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Ronald G Crystal

Publications and source records attributed to Ronald G Crystal.

At least 73 records · Page 4Linked to original sources
PubMed2004

Cytoplasmic dynein mediates adenovirus binding to microtubules.

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% +/- 3.5% to 80.7% +/- 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.

Adenosine Triphosphate↗
PubMed2004

Macaque model for severe acute respiratory syndrome.

Rhesus and cynomolgus macaques were challenged with 10(7) PFU of a clinical isolate of the severe acute respiratory syndrome (SARS) coronavirus. Some of the animals developed a mild self-limited respiratory infection very different from that observed in humans with SARS. The macaque model as it currently exists will have limited utility in the study of SARS and the evaluation of therapies.

Animals↗
PubMed2004

Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Stat1.

Intranasal inhalation of the severe acute respiratory syndrome coronavirus (SARS CoV) in the immunocompetent mouse strain 129SvEv resulted in infection of conducting airway epithelial cells followed by rapid clearance of virus from the lungs and the development of self-limited bronchiolitis. Animals resistant to the effects of interferons by virtue of a deficiency in Stat1 demonstrated a markedly different course following intranasal inhalation of SARS CoV, one characterized by replication of virus in lungs and progressively worsening pulmonary disease with inflammation of small airways and alveoli and systemic spread of the virus to livers and spleens.

Animals↗
PubMed2004

Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses.

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing beta-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the beta-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing beta-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to beta-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-beta-galactosidase antibody levels following vector administration. However, cellular responses to beta-galactosidase were significantly enhanced, with the frequency of CD4(+) as well as the CD8(+) beta-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing beta-galactosidase: BALB/c mice implanted with the CT26 syngeneic beta-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines.

Adenoviridae↗
PubMed2004

Coexpression of herpesviral thymidine kinase reporter gene and VEGF gene for noninvasive monitoring of therapeutic gene transfer: an in vitro evaluation.

UNLABELLED: Coexpression of a reporter gene and a therapeutic gene may allow for noninvasive monitoring of cardiac gene therapy. We sought to evaluate the usefulness of an adenoviral vector expressing mutant herpesviral thymidine kinase reporter gene (HSV1-sr39tk) and vascular endothelial growth factor (VEGF) 121 in independent expression cassettes (Ad4tk). METHODS: Accumulation of 14C-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FIAU) and 9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) as reporter probes, and secretion of VEGF into medium, were determined for Ad4tk-infected H9c2 rat cardiac cells in vitro. RESULTS: In vitro tracer uptake increased with increasing vector concentration and over time. It was comparable to cells infected with adenovirus expressing only wild-type HSV1-tk (reporter probe: 14C-FIAU) or mutant HSV1-sr39tk (reporter probe: 18F-FHBG). No significant uptake was observed in cells infected with adenovirus expressing VEGF alone. With increasing vector concentration, Ad4tk-infected cells increasingly released VEGF into medium. VEGF production correlated significantly with cellular reporter probe uptake (r = 0.93; P = 0.0003). CONCLUSION: The usefulness of a vector coexpressing HSV1-tk and VEGF for noninvasive imaging of expression of a therapeutic transgene has been demonstrated in vitro. This approach may allow for future in vivo monitoring of cardiac angiogenesis gene therapy.

Animals↗
PubMed2003

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis.

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.

Animals↗
PubMed2003

Adenovirus E4 gene promotes selective endothelial cell survival and angiogenesis via activation of the vascular endothelial-cadherin/Akt signaling pathway.

The early 4 region (E4) of the adenoviral vectors (AdE4(+)) prolongs human endothelial cell (EC) survival and alters the angiogenic response, although the mechanisms for the EC-specific, AdE4(+)-mediated effects remain unknown. We hypothesized that AdE4(+) modulates EC survival through activation of the vascular endothelial (VE)-cadherin/Akt pathway. Here, we showed that AdE4(+), but not AdE4(-) vectors, selectively stimulated phosphorylation of both Akt at Ser(473) and Src kinase in ECs. The phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin abrogated AdE4(+) induction of both phospho-Akt expression and prolonged EC survival. Regulation of phospho-Akt was found to be under the control of various factors, namely VE-cadherin activation, Src kinase, tyrosine kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Downstream targets of Akt signaling resulted in glycogen synthase kinase-3alpha/beta phosphorylation, beta-catenin up-regulation, and caspase-3 suppression, all of which led to AdE4(+)-mediated EC survival. Furthermore, infection with AdE4(+) vectors increased the angiogenic potential of ECs by promoting EC migration and capillary tube formation in Matrigel plugs. This selective AdE4(+)-mediated enhanced motility of ECs was also blocked by PI3K inhibitors. Taken together, these results suggest that activation of the VE-cadherin/Akt pathway is critical for AdE4(+)-mediated survival of ECs and angiogenic potential.

Adenovirus E4 Proteins↗
PubMed2003

Protective immunity evoked against anthrax lethal toxin after a single intramuscular administration of an adenovirus-based vaccine encoding humanized protective antigen.

Because of the need to develop a vaccine to rapidly protect the civilian population in response to a bioterrorism attack with Bacillus anthracis, we designed AdsechPA, a replication-deficient human serotype 5 adenovirus encoding B. anthracis protective antigen (PA) with codons optimized for expression in mammalian cells. With a single intramuscular administration to mice of 10(9) particle units of AdsechPA, a dose that can be scaled to human use, anti-PA antibodies were evoked more rapidly and at a higher level than with a single administration of the new U.S. military recombinant PA/Alhydrogel vaccine. Importantly, AdsechPA afforded approximately 2.7-fold more protection than the recombinant PA vaccine against B. anthracis lethal toxin challenge 4 weeks after a single vaccination. Even at 11 days postvaccination, AdsechPA provided some survival benefit, whereas the rPA/Alhydrogel vaccine provided none. In the context that equivalent human doses of Ad vectors have already been demonstrated to be safe in humans, a single administration of AdsechPA may provide the means to rapidly protect the civilian population against B. anthracis in response to a bioterrorism attack.

Adenoviridae↗
PubMed2003

Enhancement of spine fusion using combined gene therapy and tissue engineering BMP-7-expressing bone marrow cells and allograft bone.

STUDY DESIGN: Prospective study to assess the enhancement of spine fusion using a tissue engineering construct consisting of bone marrow cells genetically modified by adenovirus (Ad) vector-encoding bone morphogenetic protein-7 (BMP-7) seeded onto an allograft scaffold in a rat model. OBJECTIVES: To evaluate Ad transgene expression at the fusion site and the effect of AdBMP-7-treatment on fusion rates, mechanical stability, microscopic anatomy, and bone formation rates. SUMMARY OF BACKGROUND DATA: Nonunion is a major complication of spine fusion. Gene transfer may be an effective method for locally overexpressing BMP-7, a gene important for bone formation and regeneration to enhance allograft spine fusion. MATERIALS AND METHODS: Bone marrow cells were treated with AdBMP-7 or Adbetagal (encoding the marker gene beta-galactosidase), AdNull (with no gene), or no vector and implanted with allograft in a site of posterior spine fusion. Marker gene expression was assessed up to 14 days after administration. Fusions were evaluated at 8 weeks. RESULTS: Ad gene expression was maximal on day 3, waning to background levels by 14 days. With AdBMP-7 treatment, radiographic fusion rate was 70% and mechanical fusion rate was 80% versus 0% by either parameter in control groups. Fused AdBMP-7-treated spines had a 2.5-fold to 3.0-fold lower range of motion and 1.7-fold to 1.9-fold lower hysteresis than controls. Fusion masses of AdBMP-7-treated spines had the microscopic appearance of normal trabecular bone and showed a 23-fold higher uptake of fluorochrome indicating increased bone formation. CONCLUSIONS: Addition of AdBMP-7-modified marrow cells can enhance allograft spine fusion.

Adenoviridae↗
PubMed2003

Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells.

Reporter genes, including green fluorescent protein (GFP), have been used to monitor the expression of transgenes introduced into vascular cells by gene transfer vectors. Here, we demonstrate that GFP by itself can selectively induce expression of certain genes in endothelial cells. Elevation of the cytoplasmic concentration of GFP in endothelial cells, specifically, resulted in a robust upregulation of heat shock protein 70 (HSP70). GFP induced both mRNA and protein expression of HSP70 in a dose-dependent manner. GFP-mediated up-regulation of HSP70 resulted in induction of cyclooxygenase-2 (COX-2) followed by prostaglandin E2 (PGE2) production. GFP-mediated up-regulation of HSP70 is independent of mitogen-activated protein kinase and phosphatidylinositol-3-kinase signaling cascades because inhibition of these pathways had no effect on HSP70 increases. Adenoviral delivery of GFP into murine vasculature significantly enhanced blood flow, suggesting that sufficient PGE2 is produced to induce vasodilation. Identification of the molecular partners that interact with GFP will increase our understanding of the vascular-specific factors that regulate stress angiogenesis and hemostasis.

Adenoviridae↗
PubMed2003

Variability of antioxidant-related gene expression in the airway epithelium of cigarette smokers.

Cigarette smoking is the major risk factor for developing chronic bronchitis, yet only 15-20% of smokers develop this disorder. Because oxidants are the major mechanism of smoking-induced airway damage, we hypothesized that smoking is associated with upregulation of various antioxidant-related genes in the airway epithelium, but the magnitude of the response shows high inter-individual variability. Microarray analysis was used to assess levels of expression of 44 antioxidant-related genes in four categories (catalase/superoxide dismutase family; glutathione metabolism; redox balance; and pentose phosphate cycle) in bronchoscopy-obtained airway epithelium of matched cohorts (13 current smokers, 9 nonsmokers), none of whom had lung disease. There was minimal variation in gene expression levels within the same individual (right versus left lung or over time), but significant upregulation of 16/44 antioxidant-related genes in smoker epithelium compared with nonsmokers. Subgroups of smokers were identified with clusters of expression levels of antioxidant-related genes. We propose that the antioxidant-related genes demonstrating the most variability in the level of expression in smokers may be useful genetic markers in epidemiologic studies assessing susceptibility to smoking-induced chronic bronchitis.

Adult↗
PubMed2003

Monoallelic up-regulation of the imprinted H19 gene in airway epithelium of phenotypically normal cigarette smokers.

H19, a paternally imprinted gene, is postulated to have regulatory functions in normal development and oncogenesis. Loss of imprinting (LOI) of H19 is observed in human malignancies, including lung cancer. Microarray assessment of gene expression patterns in airway epithelium of healthy 20 pack-year smokers versus nonsmokers revealed that smokers have dramatically elevated H19 RNA levels without alteration of expression of other imprinted genes. Interestingly, the up-regulation of H19 was not attributable to LOI, i.e., expression of H19 in smokers was monoallelic. These observations suggest that cigarette smoking initially induces up-regulation of the active H19 allele and that there is likely progression to LOI as the burden of smoking increases and as the epithelium undergoes transition from normal to neoplastic. Overexpression and eventual LOI of H19 may represent early markers in the progression of airway epithelium toward lung cancer.

Adult↗
PubMed2003

In vivo trans-splicing of 5' and 3' segments of pre-mRNA directed by corresponding DNA sequences delivered by gene transfer.

We have developed a new paradigm of in vivo gene transfer termed "segmental trans-splicing" (STS), in which individual "donor" and "acceptor" DNA sequences, delivered in vitro or in vivo, generate pre-mRNAs with 5' and 3' splice signals, respectively, and complementary hybridization domains through which the two pre-mRNAs interact, facilitating trans-splicing of the two mRNA fragments. To demonstrate STS, we used alpha-cobratoxin, a neurotoxin that binds irreversibly to postsynaptic nicotinic acetylcholine receptors. Cells or animals receiving both donor and acceptor plasmids, but neither plasmid alone, yielded RT-PCR products with the correct sequence of mature alpha-cobratoxin mRNA, suggesting that trans-splicing had occurred. Mice receiving intravenous administration of > or = 7.5 microg donor + acceptor plasmids, but not either plasmid alone, died within 6 h. These data demonstrate that segmental trans-splicing occurs in vivo. This approach should permit the intracellular assembly of molecules hitherto too large to be accommodated within current gene transfer vectors.

Animals↗
PubMed2003

Active, specific immunotherapy for lung cancer: hurdles and strategies using genetic modification.

Active immunotherapy for lung cancer has been a challenge because of the poor antigenic characterization of these tumors and their ability to escape the immune response. However, knowledge of the mechanisms of anti-tumor immunity has expanded significantly over the past decade, leading to the development of more novel, specific strategies for augmenting the immune response. Genetic manipulation of tumor cells, immune cells, or both, may help overcome some of the previously encountered difficulties of immunotherapy. Laboratory and clinical investigations are currently ongoing to evaluate the feasibility and potential benefit of these novel approaches.

Animals↗
PubMed2003

In vivo gene transfer of pigment epithelium-derived factor inhibits tumor growth in syngeneic murine models of thoracic malignancies.

OBJECTIVE: Pigment epithelium-derived factor is known to be an inhibitor of angiogenesis. We hypothesized that in vivo gene transfer of pigment epithelium-derived factor may inhibit tumor angiogenesis and growth in syngeneic models of thoracic malignancies. METHODS: An adenovirus vector encoding the human pigment epithelium-derived factor cDNA (AdPEDF) was used to transduce human lung cancer cells in vitro. Transgene expression was assessed using Western analysis. Three different murine flank tumors (2 lung, 1 colon) were then established in syngeneic mice and treated intratumorally with phosphate-buffered saline, AdPEDF, or an empty vector (AdNull). Endpoints measured included transgene expression, tumor size, and animal survival, as well as microvessel density within the tumor. Additionally, a murine pulmonary metastasis model was established by intravenous injection of a syngeneic colon adenocarcinoma cell line expressing a marker gene (beta-galactosidase). One day later, treatment (phosphate-buffered saline, AdNull, or AdPEDF) was administered intrapleurally. Tumor burden (gross and histologic inspection, lung weight, and beta-galactosidase expression) was then evaluated 13 days after vector dosing, and survival was recorded. RESULTS: AdPEDF-derived expression of pigment epithelium-derived factor was demonstrated in vitro and in vivo. In syngeneic murine lung cancer flank tumors, intratumoral administration of AdPEDF significantly inhibited tumor growth (P <.01), prolonged mouse survival (P <.01), and decreased microvessel density (P <.01) compared with control groups. In the pulmonary metastasis model, AdPEDF-treated mice exhibited significantly reduced lung lesions, lung weight (P <.0005), beta-galactosidase expression (P <.05), and animal survival was prolonged (P <.05). CONCLUSION: Gene transfer of pigment epithelium-derived factor suppresses tumor vascularization and growth, while prolonging survival in syngeneic murine models of thoracic malignancies.

Adenoviruses, Human↗
PubMed2003

Cisplatin augments cytotoxic T-lymphocyte-mediated antitumor immunity in poorly immunogenic murine lung cancer.

OBJECTIVE: Many tumors are poorly immunogenic and resistant to cytotoxic T-lymphocyte-mediated cell lysis. Because cisplatin has been demonstrated to increase tumor cell Fas receptor expression, we hypothesized that cisplatin will enhance cytotoxic T-lymphocyte tumor cell killing and augment the antitumor effect of an active immunotherapy strategy in a poorly immunogenic murine lung cancer model. METHODS: Lewis lung carcinoma cells were exposed to cisplatin in vitro, and Fas receptor expression and apoptosis in response to an agonistic anti-Fas antibody were quantified using flow cytometry. Wild-type and Fas ligand-deficient mice bearing Lewis lung carcinoma flank tumors were then treated with intraperitoneal cisplatin as well as an intratumoral injection of an adenovirus gene transfer vector encoding CD40 ligand. End points included tumor size, animal survival, and Fas expression (determined using immunofluorescence). Cytotoxicity assays were performed using splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated animals as effectors and cisplatin-treated Lewis lung carcinoma cells as targets. RESULTS: Cisplatin induced heightened expression of Fas receptor on Lewis lung carcinoma cells in vitro and in vivo and enhanced apoptosis in cells exposed to an agonistic anti-Fas antibody. In vivo, the combination of 1 dose of intraperitoneal cisplatin and intratumoral adenovirus gene transfer vector encoding CD40 ligand inhibited tumor growth and prolonged survival compared with adenovirus gene transfer vector encoding CD40 ligand alone, resulting in a higher cure rate. This effect was lost in Fas ligand-deficient mice. Splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated wild-type mice lysed cisplatin-treated Lewis lung carcinoma cells more efficiently than untreated Lewis lung carcinoma cells, an effect lost in splenocytes from Fas ligand-deficient mice. CONCLUSION: Cisplatin augments the antitumor effect of a cytotoxic T-lymphocyte-mediated immunotherapy strategy, resulting in a higher cure rate than seen with immunotherapy alone. This effect is associated with the enhanced ability of cytotoxic T lymphocytes to lyse tumor cells that have been exposed to cisplatin through Fas/Fas ligand interactions.

Adenoviridae↗
PubMed2003

Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7.

BACKGROUND: Cartilage has a limited capacity to heal. Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy. Previously we have shown that over expression of bone morphogenetic protein-7 (BMP-7) in chondrocytes by adenovirus-mediated gene transfer leads to increased matrix synthesis and cartilage-like tissue formation in vitro. In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP-7 would accelerate the formation of hyaline-like repair tissue in an equine model of cartilage defect repair. METHODS: Chondrocytes treated with adenovirus vector encoding BMP-7 (AdBMP-7) or as control, an adenovirus vector encoding an irrelevant gene (Escherichia coli cytosine deaminase, AdCD) were implanted into extensive (15 mm diameter) articular cartilage defects in the patellofemoral joints of 10 horses. Biopsies were performed to evaluate early healing at 4 weeks. At the terminal time point of 8 months, repairs were assessed for morphology, MRI appearance, compressive strength, biochemical composition and persistence of implanted cells. RESULTS: Four weeks after surgery AdBMP-7-treated repairs showed an increased level of BMP-7 expression and accelerated healing, with markedly more hyaline-like morphology than control. Quantitative real-time polymerase chain reaction (PCR) analysis of the repair tissue 8 months after surgery showed that few implanted cells persisted. By this time, the controls had healed similarly to the AdBMP-7-treated defects, and no difference was detected in the morphologic, biochemical or biomechanical properties of the repair tissues from the two treatment groups. CONCLUSIONS: Implantation of genetically modified chondrocytes expressing BMP-7 accelerates the appearance of hyaline-like repair tissue in experimental cartilage defects. CLINICAL RELEVANCE: Rehabilitation after cell-based cartilage repair can be prolonged, leading to decreased patient productivity and quality of life. This study shows the feasibility of using genetically modified chondrocytes to accelerate cartilage healing.

Adenoviridae↗