genes. The insertions are similar with respect to events at the target site in that both are accompanied by small deletions. The nature of the DNA introduced, on the other hand, is very different, a unique fragment in a spontaneously occurring mutant and a highly dispersed, repetitive fragment in a gamma radiation-induced strain. The inserts are small (285 and 58 base-pairs) and have none of the structural features or sequences related to putative mobile sequences in mammalian cells. The mechanism of transposition was further examined by cloning the unique donor fragment of the spontaneous mutant. These analyses revealed that the insert in the mutant gene was a precise duplicate of the donor DNA fragment.

Structure of mutant alleles at the aprt locus of Chinese hamster ovary cells.

To determine the types of gene structural alterations causing deficiency of adenine phosphoribosyl transferase (aprt) activity in spontaneous and chemically induced mutations of cultured somatic cells, we analyzed the restriction enzyme cleavage patterns of aprt gene sequences in mutant strains selected from Chinese hamster ovary cells. Patterns of aprt-containing fragments in Southern blots were mostly unchanged in our collection of 280 ethyl methane sulfonate-induced and spontaneous aprt- mutants, suggesting that base-pair changes or other alterations below our limit of resolution on agarose gels (approximately 50 base-pairs) are responsible for the great majority of mutations at the aprt locus. Occasionally, these mutations could be localized when they resulted in the loss or gain of a restriction enzyme site and the generation of new fragments of predictable size. Deletions of aprt-containing sequences were detected in only eight of 119 spontaneous mutants and in only one ethyl methane sulfonate-induced mutant. An insertion of 300 base-pairs near the 5' end of the aprt structural gene was found in one spontaneous aprt- strain. This insertion mutant was stable with a reversion frequency of less than 2 X 10(-7). Several unstable aprt- mutants were detected in our collection, but these had no observable alterations of aprt coding or flanking sequences.

Structure and sequence of mutations induced by ionizing radiation at selectable loci in Chinese hamster ovary cells.

The spectrum of mutations induced by ionizing radiation at two non-essential genetic loci varies markedly. Those at the adenine phosphoribosyl transferase (aprt) locus predominantly have no detectable alterations of gene structure on Southern blots, while those at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus are largely massive deletions eliminating all coding sequence. Insertion mutations were detected at both loci. To characterize the sequence alterations producing the minor changes at the aprt locus, two mutant genes were cloned from lambda genomic libraries and sequenced. One of these mutants proved to be a 20 base-pair deletion formed between two short (3 base-pair) direct repeat sequences, while the second was the result of a 58 base-pair insertion accompanied by a 13 base-pair deletion.

Intracerebral adenovirus-mediated p53 tumor suppressor gene therapy for experimental human glioma.

Malignant gliomas of astrocytic origin are good candidates for gene therapy because they have proven incurable with conventional treatments. Although mutation or inactivation of the p53 tumor suppressor gene occurs at early stages in gliomas and is associated with tumor progression, many tumors including high-grade glioblastoma multiforme carry a functionally intact p53 gene. To evaluate the effectiveness of p53-based therapy in glioma cells that contain endogenous wild-type p53, a clinically relevant model of malignant human glioma was established in athymic nu/nu mice. Intracerebral, rapidly growing tumors were produced by stereotactic injection of the human U87 MG glioma cell line that had been genetically modified for tracking purposes to express the Escherichia coli lacZ gene encoding beta-galactosidase. Overexpression of the p53 gene by adenovirus-mediated delivery into the tumor mass resulted in rapid cell death with the eradication of beta-galactosidase-expressing glioma cells through apoptosis. In long-term experiments, the survival of mice treated with the p53 adenoviral recombinant was significantly longer than that of mice that had received control adenoviral recombinant. During the observation period of 1 year, a complete cure was achieved in 27% of animals after a single injection of p53 adenoviral recombinant, and 38% of the animals were tumor free in the group receiving multiple injections of p53 adenoviral recombinant into a larger tumor mass. These experiments demonstrate that overexpression of p53 in gliomas, even in the presence of endogenous functional wildtype p53, leads to efficient elimination of tumor cells. These results point to the potential therapeutic usefulness of this approach for all astrocytic brain tumors.

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells.

Recombinant adenovirus (AVR) promises to be an efficient vector in gene therapy for neuromuscular diseases, but in preclinical experiments the expression of therapeutic genes is shorter lived in immunocompetent animals than in immunocompromised hosts. Interferons (IFN), which are known to have a role both in early antiviral activity and in late cytotoxic immunoreaction against the virus or transduced cells, may influence the efficiency of gene transfer. In this study we investigated the role of IFNs in determining the efficiency of gene transfer by AVR. AVRs expressing beta-galactosidase (beta-gal) from either a cytomegalovirus (CMV) or a troponin-I promoter were used. Muscle cells were infected by AVR after exposure to various IFNs. The alphaIFN treatment significantly reduced (up to fivefold) the CMV promoter-driven gene expression in muscle cells in vitro and in immature muscles in vivo, while the least effective inhibitor was betaIFN. The decrease in gene expression by IFNs was more pronounced with the CMV-driven transgene than troponin-I promoter-driven one and was due to a decrease in transcript level. Intrinsic IFNs that are triggered by AVR administration can decrease the efficiency of gene transfer in muscle cells. Therefore the use of muscle specific promoters in AVR and/or IFN inhibitory agents will likely improve the prospects of effective gene therapy by AVR.

Expression of the primary coxsackie and adenovirus receptor is downregulated during skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells.

Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.

Study of adenovirus-mediated dystrophin minigene transfer to skeletal muscle by combined microscopic display of adenoviral DNA and dystrophin.

In situ DNA hybridization of an E4 adenoviral sequence amplified by in situ polymerase chain reaction (PCR) was used to mark adenovirus-containing myonuclei in muscles of immunocompetent and immunosuppressed mdx mice following intramuscular injection of adenoviral recombinants. The adenoviral recombinants contained a 6.3-kb dystrophin cDNA (minigene) driven by a cytomegalovirus (CMV) promoter/enhancer and thus, immunostaining for dystrophin of the same sections permitted correlation of adenoviral recombinant-containing myonuclei with dystrophin positivity of the same muscle fiber segments. As early as 2 hr post-injection of adenoviral recombinant, an appreciable number of adenoviral recombinant-positive (AVR+) myonuclei, and some partial dystrophin positive (pdys+) fibers were observed. Some fully dystrophin-positive (dys+) muscle fibers were present as early as 6 hr. The maximum number of fibers containing AVR+ myonuclei (observed by 72 hr) was maintained until 60 days in immunosuppressed, but not in immunocompetent, animals. In immunocompetent animals, the maximum number of dys+ fibers was observed at 10 days. The vast majority of these fibers contained AVR+ myonuclei; however, by 60 days, dys+ fibers disappeared with some AVR+ myonuclei persisting. Our studies suggest that widespread delayed inactivation of the dystrophin expression cassette is probably unlikely. Thus, optimization of immunosuppression could assure successful long-term dystrophin gene transfer for gene therapy.

Impairment of force generation after adenovirus-mediated gene transfer to muscle is alleviated by adenoviral gene inactivation and host CD8+ T cell deficiency.

Recombinant adenovirus vectors (AdV) hold promise as a means of delivering therapeutic genes to muscle in diseases such as Duchenne muscular dystrophy (DMD). However, we have previously shown that the use of AdV is hampered by the development of reduced force-generating capacity, which occurs within 1 week and is progressive up to at least 1 month after AdV delivery in immune-competent animals. Determinations of muscle force production provide a sensitive and clinically important measure of potential adverse effects of AdV-mediated gene transfer on muscle cell function. In the present study, we investigated the role of AdV-related gene expression and host T lymphocyte responses in the genesis of muscle dysfunction following AdV injection of muscle. We report that UV-irradiation of AdV particles, which reduced AdV transcriptional activity without impairing infectivity (as confirmed by in situ polymerase chain reaction), significantly reversed early (4 days post-injection) AdV-induced contractile impairment in immune-competent mice as well as in mice lacking effective CD8+ T cell activity. The superimposed additional reduction in force-generating capacity normally found between 4 and 30 days post-AdV delivery in immune-competent mice, along with the associated loss of transgene (beta-galactosidase) expression, was largely abrogated by the absence of an intact CD8+ T lymphocyte response. Furthermore, short-term administration of a neutralizing antibody against CD4+ T cells significantly prolonged transgene expression and showed a trend toward mitigation of AdV-induced reductions in force-generating capacity. Cellular infiltration and humoral immune responses against the vector and transgene product were also blunted to varying degrees in the setting of CD8+ or CD4+ T cell deficiency. We conclude that AdV-related gene expression has an early negative (probably toxic) effect on muscle cell function that is independent of CD8+ T cell-mediated immunity. In contrast, further progression of contractile impairment and the accompanying loss of transgene expression from AdV-injected muscle are largely dependent upon the activity of CD8+ T cells. These results have implications for the design of future generation vectors and the potential need for immunosuppressive therapy after AdV-mediated gene transfer to muscle.

Modulation of Starling forces and muscle fiber maturity permits adenovirus-mediated gene transfer to adult dystrophic (mdx) mice by the intravascular route.

Duchenne muscular dystrophy (DMD) and other inherited myopathies lead to progressive destruction of most skeletal muscles in the body, including those responsible for maintaining respiration. DMD is a fatal disorder caused by defects in the dystrophin gene. Recombinant adenovirus vectors (AdV) are considered a promising means for therapeutic delivery of a functional dystrophin gene to DMD muscles. If AdV-mediated dystrophin gene replacement in DMD is to be successful, development of a systemic delivery method for targeting the large number of diseased muscles will be required. In this study we investigated two major factors preventing efficient AdV-mediated gene transfer to skeletal muscles of adult animals after intravascular AdV administration: (1) an inability of AdV particles to breach the endothelial barrier and enter into contact with myofibers, and (2) a relatively nonpermissive myofiber population for AdV infection due at least in part to insufficient levels of the coxsackie/adenovirus attachment receptor (CAR). On the basis of established principles governing the transendothelial flux of macromolecules, we further hypothesized that an alteration in Starling forces (increased hydrostatic and decreased osmotic pressures) within the intravascular compartment would facilitate AdV transendothelial flux via convective transport. In addition, experimental muscle regeneration was employed to increase the prevalence of immature myofibers in which CAR expression is upregulated. Here we report that by employing the above-described strategy, high-level heterologous reporter gene expression was achievable in hindlimb muscles of normal rats as well as dystrophic (mdx) mice (genetic homolog of DMD) after a single intraarterial injection of AdV. Microsphere studies confirmed enhanced transport into muscle of fluorescent tracer particles in the size range of AdV, and there was a high concordance between CAR upregulation and myofiber transduction after intraarterial AdV delivery. Furthermore, in mdx mice examined 10 days after intraarterial AdV delivery, the aforementioned procedures had no adverse effects on the force-generating capacity of targeted muscles. These findings have implications for eventual AdV-mediated gene therapy of generalized skeletal muscle diseases such as DMD using a systemic intraarterial delivery approach.

Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles.

Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.

High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.

Replication-deficient adenovirus vectors (AdV) have been successfully used to transfer a truncated human dystrophin cDNA to skeletal muscle of dystrophin-deficient mdx mice. A dystrophin-deficient golden retriever dog model (GRMD) has been identified, which, unlike the mouse model, leads to a clinicopathological phenotype similar to that of Duchenne muscular dystrophy (DMD). We show for the first time that high-level dystrophin expression in skeletal muscle of GRMD dogs can be achieved by AdV-mediated gene transfer. However, a humoral and cellular immune response of the host against antigens of viral and transgene origin (similar to that occurring in mdx mice after AdV-mediated dystrophin gene transfer) leads to a decline of dystrophin expression over a 2-month period. Immunosuppression by cyclosporin significantly prolonged transgene expression. The GRMD model may help to solve the open questions pertaining to dystrophin gene transfer such as systemic delivery and improvement of muscle function before human trials for gene replacement therapy in DMD may be considered.

Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products.

Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer.

Differential effects of dystrophin and utrophin gene transfer in immunocompetent muscular dystrophy (mdx) mice.

Duchenne muscular dystrophy (DMD) is a fatal disease caused by defects in the gene encoding dystrophin. Dystrophin is a cytoskeletal protein, which together with its associated protein complex, helps to protect the sarcolemma from mechanical stresses associated with muscle contraction. Gene therapy efforts aimed at supplying a normal dystrophin gene to DMD muscles could be hampered by host immune system recognition of dystrophin as a "foreign" protein. In contrast, a closely related protein called utrophin is not foreign to DMD patients and is able to compensate for dystrophin deficiency when overexpressed throughout development in transgenic mice. However, the issue of which of the two candidate molecules is superior for DMD therapy has remained an open question. In this study, dystrophin and utrophin gene transfer effects on dystrophic muscle function were directly compared in the murine (mdx) model of DMD using E1/E3-deleted adenovirus vectors containing either a dystrophin (AdV-Dys) or a utrophin (AdV-Utr) transgene. In immunologically immature neonatal animals, AdV-Dys and AdV-Utr improved tibialis anterior muscle histopathology, force-generating capacity, and the ability to resist injury caused by high-stress contractions to an equivalent degree. By contrast, only AdV-Utr was able to achieve significant improvement in force generation and the ability to resist stress-induced injury in the soleus muscle of immunocompetent mature mdx animals. In addition, in mature mdx mice, there was significantly greater transgene persistence and reduced inflammation with utrophin compared to dystrophin gene transfer. We conclude that dystrophin and utrophin are largely equivalent in their intrinsic abilities to prevent the development of muscle necrosis and weakness when expressed in neonatal mdx animals with an immature immune system. However, because immunity against dystrophin places an important limitation on the efficacy of dystrophin gene replacement in an immunocompetent mature host, the use of utrophin as an alternative to dystrophin gene transfer in this setting appears to offer a significant therapeutic advantage.

Expression of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) mRNA in adult human astrocytes: comparison with adult microglia and fetal astrocytes.

Astrocytes and microglia are cell populations which are implicated as being capable of regulating and effecting immune responses within the central nervous system (CNS). These functions are postulated to be mediated at least in part by production of soluble protein molecules termed cytokines. In this study, we utilized dissociated cultures of glial cells prepared from adult and fetal CNS tissue and a combined in situ hybridization-immunocytochemical technique in order to compare expression of TNF alpha and IL-6 mRNA between adult and fetal astrocytes and between adult astrocytes and microglia. Our results, using digoxygenin-labeled riboprobes, indicate that in contrast to fetal astrocytes only rare adult astrocytes express TNF alpha and IL-6 transcripts under our serum-supplemented basal culture conditions. Activation with LPS and IFN gamma increased the proportion of adult astrocytes expressing detectable TNF alpha and IL-6 mRNA signals; however, the proportion was significantly less than for microglia contained in the same cultures. These results suggest that microglia rather than astrocytes are more likely to be sources of these cytokines within the adult human CNS. Further studies of cytokine expression by glial cells will need to consider both the age and species of the glial cells used.