Search PubMed⌕ Search

Biomedical subjects

R G Crystal

Publications and source records attributed to R G Crystal.

At least 145 records · Page 8Linked to original sources
PubMed1996

Clinical features of individuals with PI*SZ phenotype of alpha 1-antitrypsin deficiency. alpha 1-Antitrypsin Deficiency Registry Study Group.

This report describes the clinical characteristics of a group of 59 individuals with the PI*SZ phenotype and alpha 1-antitrypsin (alpha 1-AT) deficiency, identified during recruitment of a registry for subjects with severe alpha 1-antitrypsin deficiency. Currently, 1,129 individuals with levels of alpha 1-AT of 11 microM or below have been enrolled in this registry. Individuals with the SZ phenotype whose alpha 1-AT levels are at or below 11 microM will be followed in the registry; those whose levels exceeded 11 microM had baseline studies and are included in this report. Baseline pulmonary function tests included spirometry before and after an inhaled bronchodilator, diffusing capacity for carbon monoxide (DLCO), and chest roentgenograms. Among nonsmokers, subjects with the SZ phenotype demonstrated airflow obstruction less frequently than those with with the ZZ phenotype. Among ex- and current smokers, the frequency and severity of airflow obstruction was similar between SZ and ZZ subjects. Individuals with the SZ phenotype reported respiratory symptoms less frequently than did ZZ subjects. Overall, airflow obstruction was less common and milder among PI*SZ than PI*ZZ subjects. Cigarette smoking correlated more strongly with airflow obstruction among PI*SZ than PI*ZZ subjects. These observations indicate that in smokers, the PI*SZ phenotype confers a significant risk of the development of chronic obstructive pulmonary disease (COPD). Of itself, except in rare instances in nonsmoking individuals, the PI*SZ phenotype may confer little or no added risk of developing COPD.

Adult↗
PubMed1996

Activated neutrophils secrete stored alpha 1-antitrypsin.

Neutrophil elastase (NE), a potent serine protease, is stored in primary granules of neutrophils and released following neutrophil activation. Alpha-1-antitrypsin (alpha 1-AT), the major inhibitor of NE, is synthesized by mature neutrophils. In the context of the maintenance of tissue homeostasis, we hypothesized that neutrophils may be able to store alpha 1-AT, thus having it available for release concordantly with NE. Immunofluorescence and quantitative flow-cytometric studies of neutrophils and monocytes labeled with fluorescein-conjugated alpha 1-AT-antibody demonstrated larger amounts of cytoplasmic alpha 1-AT in neutrophils than in monocytes. [35S]methionine-labeling and anti-alpha 1-AT immunoprecipitation analysis showed that although both neutrophils and monocytes synthesize alpha 1-AT, the proportion of newly synthesized intracellular alpha 1-AT was much higher in neutrophils than in monocytes. Flow-cytometric analysis showed that in the presence of surface stimulation with cytochalasin B followed by formyl-methionyleucylphenylalanine (fMLP), mean intracellular alpha 1-AT was decreased in stimulated neutrophils compared with that in resting cells, suggesting that the stored alpha 1-AT was rapidly released following surface triggering. Evaluation of surface-stimulated neutrophils by [35S]methionine labeling and anti-alpha 1-AT immunoprecipitation demonstrated increased secretion of alpha 1-AT compared with that of resting neutrophils, with some of the secreted alpha 1-AT capable of forming complexes with NE. Thus, neutrophils respond to surface stimulation not only by secreting NE but also by secreting its inhibitor, alpha 1-AT, suggesting that these cells have an inherent mechanism for damping the local effects of NE, their most powerful proteolytic enzyme.

Cytochalasin B↗
PubMed1996

Direct in vivo gene transfer to canine myocardium using a replication-deficient adenovirus vector.

BACKGROUND: Direct myocardial gene transfer is a mordality that involves the introduction of genetic information into myocardial tissue to achieve a therapeutic effect. This study was designed to characterize the temporal and spatial limits of gene expression and to determine the safety of direct myocardial gene transfer in a large animal model using replication-deficient adenovirus vectors. METHODS: Mongrel dogs underwent left thoracotomy and direct myocardial injections (100 microL/injection) of adenovirus vectors (10(9) pfu) carrying the DNA for the reporter enzyme chloramphenicol acetyl transferase or the angiogenic protein vascular endothelial growth factor. Two to 14 days after vector administration, regional protein expression was evaluated in myocardium and distant organs. Left ventricular function, assessed by echocardiography, and routine hematologic and biochemical indices were evaluated before and after vector administration. RESULTS: Peak levels of chloramphenicol acetyl transferase activity were detected 2 days after vector administration, and levels above baseline persisted for at least 14 days. Local chloramphenicol acetyl transferase activity was detected at distances at least as far as 1.5 cm from the site of injection. Chloramphenicol acetyl transferase activity in distant organs was less than 0.1% of that in injected myocardium 7 days after vector administration. Localized expression of vascular endothelial growth factor was achieved for up to 7 days after a single vector administration. Cardiac function and laboratory values were unchanged during the study. CONCLUSIONS: Adenovirus-mediated direct myocardial gene transfer can be accomplished safely in a large animal model, providing high levels of protein expression in a greater spatial distribution than previously reported, with minimal transfection of distant organs. Sustained and localized expression of a potent angiogenic mediator has been accomplished, which may provide an innovative strategy to stimulate angiogenesis in ischemic myocardium.

Adenoviridae↗
PubMed1996

Safety and efficacy of in vivo gene transfer into the porcine heart with replication-deficient, recombinant adenovirus vectors.

Gene transfer with replication-deficient recombinant adenovirus (Ad) vectors may provide a novel approach to the treatment of some cardiac disorders. The relative efficiency of intramyocardial vs intracoronary Ad vector injection in transducing myocardial cells remains to be determined. Further, Ad vectors are associated with localized inflammation, and this could be associated with clinically significant side-effects. Female minipigs underwent open chest surgery and the Ad vector AdCMV.NLS beta-gal was injected into the circumflex coronary artery (IC; 2 x 10(10) p.f.u.; n = 5) or the posterobasal wall of the left ventricle (i.m.; 5 x 10(9) p.f.u., n = 4; 2 x 10(10) p.f.u., n = 18). The minipigs were killed after 2-31 days and the hearts examined for evidence of beta-galactosidase activity. Minipigs underwent epicardial echocardiography immediately before, within 15 min following the i.m. injection of AdCMV.NLS beta-gal and again at the time of death. Blood samples for white blood cell count, alkaline phosphatase, total bilirubin, blood urea nitrogen, creatinine and electrolytes were obtained before i.m. and i.c. injection of the Ad vector and before death. Intramuscular injection of the Ad vector was more efficient than i.c. infusion in infecting cells in a localized area of the heart. Myocardial beta-gal activity peaked at 3-6 days after i.m. injection and returned to its control value within 1 month. Although inflammatory cells were present at the injection site, echocardiograms did not show any evidence of either segmental or global left ventricular dysfunction. No minipigs died and all blood tests remained within normal limits following either i.m. or i.c. exposure to the Ad vector. In summary, direct i.m. administration of replication-deficient, recombinant Ad vectors provides a safe and effective approach for short-term gene transfer into the heart of large mammals.

Adenoviruses, Human↗
PubMed1995

Adenovirus-mediated gene transfer of dopamine D2 receptor cDNA into rat striatum.

A robust feature of mammalian aging associated with diminished motor control is the loss of dopamine D2 receptors from the neostriatum. Decline in this neurotransmitter receptor is also observed in neurodegenerative disorders, such as Huntington's disease and late-stage Parkinson's disease. We have constructed a replication-deficient adenoviral vector to transfer rat dopamine D2 receptor cDNA to brain as a possible therapeutic strategy. Using tissue culture cells infected with this vector, we detected dopamine D2 receptor mRNA by Northern analysis and functional receptor protein in membrane preparations as specific binding of the dopamine D2 receptor ligand, [3H]spiperone. In vivo demonstration involved autoradiographic analysis of [3H]spiperone binding in rat striatum following injection of the adenoviral vector. Dopamine D2 receptor expression was amplified markedly above normal concentrations in the injection site, whereas no increased expression was observed in sites receiving control treatments. These results demonstrate the potential of gene therapy using adenoviral vectors to transfer neurotransmitter receptor proteins to the brain to reverse deficiencies in specific neurodegenerative disorders.

Adenoviridae↗
PubMed1995

Retrograde transfer of replication deficient recombinant adenovirus vector in the central nervous system for tracing studies.

We assessed the application of a replication deficient recombinant adenovirus vector as a retrograde tracer in neural pathway studies. The adenovirus vector, Ad. RSV betagal, containing the intracellular marker gene, beta-galactosidase, was injected directly into the laterodorsal striatum of rats. The retrograde transport of the vector from the injection site was clearly visible in the cerebral cortex, thalamic nucleus, and substantia nigra. No evidence for anterograde transport of the vector was found. When the vector was injected into the genu of the corpus callosum, little uptake of the vector by fibers was noted which suggested that uptake by fibers-of-passage should not be a problem in tracing studies. The present study demonstrates that adenoviral vectors can be useful retrograde tracers in the study of afferent connections within the central nervous system.

Adenoviruses, Human↗
PubMed1995

Transfer of genes to humans: early lessons and obstacles to success.

Enough information has been gained from clinical trials to allow the conclusion that human gene transfer is feasible, can evoke biologic responses that are relevant to human disease, and can provide important insights into human biology. Adverse events have been uncommon and have been related to the gene delivery strategies, not to the genetic material being transferred. Human gene transfer still faces significant hurdles before it becomes an established therapeutic strategy. However, its accomplishments to date are impressive, and the logic of the potential usefulness of this clinical paradigm continues to be compelling.

Adenoviridae↗
PubMed1995

Identification of a 27 bp 5'-flanking region element responsible for the low level constitutive expression of the human cytosolic phospholipase A2 gene.

The cytosolic phospholipase A2 (cPLA2) gene codes for an enzyme that liberates arachidonic acid from membrane phospholipids, and thus plays a pivotal role in the production of the prostaglandin and leukotriene mediators of inflammation, as well as in a variety of cell signalling pathways. After preliminary studies demonstrated the cPLA2 gene is expressed in a variety of human tissues and was localized to the q arm of chromosome 1 between markers F13B and D1S74, we cloned and characterized the 5'-flanking region of this gene in order to identify the elements controlling its low level constitutive expression. The 5'-flanking region has features typical of a housekeeping gene with no TATA box or CAAT box, although atypical in that it is not GC rich, has no SP1 sites, and has a long run of CA repeats. Analysis of fragments of the 5'-flanking region demonstrated that 541 bp 5' to exon 1 supported reporter gene activity at a level 30% of the SV40 promoter. Interestingly, similar activity was observed by deleting most of the 5'-flanking region down to a 27 bp region containing a sequence with homology to the initiator sequence in the terminal deoxynucleotidyl transferase gene and a polypyrimidine tract similar to the initiator element of the mouse ribosomal protein gene. Within this 27 bp region, a 10 bp fragment (-17 to -8 bp) within the polypyrimidine tract is critical for the baseline expression of the human cPLA2 gene. While the 5'-flanking region contains a putative composite AP-1 and glucocorticoid response element, this region does not respond to tumor necrosis factor-alpha (TNF) and/or glucocorticoids in a cell line (HEp-2) that exhibits upregulation of cPLA2 mRNA transcript levels by TNF. The observations that the expression of the cPLA2 gene is tightly controlled at a relatively low level is consistent with the evolving concept that modulation of expression of this critical enzyme is primarily at the post-translational level.

Base Sequence↗
PubMed1995

In vivo angiogenesis induced by recombinant adenovirus vectors coding either for secreted or nonsecreted forms of acidic fibroblast growth factor.

In vivo gene transfer of angiogenic growth factors represents a potential approach to the treatment of ischemic diseases. The present study examined the in vitro and in vivo effects of two replication-deficient recombinant adenovirus (Ad) vectors coding for human acidic fibroblast growth factor (aFGF1-154). One vector codes for the nonsecreted form of the peptide (AdCMV.aFGF1-154), and the other vector codes for a recombinant, secreted form (AdCMV.sp+aFGF1-154). AdCMV.NLS beta gal, an adenovirus vector coding for beta-galactosidase (beta-Gal), was used as a control. Assessment of proliferation of starved human umbilical vein endothelial cells infected with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154 (20 pfu/cell) showed approximately 6- and 10-fold increase in cell number over control, respectively. Infection with AdCMV.sp+aFGF1-154 and with AdCMV.aFGF1-154 enhanced endothelial cell differentiation into capillary-like structures in vitro. However, this effect was significantly more pronounced with AdCMV.sp+aFGF1-154 than with AdCMV.aFGF1-154. Angiogenesis in vivo was assessed by injecting subcutaneously into mice 750 microliters of reconstituted basement membrane proteins (Matrigel) and the Ad vectors (2 x 10(8) pfu). After 14 days, there was histologic evidence of neovascularization in the animal's tissue surrounding the Matrigel plugs with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154. Further, the hemoglobin content of the Matrigel plugs with AdCMV.aFGF1-154 and with AdCMV.sp+aFGF1-154 was, respectively, 2.3- and 2.6-fold higher than with AdCMV.NLS beta gal. Together, these observations support the concept that adenovirus vectors coding for various forms of acidic FGF1-154 may be used to induce angiogenesis in vivo and may provide a new therapeutic approach to ischemic diseases.

Adenoviridae↗
PubMed1995

Modification of nasal epithelial potential differences of individuals with cystic fibrosis consequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector.

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) manifest on the nasal epithelial surface of individuals with cystic fibrosis (CF) by Na+ hyperabsorption and diminished beta-agonist-induced Cl- conductance, resulting in an abnormal bioelectric phenotype across the nasal epithelium. A clinical trial was conducted to determine if a replication-deficient, recombinant adenovirus vector containing a normal copy of the CFTR cDNA (AdCFTR) could, when administered to the nasal epithelium, correct the abnormal bioelectric phenotype. Nine individuals with CF received 2 x 10(5) to 2 x 10(8.5) plaque forming units of AdCFTR to the epithelium of one nostril. Measurements made included: baseline electrical potential difference (PD) between the surface of the nasal epithelium and the interstitial fluid, change in PD in response to amiloride, which inhibits apical Na+ channels, and change in PD in response to isoproterenol in a low Cl- solution, a measure of cAMP-regulated Cl- conductance. The functional integrity of the epithelium was evaluated by the PD response to ATP. Each individual served as their own control with measurements made in the nostril to be treated before AdCFTR administration, and in the contralateral untreated nostril. On the average, in the treated nostril over 2 weeks after the local administration of the adenovirus vector compared to measurements made in the same nostril before treatment, baseline PD decreased toward normal (-53.3 +/- 4.0 to -34.6 +/- 3.4, p = 0.01), response to amiloride decreased toward normal (36.9 +/- 4.7 to 19.7 +/- 3.0, p = 0.02), and response to low Cl- and isoproterenol increased toward normal (-4.5 +/- 1.5 to -9.1 +/- 2.1, p = 0.05). There were no changes in response to ATP (-15.3 +/- 2.7 to -15.8 +/- 1.9, p = 0.39), suggesting that the epithelium remained functionally intact. Importantly, there were no significant changes in measurements made in the untreated nostril. While limited to the nasal epithelium, these data suggest an adenovirus vector can safely deliver sufficient CFTR cDNA function to improve the abnormal CF bioelectric phenotype.

Adenoviridae↗
PubMed1995

In vitro and in vivo transfer and expression of human surfactant SP-A- and SP-B-associated protein cDNAs mediated by replication-deficient, recombinant adenoviral vectors.

Congenital pulmonary alveolar proteinosis (CPAP) is a fatal disease of full-term infants that is unresponsive to current medical therapy. It is now recognized that at least some forms of this disorder are associated with a deficiency of SP-B, one of the surfactant-associated proteins, as well as probable aberrations in the surfactant-associated proteins SP-A and SP-C. Given these developments, it is logical to hypothesize that CPAP may be amenable to gene therapy, in which the human SP-B cDNA, and possibly the cDNAs of the other surfactant associated proteins, are transferred to the epithelium of the lower respiratory tract. We constructed replication-deficient, recombinant adenovirus vectors in which a constitutive viral promoter drives the expression of the DNAs for the surfactant-associated proteins, SP-B (AdCMV.SP-B) and SP-A (AdCMV.SP-A). Following infection of the human lung A549 epithelial cell line with these vectors in vitro, the appropriately sized mRNAs for these cDNAs were detected, whereas cells infected with a control virus or uninfected cells produced none. Western blots demonstrated expression of these proteins, including appropriate processing of the hydrophobic protein, SP-B. Following in vivo intratracheal infection of rats with these vectors, Northern analysis of the lungs revealed appropriately sized mRNAs for these cDNAs whereas rats infected with control virus or uninfected rats show no hybridization with the human surfactant-associated protein probes. In the AdCM-V.SP-A-infected rats, Western blots confirmed the overproduction of the human SP-A protein in both the bronchoalveolar lavage and lung homogenates compared to controls. Thus, it is feasible to utilize adenovirus vectors to transfer and express the human surfactant associated protein cDNAs in vitro and in vivo, presenting a possible mode of therapy for CPAP, as well as other surfactant deficiency states such as the neonatal respiratory distress syndrome and possibly the adult respiratory distress syndrome.

Adenoviridae↗
PubMed1995

In vivo adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene to human colon carcinoma-derived tumors induces chemosensitivity to 5-fluorocytosine.

To evaluate the concept that in vivo transfer of the Escherichia coli cytosine deaminase gene will confer sensitivity of a solid tumor to the prodrug 5-fluorocytosine (5FC), we constructed an adenovirus vector (AdCMV.CD) carrying the cytosine deaminase gene driven by the cytomegalovirus (CMV) promoter, infected HT29 colon carcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CD produced a functional cytosine deaminase protein in HT29 cells in vitro as evidenced by the ability of lysates from the infected cells to convert [3H]5FC to its active metabolite 5-fluorouracil (5FU). The AdCMV.CD vector effectively suppressed HT29 cell growth in vitro in the presence of 5FC in a dose-dependent manner. Infection with AdCMV.CD, when as few as 10% of cells expressed the cytosine deaminase gene, was associated with a bystander effect when combined with 5FC in cell mixing studies. Further, this bystander effect was not dependent on cell-to-cell contact as demonstrated by suppression of [3H]thymidine incorporation in HT29 cells when supernatant from AdCMV.CD-infected cells treated with 5FC was transferred cells. Consistent with these in vitro observations, when AdCMV.CD was directly injected into established subcutaneous HT29 tumors in nude mice receiving 5FC, there was a four-fold reduction in tumor size at day 15 compared to controls, and a five-fold reduction at day 28. These observations suggest that adenovirus-mediated gene transfer of the E. coli cytosine deaminase gene and concomitant administration of 5FC may have potential as a strategy for local control of the growth of tumor cells susceptible to 5FU.

Adenoviridae↗
PubMed1995

Human CC10 gene expression in airway epithelium and subchromosomal locus suggest linkage to airway disease.

The CC10 gene encodes the Clara cell 10-kDa protein, which is expressed in airway epithelial cells. Quantification of CC10 gene expression in freshly isolated human proximal airway epithelial cells demonstrated very high mRNA levels, approximately fivefold greater than gamma-actin mRNA, and in situ hybridization localized CC10 mRNA to nonciliated airway epithelial cells. Sequence analysis demonstrated that the human CC10 gene is comprised of three short exons separated by a long first and short second intron, and with a 5' flanking region typical of a regulated gene. Three Alu repeats were observed in intron 1 and one in intron 2. Two polymorphic regions within the introns were identified. First, a microsatellite was localized 5' to the third Alu repeat in intron 1 with a variable number of 4- and 5-base pair (bp) repeats and a heterozygosity of 0.71. Second, in 3% of the 168 chromosomes examined, there was the insertion of a human-specific Alu repeat in intron 2, 45 bp 3' to the exon 2-intron junction. In three Centre d'Etude du Polymorphisme Humain families, meiotic breakpoint analysis using these two polymorphic loci localized the CC10 gene to 11 p12-q13 between markers D11S16 and D11S97, a region recently linked to atopy and to the beta-subunit of the high-affinity immunoglobulin E receptor. The observations in the present study of high-level expression of the CC10 gene in the epithelium of conducting airways and a subchromosomal localization of the gene to a region potentially linked to inflammatory airway disease, together with the reported anti-inflammatory and immune-modulating properties of the protein, suggest the CC10 gene product may be important in modulating inflammation within the airways. If so, the highly heterozygous microsatellite described in the present study should facilitate analysis of a possible linkage of the CC10 gene with an inherited susceptibility to asthma.

Asthma↗
PubMed1995

VEGF165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo.

To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF165 (where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF165, a secreted endothelial cell-specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HUVECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF165 (5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV.beta gal (where beta gal is beta-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF165-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF165. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF165 (20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.beta gal did not differentiate. To evaluate the ability of AdCMV.VEGF165 to function in vivo, either AdCMV. VEGF165 or AdCMV.beta gal (2 x 10(10) pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF165 up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.beta gal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF165, whereas no significant angiogenesis was observed in response to AdCMV.beta gal. Furthermore, the Matrigel plugs with AdCMV.VEGF165 demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.beta gal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF165 in the treatment of ischemic diseases.

Adenoviridae↗