Biomedical subjects

R G Crystal

Publications and source records attributed to R G Crystal.

At least 289 records · Page 16Linked to original sources

PubMed1988

Alpha-1-antitrypsin augmentation therapy for alpha-1-antitrypsin deficiency.

Alpha-1-antitrypsin (A1AT) deficiency is a genetic disorder characterized by low serum levels of A1AT and a high risk for the development of emphysema. A1AT is the principal inhibitor of neutrophil elastase, such that a deficiency of A1AT results in insufficient anti-elastase protection in the lower respiratory tract, thus allowing neutrophil elastase to destroy alveolar structures. The goal of A1AT augmentation therapy in A1AT deficiency is to raise lung A1AT levels and anti-neutrophil elastase capacity to levels that will provide adequate protection against neutrophil elastase, thereby preventing the lung from further elastase-mediated degradation. Studies with intravenous administration of human A1AT (60 mg/kg at weekly intervals) demonstrate that serum A1AT levels increased from an average 33 +/- 8 mg/dl pre-infusion to a steady-state trough level of 117 +/- 4 mg/dl, well above the projected threshold protective serum level of A1AT. The infused A1AT diffused into the lung and significantly augmented the epithelial lining fluid A1AT levels, rising from an average 0.44 +/- 0.16 microM (pre-infusion) to 2.62 +/- 1.29 microM at the nadir level just prior to the next infusion. Of critical importance is the fact that the A1AT that diffused into the lung was active as an inhibitor or neutrophil elastase, resulting in significant augmentation of epithelial lining fluid anti-neutrophil elastase capacity and normalization of the lung anti-elastase protection. In the over 800 weekly infusions administered, no significant adverse reactions have occurred. These findings demonstrate that long-term augmentation therapy with weekly infusions of A1AT is a rational, safe, and biochemically effective therapy for A1AT deficiency.

Humans↗

PubMed1988

Demonstration by in situ hybridization of dissimilar IL-1 beta gene expression in human alveolar macrophages and blood monocytes in response to lipopolysaccharide.

IL-1 beta is the major form of IL-1 produced by mononuclear phagocytes. To evaluate the possible mechanisms underlying the observation that mature populations of human mononuclear phagocytes as alveolar macrophages are relatively poor IL-1 producers compared with blood monocytes, the expression of the IL-1 beta gene mRNA transcripts was evaluated in LPS-stimulated normal autologous blood monocytes and alveolar macrophages by using a IL-1 beta cDNA probe. Although Northern analysis demonstrated that stimulated monocytes and alveolar macrophages both express 1.8-kb IL-1 beta mRNA transcripts, cytoplasmic dot blot analysis showed that the total IL-1 beta mRNA content in alveolar macrophages was only 38 +/- 5% of that in blood monocytes. In situ hybridization with antisense and sense IL-1 beta RNA probes demonstrated that whereas most of stimulated blood monocytes contained IL-1 beta mRNA transcripts, a significant proportion of autologous alveolar macrophages stimulated in an identical fashion did not express the IL-1 beta gene. Within 4 h after LPS stimulation, IL-1 beta mRNA transcripts were detected in 81 +/- 6% monocytes, whereas only 16 +/- 9% of alveolar macrophages were positive, and by 18 h this had increased only to 43 +/- 15%. Quantification of the size distribution of the IL-1 beta mRNA expressing mononuclear phagocytes demonstrated that, among the population of alveolar macrophages, the cells expressing this gene were not confined to those that were "monocyte-like." These observations demonstrate that there is a heterogeneity among population of mononuclear phagocytes in their ability to express the gene for IL-1 beta, which could explain the differences observed in IL-1 production.

Gene Expression Regulation↗

PubMed1988

Sarcoidosis is not associated with a generalized defect in T cell suppressor function.

In pulmonary sarcoidosis, the marked expansion of CD4+ (helper/inducer) T cells in the alveolar structures of the lung is maintained by local IL-2 release by activated CD4+ HLA-DR+ T cells without concomitant expansion and activation of CD8+ (suppressor/cytotoxic) T cells, suggesting that sarcoid may be associated with a generalized abnormality of CD8+ T cells. Consistent with this concept, evaluation of the expression of the IL-2R on fresh lung T cells from individuals with active sarcoidosis demonstrated that 7 +/- 1% of sarcoid lung CD4+ T cells are spontaneously expressing the IL-2R compared with only 1 +/- 1% lung CD8+ T cells (p less than 0.01). However, stimulation of purified sarcoid blood CD8+ T cells with the anti-T3/TCR complex mAb OKT3 was followed by the normal expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). In addition, lung sarcoid CD8+ T cells responded to OKT3 similarly to normal lung CD8+ T cells and to autologous blood CD8+ T cells as regards expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). Finally, using CD4+ cells activated with allogenic Ag to induce, in coculture, fresh autologous CD8+ cells to suppress proliferation of fresh autologous CD4+ cells to the same Ag, sarcoid CD8+ T cells suppressed CD4+ cell proliferation in a normal fashion (p greater than 0.1). These results demonstrate that sarcoid CD8+ (suppressor/cytotoxic) T cells are competent to respond to a proliferation signal normally and can be induced to normally suppress CD4+ T cell proliferation to Ag, suggesting that the expansion of activated CD4+ T cells in pulmonary sarcoidosis is not due to a generalized abnormality of CD8+ T cells or of their suppressor T cell function.

Adult↗

PubMed1988

Myelomonocytic cell lineage expression of the neutrophil elastase gene.

Human neutrophil elastase (NE) functions as a powerful serine protease capable of attacking a broad range of proteins. To examine the cellular site(s) of NE gene expression, a 0.65-kilobase cDNA (pPB15) complementary to the coding region of the NE gene was cloned from the cell line U937 using an oligonucleotide based on the known NE protein sequence. The sequence of pPB15 demonstrated that it coded for the 173 C-terminal residues of the 218 amino acids that comprise the mature NE protein, plus an additional 3' 60 base pairs prior to the in-frame stop codon, suggesting the NE mRNA contains sequences for a 20-residue C-terminal "pro" peptide that is not found in the mature protein. Northern analysis using 32P-labeled pPB15 as a probe revealed that neutrophils do not contain detectable NE mRNA transcripts despite the fact that this cell carries large amounts of this protein. Furthermore, resting and activated blood monocytes also contained no detectable NE mRNA transcripts, although these cells also carry detectable NE. In contrast, bone marrow precursor cells contained NE transcripts, suggesting the NE gene is expressed in blood precursor cells. In this regard, evaluation of HL-60 cells, a human cell line with myelomonocytic lineage features, demonstrated NE transcripts in resting cells and increased NE mRNA levels when the cells were induced toward the myelocytic lineage with dimethyl sulfoxide. However, when the HL-60 cells were induced toward the monocytic lineage with phorbol 12-myristate 13-acetate, NE transcripts were lost even though transcripts for interleukin-1 beta were plentiful. Together, these observations are consistent with the concept that the NE gene is not expressed in the blood cells that carry the protein, but in bone marrow precursors that express NE transcripts about the time of commitment to the myelocytic series.

Amino Acid Sequence↗

PubMed1988

T lymphocytes compartmentalized on the epithelial surface of the lower respiratory tract express the very late activation antigen complex VLA-1.

T lymphocytes on the epithelial surface of the lower respiratory tract are thought to represent a relatively compartmentalized population of T cells that exchanges slowly with the blood. Since the lung is chronically burdened with antigens, "resident" T cells likely have a history of past activation. To evaluate this concept, we analyzed resident lung T cells for VLA-1 expression, which is indicative of a history of past stimulation. Lung lavage and blood T cells were evaluated in 13 normal nonsmokers using the monoclonal antibodies Leu4 (pan T cells), Leu3 (helper/inducer T cells), Leu2 (suppressor/cytotoxic T cells), TS2/7 (alpha 1 subunit of VLA-1), and A-1A5 (beta subunit of VLA-1) using immunofluorescence and immunoprecipitation. In contrast to normal blood T cells which did not express VLA-1, lung T cells expressed the 210-kDa alpha 1 and 130-kDa beta subunits of the VLA-1 complex, the same as blood T cells activated in culture for 3 weeks. Two-color immunofluorescence with Leu4 and TS2/7 showed that 19 +/- 6% of the lung T cells were VLA-1+, suggesting that a significant proportion of T lymphocytes on the alveolar epithelial surface are in a separate compartment from the VLA-1 blood cells. In sarcoidosis, a disease characterized by exaggerated numbers of active Leu3+ T cells in the lower respiratory tract, increased numbers of lung Leu3+ T cells expressing VLA-1 were present on the epithelial surface of the lung (P less than 0.05 compared to normals). These observations are consistent with compartmentalized, chronically stimulated T lymphocytes on the alveolar epithelial surface that exchange with the systemic immune system very slowly.

Antibodies, Monoclonal↗

PubMed1988

Does de novo immunoglobulin synthesis occur on the epithelial surface of the human lower respiratory tract?

Although synthesis of immunoglobulins by cells in the human lower respiratory tract has never been directly demonstrated, local production of immunoglobulins by cells within lung parenchyma is thought to play an important role in protecting the normal human lung against a variety of pathogenic organisms, and may contribute to the development of some interstitial lung diseases. To determine whether or not the epithelial surface of the human lower respiratory tract is a site of immunoglobulin synthesis, immunoglobulin production by cells recovered by bronchoalveolar lavage and lung parenchymal tissue from normal subjects and patients with idiopathic pulmonary fibrosis (IPF) or sarcoidosis was evaluated using a reverse hemolytic plaque assay and by measuring incorporation of [35S]methionine into immunoglobulins. Although the reverse hemolytic plaque assay demonstrated immunoglobin-releasing cells to be present on the surface of the lower respiratory tract of normal subjects, and present in increased numbers in patients with IPF or sarcoidosis, these plaque-forming cells were not actively synthesizing immunoglobulins, since preincubation of lavage cells in order to remove immunoglobulin bound to the surface of the cells reduced the number of plaque-forming cells, and inhibition of protein synthesis by cycloheximide did not further reduce the number of plaque-forming cells. Furthermore, direct evaluation of immunoglobulin synthesis using radiolabeling, immunoprecipitation and SDS-polyacrylamide gel electrophoresis failed to detect de novo IgG production by cells recovered from the alveolar surface of 15 of 17 subjects, IgM by 17 of 17, and IgA by 16 of 17.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1988

Colchicine suppresses the release of fibroblast growth factors from alveolar macrophages in vitro. The basis of a possible therapeutic approach ot the fibrotic disorders.

Fibrosis is the accumulation of fibroblasts and the connective tissue products secreted by these cells, usually subsequent to tissue injury. While fibrosis can be useful in preserving the general structural integrity of a tissue, it often alters cell-cell and cell-connective tissue interactions, which leads to loss of tissue function. On the basis of the concept that mononuclear phagocytes can direct the development of fibrosis through the release of specific mediators that stimulate fibroblast proliferation, we propose a therapeutic strategy to prevent fibrosis by preventing the release of these specific mediators. The present study demonstrated that colchicine, a widely used and well-tolerated drug, can block alveolar macrophage release of 2 mediators associated with the development of fibrosis in interstitial lung diseases, fibronectin, and the alveolar-macrophage-derived growth factor (AMDGF). Colchicine blocked the spontaneous release of fibronectin by alveolar macrophages obtained from patients with fibrotic lung disease by 23 +/- 4% after 24 h and by greater than 90% after 72 h. AMDGF release was blocked by 68 +/- 10% after 4 h (p less than 0.01, all comparisons). The effect of colchicine was not due to nonspecific toxicity since [14C]proline tracer studies demonstrated that macrophages treated with colchicine were capable of de novo protein synthesis and the secretion of several protein products, despite the fact that fibronectin and AMDGF release were suppressed. The effect of colchicine on the spontaneous release of both fibronectin and AMDGF could be observed at concentrations less than 10 ng/ml, levels that can be achieved in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Colchicine↗

PubMed1988

Evaluation of the S-type of alpha-1-antitrypsin as an in vivo and in vitro inhibitor of neutrophil elastase.

S-type alpha 1-antitrypsin (alpha 1AT) is a deficiency haplotype that differs from the common normal M1 (val213) alpha 1AT haplotype by a single amino acid (glu264 to val264). To evaluate the adequacy of the antineutrophil elastase protection associated with the S homozygous state, alpha 1AT plasma and lung epithelial lining fluid (ELF) levels and antineutrophil elastase function were analyzed in 9 PISS subjects. The plasma alpha 1AT levels of SS subjects were intermediate between that of M1M1 and ZZ subjects (p less than 0.001, all comparisons) and the plasma neutrophil elastase inhibitory capacity paralleled the differences in alpha 1AT concentration (p less than 0.001, all comparisons). The association rate constant for neutrophil elastase of the purified S protein was less than that of the normal molecule (S-type, 7.1 +/- 0.1 X 10(6) M-1 s-1; M1-type, 9.6 +/- 0.2 X 10(6) M-1 s-1; p less than 0.001), but much greater than that for the Z molecule (p less than 0.001). Exposure of the purified S protein to increasing oxidant burdens resulted in a dose-dependent reduction in the ability of the molecule to inhibit neutrophil elastase in a fashion parallel to that of the M1 and Z proteins. Quantification of ELF alpha 1AT levels and antineutrophil elastase capacity demonstrated that the SS ELF parameters were, as in plasma, intermediate between M1 homozygotes and Z homozygotes. Using the association rate constant together with the quantification of ELF alpha 1AT levels, the "in vivo lung inhibition time" was estimated, yielding an assessment of the relative antineutrophil elastase screen of the PISS lower respiratory tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1988

Accumulation of Langerhans' cells on the epithelial surface of the lower respiratory tract in normal subjects in association with cigarette smoking.

Langerhans' cells are a defined subpopulation of the mononuclear phagocyte system known to accumulate in the lung in histiocytosis X, an interstitial lung disorder strongly linked to cigarette smoking. To evaluate the hypothesis that cigarette smoking itself may be associated with the accumulation of Langerhans' cells in the lung, normal nonsmokers (n = 5) and normal smokers (n = 10) were evaluated by bronchoalveolar lavage for the presence of Langerhans' cells as identified by the OKT6 monoclonal antibody and by transmission electron microscopy. While the OKT6 antibody identified 0.1 +/- 0.1% of the cells recovered from nonsmokers, it labeled 1.1 +/- 0.3% of those recovered from smokers (p less than 0.01). Furthermore, while electron microscopy demonstrated no Langerhans' cells among the lavage cells from nonsmokers, 0.4 +/- 0.1% of the cells recovered from normal smokers contained characteristic intracytoplasmic Birbeck granules, positively identifying them as Langerhans' cells. We conclude that cigarette smoking is associated with an expansion in the population of Langerhans' cells on the epithelial surface of the lower respiratory tract. While the mechanisms underlying this accumulation are unknown, it is possible that the properties of these cells contribute to the derangements of the pulmonary parenchyma found in cigarette smoking and establish a biologic link to the already observed epidemiologic association between histiocytosis X and cigarette smoking.

Adult↗

PubMed1988

Functional significance of anti-T-lymphocyte antibodies in sarcoidosis.

Pulmonary sarcoidosis is a chronic disorder characterized by the activation of helper/inducer T-cells in the lung without a concomitant increase in suppressor/cytotoxic T-cells. It is known that patients with sarcoidosis have circulating anti-T-cell antibodies, primarily of the IgM class. To evaluate a functional role for these antibodies in enhancing lung helper T-cell processes in pulmonary sarcoidosis, we evaluated serum and lavage fluid of patients with active sarcoidosis for the presence of anti-T-cell antibodies, the T-cell subset specificity of these antibodies, and the possible stimulatory or inhibitory effects of these antibodies on T-cells relevant to the exaggerated helper T-cell processes in sarcoidosis. Indirect immunofluorescence studies demonstrated that sarcoid patients had anti-T-cell antibodies of the IgM type reacting with autologous as well as with nonautologous normal T-cells. IgM recovered in sarcoid lavage fluid also reacted with T-cells, thus demonstrating the autoantibodies at the site of disease. Two-color immunofluorescence and flow cytometry showed that these sarcoid autoantibodies bound to mostly Leu2+ suppressor/cytotoxic T-cells, but also to a small proportion of Leu3+ helper/inducer T-cells. Incubating lymphocytes with sarcoid serum or IgM purified from sarcoid serum did not stimulate T-cell proliferation. Furthermore, when Leu2+ T-cells were stimulated with irradiated allogenic B-cells, increasing concentrations of sarcoid serum had no inhibitory effects on the activation and proliferative response of the Leu2+ T-cells. Likewise, the purified IgM anti-T-cell antibodies had no inhibitory effects on the mitogenic response of Leu2+ T-cells to the anti-T-cell antigen receptor-associated T3 complex antibody OKT3.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1988

High levels of transforming growth factor-beta are present in the epithelial lining fluid of the normal human lower respiratory tract.

Transforming growth factor-beta (TGF-beta), a mediator capable of modulating a broad range of effects on the behavior of many normal cells, was found in high concentrations in the epithelial lining fluid (ELF) of the normal human lower respiratory tract. Although plasma contained small amounts of TGF-beta, the concentrations of TGF-beta in normal ELF were in the 200 to 300 pM range, more than 15-fold higher. This ELF TGF-beta had similar physical characteristics to purified human platelet TGF-beta, competed with platelet TGF-beta for its receptor on A549 carcinoma cells, and stimulated the anchorage-independent growth of NRK cells in soft agar in the presence of epidermal growth factor. Furthermore, ELF TGF-beta suppressed diploid lung fibroblast proliferation in a dose-dependent fashion similar to platelet TGF-beta. In the context of these observations and with the known biologic properties of this molecule, TGF-beta in ELF has the potential to play a role in a variety of cellular processes in the lower respiratory tract.

Adult↗

PubMed1988

Clinical features and history of the destructive lung disease associated with alpha-1-antitrypsin deficiency of adults with pulmonary symptoms.

Alpha-1-antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized in adults by a high risk for the development of severe destructive lung disease at an early age. The present study was designed to draw conclusions concerning the characteristics of a referral population of 124 patients with alpha 1AT deficiency and symptomatic emphysema. Typically, the alpha 1AT level was 30 mg/dl, and the alpha 1AT phenotype was almost always PiZZ. The individuals in this population were most often male, caucasian, and ex-smokers, and they had become dyspneic between 25 and 40 yr of age. Most routine blood tests were normal. The chest radiographs and ventilation-perfusion studies typically showed abnormalities with a lower zone distribution, and about one third of the study population had evidence suggestive of pulmonary hypertension. Lung function tests were typical for emphysema; the FEV1 and DLCO were the parameters most dramatically reduced, and the annual rate of decline of those parameters was greater than that of the general population. The cumulative probability of survival of this population indicated a significantly shortened lifespan with a mean survival of 16% at 60 yr of age compared with 85% for normal persons.

Adolescent↗

PubMed1988

Differential expression of the tumor necrosis factor/cachectin gene by blood and lung mononuclear phagocytes.

Tumor necrosis factor (TNF), also called cachectin, is a mononuclear phagocyte-derived mediator with a broad range of biologic activities contributing to antineoplastic and antiviral defenses as well as mediating a variety of processes associated with acute and chronic inflammatory states, including endotoxin-induced shock. To evaluate the relative capacity of human tissue macrophages to produce this mediator, alveolar macrophages and blood monocytes from the same normal individuals were activated with lipopolysaccharide (LPS) and evaluated for TNF release and TNF mRNA transcript levels. Resting alveolar macrophages did not express TNF mRNA transcripts or release TNF. However, when activated, alveolar macrophages expressed TNF transcripts and synthesized and released TNF as evidenced by the presence of a 28 kDa mediator in LPS-activated alveolar macrophage supernatants that had cytotoxic activity for L-929 cells that was abrogated by anti-TNF antibodies and that coeluted with a pure TNF standard on a molecular sieve column. Interestingly, activated alveolar macrophages released severalfold more TNF than did autologous blood monocytes stimulated in a similar fashion and, in parallel, the alveolar macrophages expressed more TNF mRNA transcripts than activated blood monocytes. Thus, the ability to express the TNF gene and to release TNF apparently increases during maturation of blood monocytes into alveolar macrophages, suggesting that the release of TNF in the local milieu by activated tissue macrophages may be much more significant than the release of this mediator by circulating blood monocytes.

Adult↗

PubMed1988

Evaluation of the in vitro and in vivo effects of cyclosporine on the lung T-lymphocyte alveolitis of active pulmonary sarcoidosis.

Pulmonary sarcoidosis is a granulomatous disorder characterized by the accumulation of activated helper/inducer T-lymphocytes in the lower respiratory tract, a process thought to be central to the pathogenesis of the disease. Because cyclosporine, a fungus-derived cyclic peptide, has specific inhibitory effects on T-lymphocyte activation, it should suppress activated sarcoid lung T-cells, and thus it should theoretically be an ideal therapeutic agent for sarcoidosis. This was confirmed in vitro: the addition of cyclosporine to T-cells recovered from the lungs of patients with active sarcoid suppressed the spontaneous release of interleukin-2 (IL-2) and monocyte chemotactic factor by these cells and inhibited their exaggerated spontaneous replication. In contrast, the oral administration of conventional doses (10 mg/kg/day) of cyclosporine to eight of these patients over a 6-month period was not accompanied by suppression of sarcoid lung T-cell activation. On the average, the spontaneous release of IL-2 and monocyte chemotactic factor, the proliferation of lymphocytes, and the number of helper/inducer T-cells present in the lungs of these subjects remained elevated and similar to their pretherapy values. Consistent with this lack of effect on sarcoid lung T-cell activation, no improvement in lung function was observed over the trial period. Thus, although cyclosporine is effective in vitro in suppressing the exaggerated activation of sarcoid lung T-cells, it does not do so in vivo, suggesting this agent will not be useful in the therapy of active pulmonary sarcoidosis, at least when administered in a conventional fashion.

Adult↗

PubMed1988

Spontaneous expression of the interleukin 2 receptor gene and presence of functional interleukin 2 receptors on T lymphocytes in the blood of individuals with active pulmonary sarcoidosis.

Current concepts of the pathogenesis of sarcoidosis suggest that the expanded numbers of activated T-helper/inducer cells at sites of disease activity result, at least in part, from their proliferation in the local milieu. Normal clonal proliferation of T cells involves activation and expression of the IL 2 receptor (IL 2R) gene. Thus, knowing that IL 2R mRNA transcripts are relatively long lived, we hypothesized that sarcoid blood T cells may contain IL 2R mRNA transcripts and express functional surface IL 2R, although the cells are probably activated elsewhere. Northern analysis using a 32P-labeled cDNA probe for the IL 2R p55 protein demonstrated that blood T cells of patients with active sarcoidosis, but not of normal patients, express 3.5- and 1.5-kb IL 2R mRNA transcripts, the same as those observed in normal T cells activated in vitro. Consistent with this, using flow cytometry and an MAb directed against the IL 2R p55 protein (2A3), we observed detectable levels of IL 2R surface protein on increased numbers of blood T cells of active sarcoidosis patients (4.7 +/- 0.9%) compared with blood T cells of normal patients (0.9 +/- 0.2%). Importantly, when the sarcoid blood T cells were exposed to IL 2 in vitro, they proliferated at a rate greater than that of normal blood T cells under the same conditions, suggesting that the IL 2R spontaneously expressed by sarcoid blood T cells were functionally active. In the context of the known compartmentalization of spontaneous IL 2 production and T cell proliferation at sites of disease in active pulmonary sarcoidosis, these IL 2R positive blood T cells would probably have a proliferative advantage if they trafficked to sites of active sarcoidosis, such as the lower respiratory tract.

Adult↗

PubMed1988

Bias toward use of a specific T cell receptor beta-chain variable region in a subgroup of individuals with sarcoidosis.

To evaluate the concept that biases in the usage of T cell antigen receptor beta variable (V) regions may be manifested in T lymphocytes that accumulate in nonmalignant, T cell-mediated human disorders, a V beta 8-specific antibody (anti-Ti3A, 5REX9H5) was used to evaluate lung and blood T cells in pulmonary sarcoidosis, a chronic granulomatous disorder of unknown etiology. Whereas normal patients had less than 5% Ti3A+ lung (n = 7) and/or blood (n = 9) lymphocytes, strikingly, a subgroup (8 of 21) with active pulmonary sarcoidosis had greater than 7% Ti3A+ lung and/or blood T cells and a higher proportion of Ti3A+ lymphocytes in the lung compared with blood. Dual-color flow cytometry demonstrated compartmentalization of Ti3A+ CD4+ lymphocytes to lung and Ti3A+ CD8+ lymphocytes to blood. Analysis with a 32P-labeled V beta 8 probe revealed that sarcoid lung T lymphocytes contained higher amounts of V beta 8+ mRNA than autologous blood T cells. However, Southern analysis of sarcoid lung and blood T cell DNA demonstrated no evidence of clonal rearrangements of V beta 8 genes. These observations demonstrate a clear bias toward the use of at least one V beta region in sarcoidosis, and suggests T cells accumulate secondary to external selective pressure, rather than in a random polyclonal fashion or by clonal expansion of one or few T cell clones.

Antibodies, Monoclonal↗

PubMed1988

Evaluation of fibronectin gene expression by in situ hybridization. Differential expression of the fibronectin gene among populations of human alveolar macrophages.

Populations of alveolar macrophages recovered from the lower respiratory tract secrete fibronectin, a multi-functional glycoprotein capable of influencing cell migration, attachment, differentiation, and proliferation. Using in situ hybridization of 35S-labeled antisense and sense RNA fibronectin probes, the present study demonstrates that most, but not all, normal alveolar macrophages contain fibronectin mRNA transcripts, and that among those macrophages expressing this gene, the relative amount of fibronectin mRNA transcripts varies from cell to cell. Interestingly, while 66 +/- 3% of normal alveolar macrophages contain fibronectin mRNA transcripts, this is increased to 82 +/- 2% (P less than 0.01) of alveolar macrophages recovered from the lungs of individuals with idiopathic pulmonary fibrosis (IPF), a chronic inflammatory disorder associated with exaggerated amounts of fibronectin in the lower respiratory tract. Furthermore, of the macrophages expressing the fibronectin gene, those from IPF patients contain more fibronectin mRNA transcripts than those from normals. Consistent with this observation, evaluation of tissue samples from IPF patients demonstrated that of all cells present, alveolar macrophages showed the greatest numbers of fibronectin mRNA transcripts per cell. These observations demonstrate that there can be marked cell-to-cell variation in the expression of the gene for a macrophage product such as fibronectin, suggesting that there are processes that modulate similar cells in the same anatomic compartment to vary their expression of the same gene.

Adult↗

PubMed1988

Characterization of the gene and protein of the common alpha 1-antitrypsin normal M2 allele.

The normal M2 variant of alpha 1-antitrypsin (alpha 1AT) was cloned from a genomic DNA library of an individual homozygous for this allele. Sequencing of all coding exons of the M2 gene revealed it was identical to the common M1(Val213) gene except for two bases (M1(Val213) CGT Arg101, M2 CAT His101; M1(Val213) GAA Glu376 M2 GAC Asp376). Analysis of the sequence of the M1(Val213) and M2 genes around residue 101 revealed the M1 Arg101----M2 His101 caused a loss of the cutting site for the restriction endonuclease RsaI. Using this enzyme, as well as 19-mer oligonucleotides probes centered at residues 101 and 376, evaluation of genomic DNA from 22 M1 alleles and 14 M2 alleles revealed that residue 101 was Arg in all M1 alleles and His in all M2 alleles, while residue 376 was Glu in all M1 alleles and Asp in all M2 alleles. Despite the differences in sequence at two amino acids, the M1(Val213) and M2 proteins function similarly as assessed by quantification of the association rate constant of each for their natural substrate neutrophil elastase. In the context that there are two mutations separating the M1(Val213) and M2 alleles, it is likely that there is another alpha 1AT variant that was an intermediate in the evolution of these genes.

Alleles↗