Biomedical subjects

R G Crystal

Publications and source records attributed to R G Crystal.

At least 307 records · Page 17Linked to original sources

PubMed1988

Emphysema associated with complete absence of alpha 1- antitrypsin in serum and the homozygous inheritance [corrected] of a stop codon in an alpha 1-antitrypsin-coding exon.

Homozygous inheritance of the null bellingham alpha 1-antitrypsin (alpha 1AT) gene is associated with early-onset emphysema, resulting from the lack of alpha 1AT to protect the lung from neutrophil elastase. Cloning and sequencing of the null bellingham gene demonstrated that the promoter region, coding exons, and all exon-intron junctions were normal except for a single base substitution in exon III, causing the normal lys217 (AAG) to become a stop codon (TAG). Evaluation of genomic DNA of family members by using oligonucleotides directed toward this region demonstrated that the index case had inherited this mutation in a homozygous fashion. Although the consequences to the individual (i.e., emphysema) are identical to those associated with the common homozygous Z mutation, the homozygous null bellingham form of alpha 1AT deficiency has a very different genetic basis.

Adult↗

PubMed1988

Molecular genetics in pulmonary emphysema.

A vast array of new methods in molecular genetics are providing insights into problems at the cell and molecular level. Their applications in lung disease is unbounded. However, they are only methods. They are the right methods for certain kinds of questions and the wrong methods for other kinds of questions.

Gene Expression Regulation↗

PubMed1987

alpha 1-Antitrypsin nullGranite Falls, a nonexpressing alpha 1-antitrypsin gene associated with a frameshift to stop mutation in a coding exon.

alpha 1-Antitrypsin (alpha 1-AT) deficiency is a hereditary disorder associated with serum alpha 1-AT levels less than 35% of normal. There are two categories of alpha 1-AT genes that cause this state: the deficient alleles, in which alpha 1-AT is present in serum but in low levels, and the null alleles, in which no alpha 1-AT in serum can be attributed to the gene. The present study defines the molecular basis for the alpha 1-AT gene nullGranite Falls, identified and cloned from genomic DNA of an individual with severe alpha 1-AT deficiency and emphysema resulting from the heterozygous inheritance of the nullGranite Falls and Z alpha 1-AT genes. Sequencing of the 5'-flanking region, all five coding exons, and all exon-intron junctions of nullGranite Falls demonstrated it was identical with the common normal M1(Ala213) alpha 1-AT gene, except for two bases: a single deletion in the codon for amino acid Tyr160 of the mature protein and a single base substitution 168 base pairs 5' to exon I. Although no role for the promoter region mutation could be assigned, the coding exon deletion [Tyr(TAC)----(TA-)] resulted in a frameshift causing a stop coding to be formed approximately 44% from the N terminus of the precursor protein. Using oligonucleotide probes to evaluate the family of the index case demonstrated the deletion----frameshift/stop mutation was inherited in an autosomal co-dominant fashion. Thus, although the molecular basis for alpha 1-AT deficiency of the alpha 1-AT null haplotype such as nullGranite Falls is very different from the molecular basis of the more common deficient haplotypes such as Z, the phenotypic consequences of the two genes are similar; i.e. severe alpha 1-AT deficiency and an association of a high risk for the development of emphysema.

Adult↗

PubMed1987

Characterization of the M1(Ala213) type of alpha 1-antitrypsin, a newly recognized, common "normal" alpha 1-antitrypsin haplotype.

alpha 1-Antitrypsin (alpha 1AT) is a highly pleomorphic 52-kDa serum glycoprotein that functions as the major inhibitor of neutrophil elastase. Of these, the most common normal alpha 1AT haplotypes identified by isoelectric focusing (IEF) of serum are those of the M family, including M1, M2, and M3. In the course of studying the alpha 1AT type Z gene, we identified a restriction endonuclease BstEII polymorphism in the M1 gene that predicted the existence of a previously unidentified, but relatively common, haplotype of M, referred to as M1(Ala213) [Nukiwa, T., Satoh, K., Brantly, M. L., Ogushi, F., Fells, G. A., Courtney, M., & Crystal, R. G. (1986) J. Biol. Chem. 261, 15989-15994]. In this study we have cloned both alpha 1AT genes from an individual heterozygous for the M1(Ala213) and M1(Val213) haplotypes. Sequencing of the coding exons of both demonstrated that they are identical except for the Ala-Val difference at residue 213. The codominant transmission of the M1(Ala213) gene was demonstrated in a family study. Evaluation of 39 genomic samples of Caucasians with the IEF haplotype M1 demonstrated haplotype frequencies of 68% for M1(Val213) and 32% for M1(Ala213). alpha 1AT serum levels of individuals inheriting the M1(Ala213) gene in a homozygous fashion were in the same range as those for homozygous M1(Val213) as was the rate of association of the M1(Ala213) protein with neutrophil elastase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1987

Clonal gene therapy: transplanted mouse fibroblast clones express human alpha 1-antitrypsin gene in vivo.

A retroviral vector was used to insert human alpha 1-antitrypsin (alpha 1AT) complementary DNA into the genome of mouse fibroblasts to create a clonal population of mouse fibroblasts secreting human alpha 1AT. After demonstrating that this clone of fibroblasts produced alpha 1AT after more than 100 population doublings in the absence of selection pressure, the clone was transplanted into the peritoneal cavities of nude mice. When the animals were evaluated 4 weeks later, human alpha 1AT was detected in both sera and the epithelial surface of the lungs. The transplanted clone of fibroblasts could be recovered from the peritoneal cavities of those mice and demonstrated to still be producing human alpha 1AT. Thus, even after removal of selective pressure, a single clone of retroviral vector-infected cells that expressed an exogenous gene in vitro, continued to do so in vivo, and when recovered, continued to produce the product of the exogenous gene.

Animals↗

PubMed1987

Corticosteroid therapy suppresses spontaneous interleukin 2 release and spontaneous proliferation of lung T lymphocytes of patients with active pulmonary sarcoidosis.

Active pulmonary sarcoidosis is characterized by the alveolar accumulation of activated helper T lymphocytes that are spontaneously releasing IL 2 and proliferating at an enhanced rate. In this regard, sarcoidosis represents a "model" human disorder to test in vivo the known in vitro action of corticosteroids on suppressing the activated IL 2 gene. Comparable groups of patients with active sarcoidosis were prospectively evaluated with no therapy or treated with corticosteroids. Over 3.2 +/- 0.4 mo, the untreated group had no significant change in spontaneous lung T cell release of IL 2 or spontaneous proliferation. In contrast, over the same period, the treated group had marked reduction of spontaneous lung T cell release of IL 2 and proliferation (p less than 0.01, all comparisons before therapy). Furthermore, Northern analysis of lung T cell RNA before therapy demonstrated IL 2 mRNA transcripts, whereas no IL 2 transcripts were observed during therapy. These observations are consistent with the concept that directly, or indirectly, corticosteroids are capable of suppressing the IL 2 gene in activated T lymphocytes in vivo.

Adrenal Cortex Hormones↗

PubMed1987

Exaggerated spontaneous release of platelet-derived growth factor by alveolar macrophages from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a fibrotic lung disease characterized by an increased number of mesenchymal cells in the alveolar walls. Alveolar macrophages constitutively express low levels of c-sis, the protooncogene coding for the B chain of platelet-derived growth factor, a protein with chemotactic and mitogenic activity toward mesenchymal cells. We therefore hypothesized that alveolar macrophages in patients with idiopathic pulmonary fibrosis may release increased amounts of platelet-derived growth factor, which might help to explain the accumulation of mesenchymal cells and the fibrosis of the lower respiratory tract in the disease. Evaluation of alveolar macrophages recovered from the lungs of patients with idiopathic pulmonary fibrosis demonstrated that these cells spontaneously released four times more platelet-derived growth factor than did alveolar macrophages recovered from normal persons (P less than 0.01). That the platelet-derived growth factor molecules were potentially active was shown by their chemotactic activity for smooth-muscle cells and their ability to act as a "competence" factor for fibroblast growth. These observations suggest the possibility that the accumulation of mesenchymal cells within the alveolar walls in patients with idiopathic pulmonary fibrosis may result partly from the exaggerated release of the potent mitogen platelet-derived growth factor by mononuclear phagocytes in the lower respiratory tract.

Biological Assay↗

PubMed1987

Replacement therapy for alpha 1-antitrypsin deficiency associated with emphysema.

In patients with alpha 1-antitrypsin deficiency, the development of emphysema is believed to be caused by the unchecked action of proteases on lung tissue. We evaluated the feasibility, safety, and biochemical efficacy of intermittent infusions of alpha 1-antitrypsin in the treatment of patients with alpha 1-antitrypsin deficiency. Twenty-one patients were given 60 mg of active plasma-derived alpha 1-antitrypsin per kilogram of body weight, once a week for up to six months. After a steady state had been reached, the group had trough serum levels of alpha 1-antitrypsin of 126 +/- 1 mg per deciliter as compared with 30 +/- 1 mg per deciliter before treatment, and serum anti-neutrophil elastase capacities of 13.3 +/- 0.1 microM as compared with 5.4 +/- 0.1 microM. The alpha 1-antitrypsin level in the epithelial-lining fluid of the lungs was 0.46 +/- 0.16 microM before treatment, and the anti-neutrophil elastase capacity was 0.81 +/- 0.13 microM. Six days after infusion, alpha 1-antitrypsin levels (1.89 +/- 0.17 microM) and anti-neutrophil elastase capacities (1.65 +/- 0.13 microM) in the lining fluid were significantly increased (P less than 0.0001). Because of the chronicity of the disorder and the lack of sensitive measures of lung destruction, the clinical efficacy of this therapy could not be studied rigorously. No changes in lung function were observed in our patients over six months of treatment. The only important adverse reactions to the 507 infusions were four episodes of self-limited fever. This study demonstrates that infusions of alpha 1-antitrypsin derived from plasma are safe and can reverse the biochemical abnormalities in serum and lung fluid that characterize this disorder. Together with lifetime avoidance of cigarette smoking, replacement therapy with alpha 1-antitrypsin may be a logical approach to long-term medical treatment.

Adult↗

PubMed1987

Evaluation of alveolar macrophages in normals and individuals with active pulmonary sarcoidosis for the spontaneous expression of the interleukin-1 beta gene.

We have evaluated the hypothesis that the presence of large numbers of activated helper/inducer T lymphocytes in the lungs of individuals with active pulmonary sarcoidosis is associated with the exaggerated release of interleukin-1 (IL-1) by alveolar macrophages. Evaluation of media from unstimulated cultured sarcoid alveolar macrophages failed to detect IL-1 activity. When parallel cultures of sarcoid and normal alveolar macrophages were stimulated with lipopolysaccharide (LPS), they released similar amounts of IL-1 activity. Using a highly specific polyclonal anti-IL-1 beta antibody and flow cytometry to evaluate cell-associated IL-1 beta, analysis of fresh alveolar macrophages from patients with active sarcoidosis and normal individuals revealed no detectable cell-associated IL-1 beta, but IL-1 beta was present when macrophages from sarcoid patients and normals were stimulated with LPS. Similar observations were made using immunoblot analysis of cell lysates of the same unstimulated and stimulated macrophages. Finally, Northern analysis of alveolar macrophages for IL-1 beta mRNA transcripts demonstrated minimal, but equivalent, amounts of IL-1 beta in both normal and sarcoid macrophages, as compared to the much larger quantities present in LPS-stimulated alveolar macrophages. Thus, while alveolar macrophages of individuals with sarcoidosis are clearly capable of expressing the IL-1 beta gene, these findings suggest that altered expression of the IL-1 beta gene by alveolar macrophages does not play a central role in the exaggerated lung T-cell activation characteristic of sarcoidosis.

Gene Expression Regulation↗

PubMed1987

Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.

Alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized by reduced serum levels of alpha 1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment alpha 1AT levels in this disorder with physiologically normal human alpha 1AT, we have integrated a full-length normal human alpha 1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the alpha 1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi 2 and infected NIH 3T3. The clones produced three mRNA transcripts (5.8, 4.8, and 2.4 kilobases) containing human alpha 1AT sequences, secreted an alpha 1AT molecule recognized by an anti-human alpha 1AT antibody, with the same molecular mass (52 kDa) as normal human alpha 1AT and that complexed with and inhibited human neutrophil elastase. The psi 2 produced alpha 1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal alpha 1AT purified from human plasma and markedly longer than that of nonglycosylated human alpha 1AT cDNA-directed yeast-produced alpha 1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human alpha 1AT cDNA into non-alpha 1AT-producing cells, resulting in the synthesis and secretion of physiologically "normal" human alpha 1AT.

Animals↗

PubMed1987

Cyclooxygenase metabolites are compartmentalized in the human lower respiratory tract.

Cyclooxygenase metabolites of arachidonic acid are thought to play an important role in the regulation of diverse physiological functions in the lung. Although the concentration of these metabolites required to have effects is several orders of magnitude greater than the concentration of these mediators in the blood, it has been postulated that local concentrations within tissues are much higher. In a direct test of this hypothesis, the concentrations of the cyclooxygenase products of arachidonic acid including prostaglandin (PG) E, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane B2, were measured in a specialized tissue compartment, the epithelial surface of the lower respiratory tract. The concentration of these mediators within this compartment was 50- to 80-fold greater than concurrent blood levels and are sufficient to likely have physiological effects. Thus the epithelial surface of the lower respiratory tract represents a specialized compartment with high local levels of cyclooxygenase products of arachidonic acid.

6-Ketoprostaglandin F1 alpha↗

PubMed1987

Normal alveolar epithelial lining fluid contains high levels of glutathione.

The epithelial cells on the alveolar surface of the human lower respiratory tract are vulnerable to toxic oxidants derived from inhaled pollutants or inflammatory cells. Although these lung cells have intracellular antioxidants, these defenses may be insufficient to protect the epithelial surface against oxidants present at the alveolar surface. This study demonstrates that the epithelial lining fluid (ELF) of the lower respiratory tract contains large amounts of the sulfhydryl-containing antioxidant glutathione (GSH). The total glutathione (the reduced form GSH and the disulfide GSSG) concentration of normal ELF was 140-fold higher than that in plasma of the same individuals, and 96% of the glutathione in ELF was in the reduced form. Compared with nonsmokers, cigarette smokers had 80% higher levels of ELF total glutathione, 98% of which was in the reduced form. Studies of cultured lung epithelial cells and fibroblasts demonstrated that these concentrations of reduced glutathione were sufficient to protect these cells against the burden of H2O2 in the range released by alveolar macrophages removed from the lower respiratory tract of nonsmokers and smokers, respectively, suggesting that the glutathione present in the alveolar ELF of normal individuals likely contributes to the protective screen against oxidants in the extracellular milieu of the lower respiratory tract.

Adult↗

PubMed1987

Mechanisms of macrophage accumulation in the lungs of asbestos-exposed subjects.

Chronic asbestos exposure is associated with the accumulation of mononuclear phagocytes in the lower respiratory tract. This process can be both protective and injurious, since macrophages can aid in asbestos clearance yet also modulate structural derangements of the alveolar walls. To understand why macrophages accumulate in the lungs of asbestos-exposed persons, 2 possible mechanisms were evaluated using alveolar macrophages from subjects with histories of chronic high exposure to airborne asbestos: enhanced recruitment of blood monocytes to the lung, and an increased rate of replication of macrophages in situ. Monoclonal antibody analysis with antibodies that detect surface antigens on the majority of circulating blood monocytes but only on a minority of mature alveolar macrophages demonstrated that an increased proportion of alveolar macrophages of asbestos workers expressed monocyte lineage antigens, suggesting the presence of "young" newly recruited macrophages and thus enhanced recruitment. Culture of the alveolar macrophages from these subjects with [3H]thymidine followed by autoradiography demonstrated an increased proportion of alveolar macrophages synthesizing DNA, suggesting the macrophages are replicating at an increased rate in situ. These observations are consistent with the concept that both enhanced recruitment of blood monocytes and increased local proliferation of alveolar macrophages contribute to the accumulation mononuclear phagocytes in the lung of persons with chronic asbestos exposure.

Asbestos↗

PubMed1987

Pharmacologic suppression of the neutrophil component of the alveolitis in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease of unknown etiology characterized by inflammation of the lower respiratory tract and injury and fibrosis of the lung parenchyma. Neutrophils are a significant component of the alveolitis of IPF, and are known to have a potent armamentarium of mediators capable of damaging the lung parenchyma. To evaluate the ability of 2 pharmacologic agents, corticosteroids and cyclophosphamide, to suppress the neutrophil component of the inflammation of IPF, bronchoalveolar lavage was used to monitor those patients with active disease (greater than 10% neutrophils recovered by lavage). Those patients treated with corticosteroids alone showed no suppression in the neutrophil component of the alveolitis after 3 months and at 6 months of therapy (p greater than 0.8, both compared to before therapy). In marked contrast, patients treated with cyclophosphamide, alone or with corticosteroids, showed a significant reduction in the neutrophil alveolitis at 3 months (cyclophosphamide alone, p less than 0.01; cyclophosphamide and corticosteroids, p less than 0.02; both compared to before therapy) and at 6 months (cyclophosphamide alone, p less than 0.01; compared to before therapy). Pulmonary function tests at the beginning and end of the study were not different in any of the treatment groups. Thus, cyclophosphamide, alone or in combination with corticosteroids, is much more effective than corticosteroids alone in suppressing the neutrophil component of the inflammation of IPF. Whether or not this effect of cyclophosphamide will result in overall stabilization or improvement of the disease will require a larger, likely multicenter, trial.

Adult↗

PubMed1987

Characterization of the lower respiratory tract inflammation of nonsmoking individuals with interstitial lung disease associated with chronic inhalation of inorganic dusts.

The pneumoconioses, interstitial lung disorders resulting from the inhalation of inorganic dusts, are associated with chronic inflammatory processes in the lower respiratory tract. To characterize these inflammatory processes in relation to the pathogenesis of these disorders, we studied 39 nonsmoking individuals with long-term occupational exposures to inorganic dust and functional evidence of interstitial disease (asbestosis, n = 18; coal workers' pneumoconiosis, n = 15; silicosis, n = 6). In all 3 disorders, the inflammation was dominated by alveolar macrophages. Because a common feature of these interstitial lung diseases is concurrent injury and fibrosis of alveolar walls, we assessed whether these alveolar macrophages were spontaneously releasing mediators capable of giving rise to these changes. Alveolar macrophages from the study population were spontaneously releasing increased amounts of superoxide anion and hydrogen peroxide (both p less than 0.01 compared to normals), oxidants capable of injuring lung parenchymal cells. The alveolar macrophages were also spontaneously releasing significantly increased amounts of fibronectin and alveolar macrophage-derived growth factor (both p less than 0.01 compared to normals), mediators that act synergistically to signal fibroblast replication. Taken together, these findings define a major role for the alveolar macrophage in mediating the alveolar wall injury and fibrosis that characterize the common pneumoconioses and suggest that the alveolar macrophage is an important "target" for developing strategies designed to prevent loss of lung function in these individuals.

Adult↗

PubMed1987

Evaluation of tamoxifen as a therapy to augment alpha-1-antitrypsin concentrations in Z homozygous alpha-1-antitrypsin-deficient subjects.

Tamoxifen, an agent that binds to intracytoplasmic estrogen receptors, was evaluated as a possible means of increasing alpha-1-antitrypsin (alpha 1AT) synthesis and/or secretion and thus alpha 1AT serum levels in subjects with the homozygous form of alpha 1AT deficiency. Administration of tamoxifen (10 mg twice daily) to 30 Z homozygotes for a 30-day period was not associated with adverse reactions. However, although serum alpha 1AT levels increased significantly (p less than 0.03), the increase was minor (average pretreatment levels, 32 +/- 1 mg/dl; levels at 30 days of therapy, 35 +/- 1 mg/dl) and far below the "threshold" level of 80 mg/dl considered "protective" against an increased risk for emphysema. Thus, while the concept that increasing alpha 1AT synthesis and/or secretion is a rational goal for treating the Z homozygous form of alpha 1AT deficiency, tamoxifen will not be useful in this regard.

Adult↗

PubMed1987

Susceptibility to experimental interstitial lung disease is modified by immune- and non-immune-related genes.

To evaluate the concept that genetic factors modulate susceptibility to agents that cause interstitial lung disease, animal models of interstitial lung disease caused by bleomycin or by inhalation of organic particulates (ovalbumin or bovine gamma globulin after specific immunization) were studied in strains of mice with different genetic backgrounds. Because immune processes have been implicated in modulating the susceptibility to agents that cause interstitial lung disease, we also compared congenic, resistant strains (strains with the same background but with different H-2 haplotypes) for their sensitivity to the same agents. In bleomycin-induced disease, the degree of lung disease was different in some of the different strains of mice and, in some strains, was related to H-2 locus genes since all strains with H-2b haplotypes were high responders, whereas most of the strains with H-2a, H-2d, and H-2k haplotypes were low responders. However, some of the strains of mice with the same H-2 haplotype but otherwise different genetic backgrounds had different responses to bleomycin, suggesting that there is also a role for non-H-2 genetic factors in modulating the response to this experimental interstitial lung disease. In the ovalbumin-induced lung disease model, as in bleomycin-induced lung disease, there were different strain susceptibilities: 2 of the 3 strains in the H-2b group were high responders, as was 1 of the 3 strains in the H-2k group. Interestingly, evaluation of the congenic, resistant strains showed that on the same backgrounds the H-2-related genes were able to modulate the degree of lung lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Comparison of alpha-1-antitrypsin levels and antineutrophil elastase capacity of blood and lung in a patient with the alpha-1-antitrypsin phenotype null-null before and during alpha-1-antitrypsin augmentation therapy.

The null-null phenotype of alpha 1-antitrypsin (alpha 1AT), a phenotype characterized by no detectable alpha 1AT in serum, presents a rare opportunity to examine the contribution of alpha 1AT to the antineutrophil elastase protection of the lower respiratory tract. The subject, a 35-yr-old lifetime non-smoker with moderate emphysema, has been characterized as having alpha 1AT serum levels of zero resulting from the homozygous inheritance of alpha 1AT genes that do not express detectable alpha 1AT mRNA transcripts. Evaluation of the antineutrophil elastase capacity of the null-null serum showed it was less than 5% of normal, whereas that of the epithelial lining fluid (ELF) of the lower respiratory tract was 13% of normal. However, after 60 mg/kg of intravenously administered alpha 1AT augmentation therapy once weekly for 4 wk, the serum alpha 1AT levels peaked at greater than 300 mg/dl, trough levels just prior to the next infusion were 81 +/- 2 mg/dl, and the average serum level integrated for the month of infusions was 138 mg/dl. Consistent with this serum rise in alpha 1AT, the serum antineutrophil elastase capacity increased in parallel(r = 0.98). Importantly, evaluation of the ELF 2 and 6 days after infusion demonstrated increases of alpha 1AT levels (range, 1.4 to 2.1 microM) and antineutrophil elastase capacity (range, 1.6 to 2.5 microM), values within the lower range of normal. Furthermore, the lung ELF alpha 1AT levels rose in direct proportion to the serum alpha 1AT levels, and the ELF antineutrophil elastase capacity rose in direct proportion to the ELF alpha 1AT levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗