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Biomedical subjects

R G Crystal

Publications and source records attributed to R G Crystal.

At least 217 records · Page 12Linked to original sources
PubMed1992

Extensive posttranscriptional deletion of the coding sequences for part of nucleotide-binding fold 1 in respiratory epithelial mRNA transcripts of the cystic fibrosis transmembrane conductance regulator gene is not associated with the clinical manifestations of cystic fibrosis.

Cystic fibrosis (CF) is a recessive hereditary disorder, requiring both parental cystic fibrosis conductance transmembrane regulator (CFTR) genes to carry mutations for clinical disease to manifest, i.e., only 50% of normal CFTR gene expression is required to maintain a normal phenotype. To help define the minimum amount of normal CFTR gene expression necessary to maintain normalcy, we have capitalized on our prior observation (Chu, C.-S., B. C. Trapnell, J. J. Murtagh, Jr., J. Moss, W. Dalemans, S. Jallat, A. Mercenier, A. Pavirani, J.-P. Lecocq, G. R. Cutting, et al. 1991. EMBO [Eur. Mol. Biol. Organ] J. 10:1355-1363) that normal individuals can have up to 66% of bronchial CFTR mRNA transcripts that are missing exon 9, a region representing 21% of the sequence coding for the critical nucleotide (ATP)-binding fold 1 (NBF1) of the predicted CFTR protein. The study population included 78 individuals with no prior diagnosis of CF. Evaluation of bronchial epithelial cells (obtained by bronchoscopy) revealed that exon 9 was variably deleted in all individuals. Remarkably, there were four individuals, all greater than or equal to 35 yr, in whom bronchial epithelial cells exhibited 73, 89, 90, and 92% CFTR transcripts with inframe deletion of exon 9, respectively, despite normal sweat Cl- and no clinical manifestation of CF. In the context that only 8% or less of bronchial CFTR transcripts need exon 9 to maintain normal airway function, these observations strongly suggest that either exon 9 is not necessary for CFTR structure and/or function or that only a very small fraction of bronchial epithelial cells need to express normal CFTR mRNA transcripts with exon 9 to perform the function of CFTR sufficient to maintain a normal phenotype in vivo.

Adult↗
PubMed1992

Modulation of airway inflammation in cystic fibrosis. In vivo suppression of interleukin-8 levels on the respiratory epithelial surface by aerosolization of recombinant secretory leukoprotease inhibitor.

Based on the knowledge that neutrophil elastase (NE) in cystic fibrosis (CF) epithelial lining fluid (ELF) can induce human bronchial epithelial cells to express the gene for interleukin 8 (IL-8), an 8.5-kD neutrophil chemoattractant, we have evaluated CF ELF for the presence of IL-8, and investigated the ability of aerosolized recombinant secretory leukoprotease inhibitor (rSLPI) to suppress NE, and hence IL-8, levels on the respiratory epithelial surface in CF. Enzyme-linked immunoassay revealed 21.9 +/- 4.8 nM IL-8 in CF ELF compared with none in normals. Active NE was detectable in ELF of all individuals with CF and was significantly decreased (P < 0.03) after aerosolization of rSLPI. Human bronchial epithelial cells exposed to CF ELF recovered before rSLPI therapy expressed IL-8 mRNA transcripts, but ELF recovered after rSLPI therapy induced far less bronchial epithelial cell IL-8 gene expression. Consistent with this, rSLPI aerosol therapy caused a marked reduction in CF ELF IL-8 levels (P < 0.05) and neutrophil number (P < 0.02). There was also a clear association between CF ELF active NE and IL-8 levels (r = 0.94). These data suggest that rSLPI therapy not only suppresses respiratory epithelial NE levels, but also breaks a cycle of inflammation on the CF epithelial surface.

Adult↗
PubMed1991

Interleukin-8 gene expression in human bronchial epithelial cells.

The capacity of cells of the human bronchial epithelium to express the gene for interleukin-8 (IL-8) was evaluated in bronchial epithelium derived cell lines, HS-24 and BET-1A, using tumor necrosis factor-alpha (TNF) as a model inflammatory stimulus. As in other epithelium, TNF markedly increased the level of the 1.8-kilobase IL-8 mRNA transcripts in both bronchial epithelial cell lines. In HS-24 cells, nuclear run-on analyses showed the IL-8 gene transcription rate was dramatically increased, more than 30-fold, after TNF stimulation. The half-life of IL-8 mRNA transcripts in these cells was approximately 40 min and did not change after TNF stimulation, suggesting that TNF up-regulated IL-8 gene expression mainly at the transcriptional level. DNase I hypersensitivity site mapping of chromatin DNA in resting HS-24 cells demonstrated two hypersensitivity sites within 400 base pairs (bp) 5' to exon I and one site within exon I. However, after TNF stimulation, the exon I hypersensitivity site disappeared and a new site approximately 120 bp 5' to exon I emerged. Consistent with these observations, transfection studies with HS-24 cells using fusion genes composed of the 5'-flanking sequences of the IL-8 gene and a luciferase reporter gene demonstrated potent promoter activity in a 174-bp segment (-130 to +44 relative to the transcription start site), which also exhibited a response to TNF, while a segment from -112 to +44 showed very low promoter activity and no response to TNF. Thus, human bronchial epithelial cells can express the IL-8 gene, with expression in response to the inflammatory mediator TNF regulated mainly at the transcriptional level, and with elements within the 5'-flanking region of the gene that are directly or indirectly modulated by the TNF signal.

Bronchi↗
PubMed1991

Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin.

Consistent with the fact that the clinical disorder cystic fibrosis (CF) is manifested on epithelial surfaces, active transcription of the CF transmembrane conductance regulator (CFTR) gene and CFTR mRNA transcripts are detectable in a variety of epithelial cells, suggesting CFTR gene expression might be epithelial cell-specific. However, analysis of the CFTR gene promoter suggests it is a housekeeping gene, implying more widespread expression than only in epithelial cells. To evaluate the latter hypothesis, various human cells of non-epithelial origin, including lung fibroblasts, U-937 histiocytic lymphoma cells, K-562 erythroleukemia cells, HL-60 promyelocytic leukemia cells as well as freshly isolated blood lymphocytes, neutrophils, monocytes, and alveolar macrophages were examined for CFTR gene expression. Although Northern analysis failed to show CFTR mRNA transcripts in these cells, amplification of mRNA (after conversion to cDNA) by polymerase chain reaction combined with Southern analysis demonstrated the presence of CFTR mRNA transcripts at low levels in all cells evaluated except HL-60 cells. Comparative quantitative analysis showed fibroblasts contained 200-400 fold less CFTR mRNA transcripts than the T84 and HT-29 colon carcinoma epithelial cell lines, but had similar levels of CFTR transcripts to those of other epithelial cell lines. Nuclear transcription run-on analyses demonstrated very low level CFTR gene transcription in fibroblasts and U-937 cells, similar to that of other epithelial cells, but lower than the T84 and HT-29 colon carcinoma cell lines. Interestingly, while chromatin DNA of fibroblasts had no DNase I hypersensitivity sites in the 5' flanking region of the CFTR gene, HT-29 chromatin DNA exhibited four DNase I accessible sites in the same region, suggesting that these sites may be related to more active transcription of the CFTR gene in the intestinal epithelial cells than in fibroblasts.

Base Sequence↗
PubMed1991

Respiratory tract gene transfer. Transplantation of genetically modified T-lymphocytes directly to the respiratory epithelial surface.

To evaluate the strategy for potentially treating respiratory disorders with genetically modified T-lymphocytes, the interleukin-2 (IL-2)-dependent murine T-cell line, CTLL2, was genetically altered with the Escherichia coli beta-galactosidase (beta-gal) gene (lacZ) in vitro with a retroviral vector and the modified T-cells were transplanted directly to the respiratory epithelial surface of syngeneic C57Bl/6 mice. Southern and Northern analyses confirmed that the neomycin-selected modified T-cells contained and expressed the lacZ gene. The fate of the modified T-cells (CTLL2/lacZ) was followed by flow cytometry with T-cell surface marker Thy1.2 and fluorescent beta-gal analysis. One day after transplantation (7.5 x 10(5) CTLL2/lacZ T-cells/g of body weight), 95 +/- 3% of the Thy1.2+ T-cells recovered from respiratory epithelial lining fluid (ELF) were beta-gal+. Importantly, the modified T-cells remained in the lung for some time; at 3 days, Thy1.2+ beta-gal+ T-cells represented 63 +/- 12% of ELF Thy1.2+ T-cells and 59 +/- 6% of Thy1.2+ T-cells recovered from the whole lung. At 7 days, 33 +/- 8% of the Thy 1.2+ cells in ELF and 75 +/- 6% of the Thy1.2+ cells in whole lung were Thy1.2+ beta-gal+. In contrast, the proportion of the Thy1.2+ beta-gal+ T-cells in the spleen, the major extrapulmonary lymphatic organ, never rose above 3 +/- 1% of the total Thy1.2+ cells. The number of Thy1.2+ beta-gal+ T-cells in the lung could be modified by the systemic administration of IL-2, with whole lung Thy1.2+ beta-gal+ T-cells increasing 4.6-fold 3 days after transplantation, compared with non-IL-2-treated animals. These studies suggest that direct transplantation of genetically modified T-cells into the lung is feasible and represents a viable strategy for lung-specific gene transfer.

Animals↗
PubMed1991

Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis.

The most common mutation of the cystic fibrosis transmembrane conductance regulator gene, CFTR, associated with the clinical disorder cystic fibrosis (CF) is called "delta Phe508," a triple-base deletion resulting in loss of phenylalanine at residue 508 of the predicted 1480-amino acid CFTR protein. In the context that the lung is the major site of morbidity and mortality in CF, we evaluated airway epithelial cells for CFTR mRNA transcripts in normal individuals, normal-delta Phe508 heterozygotes, and delta Phe508 homozygotes to determine if the normal and delta Phe508 CFTR alleles are expressed in the respiratory epithelium, to what extent they are expressed, and whether there are relative differences in the expression of the normal and abnormal alleles at the mRNA level. Respiratory tract epithelial cells recovered by fiberoptic bronchoscopy with a cytology brush demonstrated CFTR mRNA transcripts with sequences appropriately reflecting the normal and delta Phe508 CFTR alleles of the various study groups. CFTR gene expression quantified by limited polymerase chain reaction amplification showed that in normal individuals, CFTR mRNA transcripts are expressed in nasal, tracheal, and bronchial epithelial cells at approximately 1-2 copies per cell, more than 100-fold greater than in pharyngeal epithelium. Importantly, allele-specific hybridization studies demonstrated that the normal and delta Phe508 CFTR alleles are expressed in the respiratory epithelium in similar amounts.

Base Sequence↗
PubMed1991

Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterised by alveolar inflammation, exaggerated release of oxidants, and subnormal concentrations of the antioxidant glutathione in respiratory epithelial lining fluid (ELF). Glutathione (600 mg twice daily for 3 days) was given by aerosol to 10 patients with IPF. Total ELF glutathione rose transiently, ELF oxidised glutathione concentrations increased, and there was a decrease in spontaneous superoxide anion release by alveolar macrophages. Thus, glutathione by aerosol could be a means of reversing the oxidant-antioxidant imbalance in IPF.

Adult↗
PubMed1991

Human neutrophils express the alpha 1-antitrypsin gene and produce alpha 1-antitrypsin.

The potent serine protease, neutrophil elastase (NE), is stored in neutrophil azurophilic granules, where it is available to degrade phagocytosed material and can be released by the cell to assist in tissue migration and help clear tissue debris. While neutrophils carry NE, they cannot produce it; the NE gene is expressed only in bone marrow granulocyte precursor cells. Protection of normal tissues from the destructive capacity of NE is provided by alpha 1-antitrypsin (alpha 1 AT), a 52-Kd serine antiprotease produced by hepatocytes and mononuclear phagocytes. In the context of the broad destructive capacity of NE, we evaluated the concept that human neutrophils may be able to modulate the extracellular activity of NE by synthesizing and secreting alpha 1AT. Immunocytochemical analysis demonstrated that the neutrophil contains alpha 1AT. Northern analysis and in situ hybridization with alpha 1AT-specific probes demonstrated the presence of alpha 1AT messenger RNA transcripts within neutrophils. [35S]methionine-labeling of neutrophils followed by immunoprecipitation of the supernatant with an anti-alpha 1AT antibody and sodium dodecyl sulfate-acrylamide gel analysis demonstrated that neutrophils can synthesize alpha 1AT de novo and secrete the synthesized molecule. In the presence of major neutrophil degranulation, the antiprotease effect of neutrophil alpha 1AT is overwhelmed, allowing the NE to act unopposed in the extracellular microenvironment. However, in conditions where small amounts of NE are released by neutrophils, at least some of the secreted newly synthesized alpha 1AT was capable of complexing with NE. Thus, despite the fact that the neutrophil cannot synthesize NE, it can synthesize and secrete alpha 1AT, the inhibitor of NE, ie, the neutrophil is capable, to some extent, of modulating NE activity in the local milieu without the help of antiproteases produced by other cells.

Gene Expression↗
PubMed1991

The cystic fibrosis gene has a "housekeeping"-type promoter and is expressed at low levels in cells of epithelial origin.

Evaluation of the expression of the cystic fibrosis (CF) gene in human epithelial cell lines demonstrated active, but low level, transcription of the gene. Analysis of 3.8 kilobases of genomic sequences 5' to exon 1 of the CF gene demonstrated no TATA promoter element, but a high G + C content, multiple transcription start sites, and several potential Sp1 binding sites. Fragments of 5'-flanking sequences from 2.2 kilobases to as small as 102 base pairs 5' to the major transcription start site supported constitutive reporter gene expression in epithelial cells, but at low levels, and independent of the length of the 5' fragment. CF gene transcription was down-regulated by phorbol myristate acetate. Importantly, evaluation of freshly isolated normal human bronchial cells also demonstrated CF gene transcription at a relatively low rate. Together, these observations suggest that although the normal CF gene promoter has characteristics of a "housekeeping"-type gene, and the gene is expressed at low levels in cells of organs that manifest the clinical disorder "cystic fibrosis," its expression can be modulated transcriptionally, implying a possible therapeutic approach for the disease.

Base Sequence↗
PubMed1991

Aerosol alpha 1-antitrypsin treatment for cystic fibrosis.

In cystic fibrosis neutrophil-dominated inflammation on the respiratory epithelial surface results in a chronic epithelial burden of the destructive enzyme, neutrophil elastase. alpha 1-antitrypsin (alpha 1AT), the main inhibitor of neutrophil elastase in lung, was given in aerosol form to 12 cystic fibrosis patients. It suppressed neutrophil elastase in the respiratory epithelial lining fluid (ELF) and restored the ELF anti-neutrophil elastase capacity when ELF alpha 1AT reached 8 mumol/l. This treatment also reversed the inhibitory effect of cystic fibrosis ELF on pseudomonas killing by neutrophils, which suggests that it may augment host defence in cystic fibrosis.

Adult↗
PubMed1991

Alpha 1-antitrypsin deficiency: pathogenesis and treatment.

Inherited defects of the gene coding for a serum protein that protects alveolar walls from proteolysis constitute one cause of emphysema, with or without liver disease. The deficiency can be reversed with intravenous administration of the antiprotease, keeping serum levels above the minimum needed for lung protection.

Alleles↗
PubMed1991

Alpha 1-antitrypsin deficiency and liver disease.

Alpha 1-antitrypsin (alpha 1AT) deficiency, one of the most common lethal hereditary disorders among Caucasians, is associated with emphysema in adults, while in children it is associated with liver disease. Produced in the liver and released into the plasma, alpha 1AT serves as the body's major inhibitor of neutrophil elastase, a powerful proteolytic enzyme capable of degrading extracellular structural proteins. The pathogenesis of the liver disease associated with alpha 1AT deficiency is not as well understood, but is clearly linked to specific mutations in coding exons of the alpha 1AT gene, and the resulting accumulation of alpha 1AT within hepatocytes. At present, therapy for the liver disease associated with alpha 1AT deficiency is symptomatic, with liver transplantation as a last resort. New strategies are being developed to suppress the accumulation of alpha 1AT by transferring the normal gene into the liver.

Humans↗
PubMed1991

Preferential usage of the T-cell antigen receptor beta-chain constant region C beta 1 element by lung T-lymphocytes of patients with pulmonary sarcoidosis.

Evaluation of the T-cells accumulating at sites of disease in active sarcoidosis suggests the accumulation process is not random, evidenced by a bias in the types of T-cells present. To evaluate the concept that this bias extends to the accumulation of T-cells with the preferential use of specific T-cell antigen receptor (TCR) beta-chain constant region elements, beta-chain mRNA transcripts of lung and blood T-cells of normal subjects and patients with pulmonary sarcoidosis were compared for the relative usage of constant region beta 1 or beta 2 elements. Quantitative evaluation of C beta 1 and C beta 2 mRNA transcripts demonstrated a C beta 1/C beta 2 usage in normal blood of 0.63 +/- 0.02, similar to that of normal lung (0.64 +/- 0.06, p greater than 0.7), and in sarcoid blood (0.59 +/- 0.03, p greater than 0.2). In contrast, the lung T-lymphocytes of patients with sarcoidosis reflected a marked bias in the usage of C beta 1 elements (C beta 1/C beta 2: 0.88 +/- 0.06, p less than 0.001 compared with sarcoid blood and normal blood; p less than 0.02 compared with normal lung). Interestingly, a subgroup of these patients (six of 18) showed a markedly exaggerated skewing in the use of C beta 1 elements (C beta 1/C beta 2 ratio greater than 1, i.e., greater than 3 standard deviations above mean), demonstrating heterogeneity among sarcoid patients with regard to specific C beta 1 usage.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Augmentation of functional prostaglandin E levels on the respiratory epithelial surface by aerosol administration of prostaglandin E.

Prostaglandin E (PGE), a cyclooxygenase metabolite normally present in high concentrations in respiratory epithelial lining fluid (ELF), is capable of suppressing mesenchymal cell proliferation mediated by polypeptide-derived growth factors. Although PGE is normally abundant in respiratory ELF, PGE levels in ELF of individuals with idiopathic pulmonary fibrosis (IPF), a fibrotic lung disorder characterized by intraalveolar mesenchymal cell accumulation and fibrosis, were found to be 50% lower than normal (p less than 0.01): that is, a relative PGE "deficiency" in ELF may enhance intraalveolar mesenchymal cell proliferation in IPF. With this background, it is rational to consider augmenting PGE levels in ELF as a future therapy for IPF. Since systemic administration of PGE is associated with significant adverse effects, in vitro and experimental animal studies were carried out to evaluate whether aerosol PGE administration could augment ELF PGE levels. Greater than 50% of a solution of PGE1 could be placed in droplets less than 3 microns mass median aerodynamic diameter without loss of function. Aerosolization of PGE1 to sheep (n = 14) resulted in a marked augmentation of ELF PGE1 levels (preaerosol 20 +/- 7 nM, 30 min postaerosol 1,150 +/- 210 nM; p less than 0.0 to 0.1). ELF PGE1 levels remained elevated for up to 2 h (p less than 0.05 compared with baseline) and returned to baseline by 3 h (p greater than 0.2). Lung interstitial fluid (lymph) PGE1 levels increased slightly, but to levels far less than ELF levels (preaerosol 7 +/- 1 nM, 30 min postaerosol 13 +/- 2 nM; p less than 0.01), and plasma PGE1 levels did not change (p greater than 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

Aerosols↗
PubMed1991

Anti-neutrophil elastase defense of the normal human respiratory epithelial surface provided by the secretory leukoprotease inhibitor.

Secretory leukoprotease inhibitor (SLPI), a 12-kD nonglycosylated serine antiprotease with a high capacity for inhibiting neutrophil elastase (NE), is produced by cells of mucosal surfaces including the human lung. The molar concentrations of SLPI in total respiratory tract epithelial lining fluid (ELF) were 56 +/- 10% that of alpha 1-antitrypsin, suggesting SLPI may be more important for the anti-NE protection of the pulmonary epithelial surface than previously thought. However, evaluation demonstrated that SLPI in respiratory ELF was only one-third functional. Studies aerosolizing recombinant SLPI (rSLPI) to sheep demonstrated that in the short term, neither aerosolization and alveolar deposition nor the lavage procedure inactivated the SLPI molecule. In vitro studies with rSLPI demonstrated that exposure to oxidants did not modify the form of the molecule, while exposure to oxidants and NE caused the molecule to be cleaved from 12 to 8 kD. Consistent with this, evaluation of SLPI in lavage fluid of individuals with cystic fibrosis (a condition with oxidants and NE on the respiratory epithelium) showed that the SLPI was degraded. However, evaluation of SLPI in normal ELF by molecular sieve analysis and Western analysis demonstrated an intact 12-kD molecule, suggesting that the partial inactivation of SLPI in normals in vivo is not because it is complexed to NE or exposed to oxidants + NE. Together, these observations demonstrate that SLPI is present in large amounts in respiratory ELF, but since the majority of the SLPI is inactive, it likely does not play a significant role in protecting the normal respiratory epithelium, except perhaps in the upper airways where the levels of SLPI are the highest.

Adult↗
PubMed1991

Risk factors for emphysema. Cigarette smoking is associated with a reduction in the association rate constant of lung alpha 1-antitrypsin for neutrophil elastase.

The increased risk of developing emphysema among individuals who smoke cigarettes and who have normal levels of alpha 1-antitrypsin (alpha 1AT) is hypothesized to result from a decrease in the antineutrophil elastase capacity of the lower respiratory tract alpha 1AT of smokers compared with nonsmokers. To evaluate this hypothesis we compared the time-dependent kinetics of the inhibition of neutrophil elastase by lung alpha 1AT from healthy, young cigarette smokers (n = 8) and nonsmokers (n = 12). alpha 1-antitrypsin was purified from lavage fluid using affinity and molecular sieve chromatography, and the association rate constant (k assoc) for neutrophil elastase quantified. The k assoc of smoker plasma alpha 1AT (9.5 +/- 0.5 X 10(6) M-1s-1) was similar to that of nonsmoker plasma (9.3 +/- 0.7 X 10(6) M-1s-1, P greater than 0.5). In marked contrast, the k assoc of smoker lower respiratory tract alpha 1AT was significantly lower than that of nonsmoker alpha 1AT (6.5 +/- 0.4 X 10(6) M-1s-1 vs. 8.1 +/- 0.5 X 10(6) M-1s-1, P less than 0.01). Furthermore, the smoker lower respiratory tract alpha 1AT k assoc was significantly less than that of autologous plasma (P less than 0.01). When considered in the context of the concentration of alpha 1AT in the lower respiratory tract epithelial lining fluid, the inhibition time for neutrophil elastase of smoker lung alpha 1AT was twofold greater than that of nonsmoker lung alpha 1AT (smoker: 0.34 +/- 0.05 s vs. nonsmoker: 0.17 +/- 0.05 s, P less than 0.01). Consequently, for concentrations of alpha 1AT in the lower respiratory tract it takes twice as long for an equivalent amount of neutrophil elastase to be inhibited in the smoker's lung compared with the nonsmoker's lung. These observations support the concept that cigarette smoking is associated with a decrease in the lower respiratory tract neutrophil elastase inhibitory capacity, thus increasing the vulnerability of the lung to elastolytic destruction and thereby increasing the risk for the development of emphysema.

Emphysema↗