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Biomedical subjects

I E Alexander

Publications and source records attributed to I E Alexander.

At least 19 recordsLinked to original sources
PubMed2006

Fibroblasts modulate cardiomyocyte excitability: implications for cardiac gene therapy.

In an earlier study exploring the potential of gene transfer to repair myocardial conduction defects, we observed that myotubes, generated by forced expression of MyoD, exhibit reduced excitability when also modified to express connexin43 (Cx43). We hypothesized that this effect was caused by gap junction-mediated coupling between myotubes and the underlying fibroblast feeder layer. This intriguing possibility has important implications for ongoing efforts to develop strategies for repairing myocardial conduction defects by gene transfer, and also provides novel insights into the electrophysiological function of naturally occurring heterologous cell coupling within the heart. Although a conductive function for fibroblasts through heterologous coupling has previously been reported, the current study provides novel evidence that fibroblasts can modulate cardiomyocyte excitability in a Cx43-dependent manner. In a co-culture study system, neonatal rat cardiomyocytes were grown on monolayers of mouse fibroblasts with genetically altered Cx43 expression and the effect on intrinsic beat frequency examined. Cardiomyocytes grown on wild-type (WT) fibroblasts expressing native levels of Cx43 beat significantly slower than cells grown on fibroblasts devoid of this molecule (germline knockout) or with dominant-negative functional suppression. Expression of Cx43 in fibroblasts from Cx43 knockout mice restored cardiomyocyte beat frequency, to rates comparable with those observed in co-culture with WT fibroblasts.

Animals↗
PubMed2004

C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression.

We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development.

Adult↗
PubMed2004

Gene therapy: applications and progress towards the clinic.

Gene therapy was originally conceived as an approach to the treatment of genetic disease, to repair or replace a faulty gene. Subsequently, gene therapy clinical trials have been undertaken for a wide range of conditions, particularly cancer and AIDS. Overall, the results from gene therapy have been disappointing. The reasons include the following: (i) low gene transfer efficiencies and (ii) shortcomings in the identification and manipulation of appropriate target cells, including progenitor cell populations required for the maintenance of long-term effects. Today, the immense potential of gene therapy remains, but more basic research is required to improve technical aspects of this form of cellular therapy.

Australia↗
PubMed2003

Cardiac gene therapy: therapeutic potential and current progress.

Cardiovascular disease remains a major cause of morbidity and mortality in modern societies. While contemporary treatment modalities are making steady inroads to reduce this disease burden there remains a pressing need to vigorously explore novel therapeutic strategies. Rapid advances in our understanding of molecular pathology and the evolution of increasingly efficient gene transfer technology offer the imminent prospect of gene-based approaches to, at least, a subset of cardiovascular pathophysiologies. Initially envisaged as a treatment strategy for inherited monogenic disorders, it is now apparent that gene therapy has broader potential that encompasses acquired polygenic diseases, including many that affect the cardiovascular system. Extensive in vitro and animal studies are providing an increasingly sound scientific basis for cautious human evaluation. This review focuses on gene therapy of diseases primarily afflicting the heart, and provides an overview of gene and vector delivery systems with particular emphasis on systems suited to individual cardiac conditions. The pathophysiology underlying these conditions and molecular targets for therapeutic intervention are also reviewed.

Cardiovascular Diseases↗
PubMed2001

Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

Animals↗
PubMed2000

Adeno-associated virus vectors show variable dependence on divalent cations for thermostability: implications for purification and handling.

Recombinant adeno-associated virus (rAAV) shows significant promise as a vector for gene transfer in pre-clinical models of human disease, and is currently being evaluated in human clinical trials. As a consequence, increasing attention is being turned to the important tasks of optimizing rAAV titer, purity, and stability. We have observed dramatic variation in divalent cation dependence for thermostability of different rAAV vectors. To further investigate this observation, the thermostability of eight different vector constructs ranging in size from 73 to 107% of wild-type genome size (4.68 kilobases) was determined in the presence and absence of divalent cations. Virions containing smaller genomes (i.e., <85% wild type) were relatively divalent cation independent for thermostability. In contrast, virions containing recombinant genomes close to, or exceeding, wild-type size (i.e., >95% wild type) were dependent on divalent cations for thermostability. Genome sequence also appeared to be a factor in the thermostability of the larger rAAV vectors. These observations are of both practical and theoretical significance. Divalent cations should be included in all buffer solutions used during rAAV purification and storage, and unnecessary heat exposure avoided. These data also demonstrate that different recombinants of a particular virus should not be assumed to possess the same thermostability profile.

Cations, Divalent↗
PubMed1999

Human fibroblasts transduced with CD80 or CD86 efficiently trans-costimulate CD4+ and CD8+ T lymphocytes in HLA-restricted reactions: implications for immune augmentation cancer therapy and autoimmunity.

Augmenting immunogenicity by genetically modifying tumor cells to express costimulatory molecules has proven to be a promising therapeutic strategy in murine tumor models and is currently under investigation in human clinical trials for metastatic cancer. However, there are significant technical and logistic problems associated with implementing strategies requiring direct gene modification of primary tumor cells. In an effort to circumvent these problems, we are developing a strategy in which the costimulatory signal required for tumor-specific T lymphocyte activation is provided by a genetically modified human fibroblast (trans-costimulation). We have evaluated the efficiency of CD80- and CD86-mediated trans-costimulation in the activation of human CD8+ and CD4+ T lymphocytes in MHC class I- and class II-restricted lymphoproliferation reactions. Our studies demonstrate that the efficiency of CD80- or CD86-mediated trans-costimulation of purified human CD8+ and CD4+ T lymphocytes is comparable to cis-costimulation under defined conditions. Moreover, a dose-response relationship consistent with the predicted two-hit kinetics of the reaction was evident in trans-costimulation reactions in which the ratio of target cells expressing either signal 1 or signal 2 was varied incrementally from 1:10 to 10:1. Importantly, the level of cell-surface CD86 required for trans-costimulation is equivalent to that constitutively expressed by human peripheral blood monocytes. These results may have significant implications for the clinical implementation of this type of cancer immunotherapy and also raise questions about the possibility of trans-costimulating autoreactive T lymphocytes in vivo.

3T3 Cells↗
PubMed1999

Inhibition of human immunodeficiency virus type 1 replication by the K10-K42 peptide of GAP31 is due to induction of rapid but nonspecific precipitation of viral and nonviral proteins.

The 33-amino acid peptide K10-K42 has previously been described as having potent anti-HIV-1 activity, and antiviral efficacy against hepatitis B and human cytomegalovirus in vitro. Although the exact mechanism of antiviral activity was unknown, it was hypothesised that the K10-K42 peptide inhibited HIV-1 by interfering with one or more of the intracellular processes of reverse transcription, integration, and/or viral gene expression. We performed a series of experiments to identify and characterize the inhibitory mechanism, and to determine whether intracellular expression of the K10-K42 peptide would potentiate its antiviral efficacy in vitro. Surprisingly, our results revealed that the antiviral activity of the K10-K42 peptide could be explained without implicating intracellular inhibition of HIV-1 replication. The activity appeared to be due to an extraordinary capacity of the K10-K42 peptide to precipitate viral and nonviral proteins in vitro. The protein-precipitating capacity of the K10-K42 peptide was sequence specific and a scrambled version of the 33-amino acid peptide did not retain the activity. Although the unusual biochemical properties of the K10-K42 peptide probably negate a number of potential therapeutic applications, they do merit further investigation. Moreover, these findings provide a plausible explanation of the mechanism by which the K10-K42 peptide can inhibit replication of viruses from families as genetically and functionally diverse as Retroviridae, Hepadnaviridae, and Herpesviridae.

Amino Acid Sequence↗
PubMed1999

Human PBMC-derived dendritic cells transduced with an adenovirus vectorinduce cytotoxic T-lymphocyte responses against a vector-encoded antigen in vitro.

Dendritic cells (DC) are among the most potent antigen-presenting cells known and play an important role in the initiation of antigen-specific T-lymphocyte responses. Several recent studies have demonstrated that DC expressing vector-encoded tumor-associated antigens can induce protective and therapeutic immunity in murine cancer models. In the current study we set out to examine in vitro the utility of adenovirus vectors in the transduction of human DC for the induction of antigen-specific T-lymphocyte responses against a defined vector-encoded antigen. DC were derived from the adherent fraction of PBMC by culture in defined medium containing GM-CSF and IL-4. A replication-defective E1/E3-deleted type 5 adenovirus vector encoding bacterial beta-galactosidase (beta-gal) under the transcriptional control of a CMV promoter was used to transduce DC at multiplicities of infection (MOI) up to 1000. While high MOI were required to achieve efficient transduction there was no significant effect on DC morphology, immunophenotype or potency in allogeneic lymphocyte proliferation assays. Furthermore, transduced DC-induced antigen-specific CTL activity against adenoviral proteins and more significantly, the vector-encoded antigen beta-gal. These data clearly demonstrate the potential of adenovirus vectors in anticancer DC vaccine strategies and provide an important link between existing animal data and human clinical application.

Adenoviridae↗
PubMed1998

Identification of a dynamic intracellular reservoir of CD86 protein in peripheral blood monocytes that is not associated with the Golgi complex.

In the process of developing a cancer immunotherapy strategy, we have identified and characterized a novel intracellular reservoir of CD86 protein in peripheral blood monocytes. This observation emerged from studies aimed at using retrovirus vectors to genetically modify tumor cells to express the costimulatory proteins CD80 and CD86. Retrovirus-mediated expression of CD80 and CD86 in T lymphoblastoid CEM cells resulted in an unexpected intracellular focal concentration of both proteins in the genetically modified cells. By extending these studies to an analysis of CD80 and CD86 expression in PBMC, we observed that endogenous CD86 expression in peripheral blood monocytes also involved a similar intracellular focal concentration of the protein. The intracellular concentration of CD86 in monocytes was not due to storage within the Golgi apparatus, and required intact microtubules to retain structural integrity. Furthermore, as the intensity of CD86 fluorescence increased on monocytes as a function of time in vitro, the intracellular focal concentration correspondingly decreased. These results are consistent with antegrade CD86 transport from an intracellular reservoir to the cell surface membrane. In this report, we detail the intracellular and membrane localization studies with tumor cell lines and PBMC, and describe the temporal relationship between intracellular storage and trafficking of CD86 to the cell surface membrane in peripheral blood monocytes. We hypothesize that this intracellular reservoir allows rapid and sustained deployment of an important costimulatory molecule to the monocyte surface membrane during initiation and maturation of the cell-mediated immune response.

Antigens, CD↗
PubMed1997

Transfer of contaminants in adeno-associated virus vector stocks can mimic transduction and lead to artifactual results.

The potential of adeno-associated virus (AAV) vectors for gene transfer and gene therapy applications is currently being intensively investigated. Although much progress has been made in defining AAV vector biology, inconsistencies remain in the literature regarding the efficiency of AAV transduction in various cell types. In the course of exploring these differences, we have identified a problem associated with the use of AAV vector stocks that results in overestimation of gene transfer efficiencies. We show here that biologically active vector-encoded proteins can contaminate AAV vector stocks, especially cell lysate preparations that have not been further purified, and can be transferred in a virion-independent manner to target cells, a phenomenon called pseudotransduction. This observation is significant because impure cell lysate stocks have been widely employed in the AAV literature, and we demonstrate here that this phenomenon can occur with commonly used reporter proteins such as beta-galactosidase and alkaline phosphatase. We conclude that although there are many potential explanations for apparently conflicting results in the literature, the possibility of pseudotransduction must be considered, especially when cell lysate stocks of AAV vectors have been employed. This artifact can be avoided by further vector purification.

Alkaline Phosphatase↗
PubMed1997

Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors.

We previously found that gene transduction by adeno-associated virus (AAV) vectors in cell culture can be stimulated over 100-fold by treatment of the target cells with agents that affect DNA metabolism, such as irradiation or topoisomerase inhibitors. Here we show that previous gamma-irradiation increased the transduction rate in mouse liver by up to 900-fold, and the topoisomerase inhibitor etoposide increased transduction by about 20-fold. Similar rates of hepatic transduction were obtained by direct injection of the liver or by systemic delivery via tail vein injection. Hepatocytes were much more efficiently transduced than other cells after systemic delivery, and up to 3% of all hepatocytes could be transduced after one vector injection. The presence of wild-type AAV, which contaminates many AAV vector preparations, was required to observe a full response to gamma-irradiation. Injection of mice with AAV vectors encoding human clotting factor IX after gamma-irradiation resulted in synthesis of low levels of human clotting factor IX for the 5-month period of observation. These studies show the potential of targeted gene transduction of the liver by AAV vectors for treatment of various hematological or metabolic diseases.

Animals↗
PubMed1997

Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration.

The ability of recombinant adeno-associated virus (AAV) vectors to integrate into the host genome and to transduce nondividing cells makes them attractive as vehicles for gene delivery. In this study, we assessed the ability of several AAV vectors to transduce airway cells in rabbits by measuring marker gene expression. AAV vectors that transferred either a beta-galactosidase (beta-gal) or a human placental alkaline phosphatase (AP) gene were delivered to one lobe of the rabbit lung by use of a balloon catheter placed under fluoroscopic guidance. We observed vector-encoded beta-gal or AP staining almost exclusively in the epithelial and smooth muscle cells in the bronchus at the region of balloon placement. The overall efficiency of transduction in the balloon-treated bronchial epithelium was low but reached 20% in some areas. The majority of the staining was in ciliated cells but was also observed in basal cells and airway smooth muscle cells. We observed an 80-fold decrease in marker-positive epithelial cells during the 60-day period after vector infusion, whereas the number of marker-positive smooth muscle cells stayed constant. Although treatment with the topoisomerase inhibitor etoposide dramatically enhanced AAV transduction in primary airway epithelial cells in culture, treatment of rabbits did not improve transduction rates in the airway. Vector readministration failed to produce additional transduction events, which correlated with the appearance of neutralizing antibodies. These results indicate that both readministration and immune modulation will be required in the use of AAV vectors for gene therapy to the airway epithelium.

Alkaline Phosphatase↗
PubMed1996

Effects of gamma irradiation on the transduction of dividing and nondividing cells in brain and muscle of rats by adeno-associated virus vectors.

Vectors based on adeno-associated virus (AAV) are under investigation for use in gene therapy applications. Critical aspects of AAV vector biology remain undefined, in particular the intracellular events and activities mediating transduction and determining host cell permissiveness for transduction. Using cultured primary human fibroblasts, we previously showed that AAV vectors preferentially, but not exclusively, transduce cells in the S phase of the cell cycle, and that transduction can be markedly enhanced by pretreatment of target cells with physical and chemical agents that perturb DNA metabolism. In this study, we tested whether similar improvements in AAV vector performance might be achievable in vivo. The adult rat brain and overlying scalp muscle were selected for vector inoculation because of the presence of well-defined populations of dividing, quiescent, and post-mitotic cells, and gamma irradiation was chosen as a reproducible means of inducing DNA repair in these cells. We find that gamma irradiation markedly enhances the transduction of dividing cell populations in the pia-arachnoid and choroid epithelium within the central nervous system, and of mature nondividing muscle cells in the scalp, whereas gamma irradiation did not increase the basal transduction level of post-mitotic neurons in the hippocampus. These data confirm that replicative cellular DNA synthesis is not required for transduction by AAV vectors and show that the mitotic state of target cells is not necessarily predictive of responsiveness to transduction-enhancing treatments. Most importantly, these data demonstrate that target cells can be manipulated in vivo to render them more permissive for AAV vector transduction.

Age Factors↗
PubMed1995

DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.

Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased > 300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.

Aphidicolin↗
PubMed1995

Fetal brain disruption sequence in sisters.

We report two female siblings with the fetal brain disruption sequence. Extensive investigation of both children failed to define a definitive aetiology but clinical and laboratory findings are consistent with a hitherto unknown storage disease. We postulate that the accumulation of a neurotoxic metabolite may be responsible for the disease phenotype observed. This is the first report of recurrence of the fetal brain disruption sequence and supports the existence of a genetic form of this condition. Previous reports have emphasized possible environmental aetiologies. Infants with fetal brain disruption sequence should be investigated exhaustively and, in the absence of definitive evidence of an environmental cause, the possibility of a genetic aetiology should be considered. In some families the recurrence risk may be as high as one in four.

Biopsy↗
PubMed1995

Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells.

Immortalized cell lines have been used to study infection and replication of adeno-associated virus (AAV) in culture, but primary cells presumably provide a better model for AAV behavior in animals. Here, we have evaluated the ability of AAV vectors to transduce primary and immortalized strains of human epithelial cells and fibroblasts. Two AAV vectors were used, one that transduced an alkaline phosphatase gene (AAV-LAPSN), and one that transduced a beta-galactosidase/neomycin phosphotransferase fusion gene (AAV-L beta geo). The transduction efficiency of the AAV-LAPSN vector, quantitated by measurement of alkaline phosphatase-positive cell foci following infection, was 10 to 60 times greater in immortalized human cells than in primary cells, and total alkaline phosphatase activity in cell lysates was 40 to 50 times greater in immortalized cells. The AAV-L beta geo vector gave similar results. In contrast, the transduction efficiency of a retrovirus vector encoding alkaline phosphatase was equivalent in primary and immortalized cells. Analysis of the quantity and state of the AAV vector genomes in cells showed that primary and immortalized cells contained comparable numbers of vector copies per cell and that the vast majority of vector DNA was not integrated into the cell genome. Additionally, the level of AAV vector-derived message paralleled the transduction efficiency. These results indicate that the block to functional transduction in primary cells occurred after virus entry and limited the abundance of vector-derived message. Data from AAV transduction in cultures of human cells containing immortalizing genes suggest that cellular changes secondary to the introduction of immortalizing genes increased permissiveness for transduction by AAV vectors. In summary, our data demonstrate that AAV vectors transduce primary human cells much less efficiently than immortalized cells and indicate the importance of using primary cells to evaluate AAV vectors for gene therapy applications.

Cells, Cultured↗