ABSTRACT
Modulation of cytokine expression represents a potentially useful approach for the treatment of idiopathic pulmonary fibrosis (IPF). To identify potential targets for such intervention, semi-quantitative reverse transcriptase-polymerase chain reaction was used to compare the expression of messenger ribonucleic acids (mRNAs) coding for 17 cytokines in lung tissue obtained from patients with IPF at the time of diagnosis and control subjects. Some cytokines were also studied at the protein level by immunohistochemical techniques. mRNAs coding for all of the cytokines evaluated were detected in both control and fibrotic lung samples. Only transforming growth factor (TGF)-beta and interleukin (IL)-10 mRNAs were quantitatively increased in lung biopsies from patients with IPF compared with those of controls, results confirmed at the protein level by immunohistochemistry. Although mRNAs for platelet-derived growth factor (PDGF)-BB and keratinocyte growth factor (KGF) were expressed in similar amounts in lungs from patients with IPF and controls, localised accumulation of both factors was also observed in IPF. Hyperplastic alveolar epithelial cells were a prominent source of cytokines, where IL-10, PDGF-BB and KGF were present in increased amounts, although increased accumulation in fibroblasts, smooth-muscle cells and matrix components was also observed (PDGF-BB, TGF-beta). These results offer new insights into the cytokines produced in the lung in idiopathic pulmonary fibrosis and suggest that modulation of the production of transforming growth factor-beta and interleukin-10 may represent a potentially useful therapeutic strategy for this disabling disease.
Subject(s)
Interleukin-10/metabolism , Lung/metabolism , Pulmonary Fibrosis/metabolism , RNA, Messenger/metabolism , Transforming Growth Factor beta/metabolism , Female , Humans , Immunohistochemistry , Lung/pathology , Male , Middle Aged , Pulmonary Fibrosis/pathology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Intravenous injection of complete Freund's adjuvant (CFA) in rats has been proposed as an experimental model for pulmonary sarcoidosis, but only some animals develop granulomas. Because the detection of the presence of granulomas and evaluation of the extent of the reaction has required histological evaluation, this model has been of limited use in following the evolution of the granulomatous process. The present study evaluated the ability of lung computed tomography (CT) scanning to identify in vivo pulmonary granulomas induced by CFA in rats. Wistar rats were injected with CFA to induce pulmonary sarcoid-like granulomas, and the presence and extent of pulmonary abnormalities, as detected by spiral CT and histopathological analysis, were compared. Spiral CT had a high sensitivity and specificity for the detection of sarcoid-like granulomas in rats injected with CFA. The extent of the pulmonary granulomatous reaction as assessed by the two techniques strongly correlated (r=0.93, p<0.01). In contrast, the mean density of lungs containing granulomas was not significantly different from that of lungs with no granulomatous reaction. Thus, lung computed tomography appears to be a valuable tool for the in vivo evaluation of the pulmonary granulomatous reaction induced by complete Freund's adjuvant in rats. With the help of computed tomography, this experimental model should be suitable for the sequential study of pulmonary sarcoid-like granulomas, particularly in response to various therapeutic strategies.
Subject(s)
Adjuvants, Immunologic/adverse effects , Freund's Adjuvant/adverse effects , Sarcoidosis, Pulmonary/chemically induced , Sarcoidosis, Pulmonary/diagnostic imaging , Tomography, X-Ray Computed , Adjuvants, Immunologic/administration & dosage , Animals , Disease Models, Animal , Freund's Adjuvant/administration & dosage , Granuloma/chemically induced , Granuloma/diagnostic imaging , Injections, Intravenous , Male , Rats , Rats, Wistar , Sensitivity and SpecificityABSTRACT
Mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and protease that confer resistance to antiretroviral agents are usually accompanied by a reduction in the viral replicative capacity under drug-free conditions. Consequently, when antiretroviral treatment is interrupted in HIV-1-infected patients harboring drug-resistant virus, resistant quasi-species appear to be most often replaced within several weeks by wild-type virus. Using a real-time PCR-based technique for the selective quantification of resistant viral sequences in plasma, we have studied the kinetics of the switch from mutant to wild-type virus and evaluated the extent to which minority populations of resistant viruses not detected by genotyping persist in these individuals. Among 12 patients with viruses expressing the V82A or L90M resistance mutation who had undergone a 3-month interruption of therapy and for whom conventional genotyping had revealed an apparent total reconversion to wild-type virus, minority populations expressing these mutations, representing 0.1 to 21% of total virus, were still detectable in 9 cases. Kinetic studies demonstrated that viruses expressing resistance mutations could be detected for >5 months after the discontinuation of treatment in some patients. Most of the minority resistant genomes detected more than 3 months after the interruption of therapy carried only part of the mutations present in the resistant viruses prior to treatment interruption and appeared to result from the emergence of existing strains selected at earlier stages in the development of drug resistance. Thus, following the interruption of treatment, viral populations containing resistance mutations can persist for several months after the time when conventional genotyping techniques detect only wild-type virus. These populations include viral strains with only some of the resistance mutations initially present, strains that presumably express better fitness under drug-free conditions.
Subject(s)
Anti-HIV Agents/therapeutic use , HIV Infections/virology , HIV-1/genetics , Drug Resistance, Microbial , Genotype , HIV Infections/drug therapy , HIV Protease , HIV-1/drug effects , Humans , Mutation , Polymerase Chain Reaction/methods , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , Time Factors , Treatment RefusalABSTRACT
The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guérin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU. Mycobacterial growth was optimal in cultures containing 5 x 10(4) cells/well, but increasing the number of cells to 2 x 10(5) cells/well resulted in complete inhibition of mycobacterial growth. This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins. The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 x 10(4) cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.
Subject(s)
Cell Count , Cell Culture Techniques/methods , Macrophages/immunology , Macrophages/microbiology , Mycobacterium bovis/growth & development , Mycobacterium bovis/immunology , Cell Adhesion/immunology , Cell Differentiation/immunology , Cell Survival/immunology , Colony Count, Microbial , Colony-Forming Units Assay , Culture Media, Conditioned/pharmacology , Enzyme Inhibitors/pharmacology , Extracellular Space/immunology , Extracellular Space/microbiology , Humans , Hydrogen-Ion Concentration , Jurkat Cells , Lysine/analogs & derivatives , Lysine/pharmacology , Macrophages/cytology , Macrophages/enzymology , Mycobacterium bovis/drug effects , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase Type II , Tumor Cells, CulturedABSTRACT
Some patients infected with human immunodeficiency virus (HIV) who are experiencing antiretroviral treatment failure have persistent improvement in CD4+ T cell counts despite high plasma viremia. To explore the mechanisms responsible for this phenomenon, 2 parameters influencing the dynamics of CD4+ T cells were evaluated: death of mature CD4+ T cells and replenishment of the CD4+ T cell pool by the thymus. The improvement in CD4+ T cells observed in patients with treatment failure was not correlated with spontaneous, Fas ligand-induced, or activation-induced T cell death. In contrast, a significant correlation between the improvement in CD4+ T cell counts and thymic output, as assessed by measurement of T cell receptor excision circles, was observed. These observations suggest that increased thymic output contributes to the dissociation between CD4+ T cell counts and viremia in patients failing antiretroviral therapy and support a model in which drug-resistant HIV strains may have reduced replication rates and pathogenicity in the thymus.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , HIV Infections/immunology , HIV-1 , Thymus Gland/immunology , Adult , Anti-HIV Agents/therapeutic use , CD4 Lymphocyte Count , Cell Death , Cells, Cultured , Cohort Studies , Fas Ligand Protein , Female , HIV Infections/drug therapy , HIV Infections/virology , Humans , Leukocytes, Mononuclear/immunology , Male , Membrane Glycoproteins , Middle Aged , Receptors, Antigen, T-Cell/analysis , Treatment Failure , Viral Load , ViremiaABSTRACT
IMPORTANCE OF T-LYMPHOCYTES IN SARCOIDOSIS: Sarcoidosis is thought to result from an uncontrolled granulomatous immune response. T-lymphocytes are an essential component of this immune reaction. The recognition of specific antigens through receptors expressed on the cell membrane activates the T-cells, resulting in the expression of effector functions that ultimately control granuloma formation. T-cells participating in sarcoid reactions are readily accessible using bronchoalveolar lavage, and have been intensively studied. SPECIFICITY OF T-CELLS: Several groups have evaluated the diversity of T-cell receptors expressed by T-cells from patients with sarcoidosis. These studies have demonstrated that oligoclonal T-cell populations are present both in the lung and blood of these patients, findings that support the conclusion that antigen-induced immune responses play a role in the pathogenesis of sarcoidosis. The identification of the antigen(s) recognized by these cells remains an important goal, and may help identify etiologic agents. CYTOKINE PRODUCTION: Considerable progress has been made in characterizing the cytokines produced by T-lymphocytes and other cells participating in the granulomatous reaction. The modulation of the activity of these mediators represents a promising approach for the development of more specific therapeutic agents.
Subject(s)
Sarcoidosis/immunology , T-Lymphocytes/immunology , Adjuvants, Immunologic , Bronchoalveolar Lavage , Cytokines/immunology , HumansABSTRACT
Because Langerhans cells (LC) in peripheral tissues are generally "immature" cells with poor lymphostimulatory activity, the contribution of immune responses initiated by LC to the pathogenesis of pulmonary LC histiocytosis (LCH) has been uncertain. In this study we demonstrate that LC accumulating in LCH granulomas are phenotypically similar to mature lymphostimulatory dendritic cells present in lymphoid organs. LC in LCH granulomas intensely expressed B7-1 and B7-2 molecules, whereas normal pulmonary LC and LC accumulating in other pathologic lung disorders did not express these costimulatory molecules. The presence of B7+ LC in LCH granulomas was associated with the expression in these lesions, but not at other sites in the lung, of a unique profile of cytokines (presence of GM-CSF, TNF-alpha, and IL-1beta and the absence of IL-10) that is known to promote the in vitro differentiation of LC into cells expressing a lymphostimulatory phenotype. Finally, LCH granulomas were the only site where CD154-positive T cells could be identified in close contact with LC intensely expressing CD40 Ags. Taken together, these results strongly support the idea that an abnormal immune response initiated by LC may participate in the pathogenesis of pulmonary LCH, and suggest that therapeutic strategies aimed at modifying the lymphostimulatory phenotype of LC may be useful in the treatment of this disorder.
Subject(s)
Cytokines/physiology , Dendritic Cells/pathology , Histiocytosis, Langerhans-Cell/pathology , Langerhans Cells/pathology , Adult , Aged , Animals , B7-1 Antigen/biosynthesis , CD40 Antigens/biosynthesis , CD40 Ligand , Cell Differentiation/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Granuloma, Respiratory Tract/immunology , Granuloma, Respiratory Tract/metabolism , Granuloma, Respiratory Tract/pathology , Histiocytosis, Langerhans-Cell/immunology , Histiocytosis, Langerhans-Cell/metabolism , Humans , Langerhans Cells/immunology , Langerhans Cells/metabolism , Ligands , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Membrane Glycoproteins/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Middle AgedABSTRACT
Normal alveolar macrophages (AM) are not efficient in inducing the proliferation of resting T lymphocytes, and, rather, tend to inhibit pulmonary immune responses. In contrast, epithelioid cells (EC), activated macrophages that play an essential role in the course of granulomatous responses, appear to stimulate T cell proliferation efficiently. The inability of macrophages to deliver potent costimulatory signals through the B7/CD28 and CD40/CD40L pathways could explain their weak accessory cell activity. Using MoAbs and immunohistochemical techniques, however, we found that essentially all AM in normal human lung tissue expressed B7-1, B7-2 and CD40 molecules, and most of these cells were strongly positive. Pulmonary macrophages in other compartments also expressed these costimulatory molecules; no differences in expression were observed comparing macrophages from smokers and non-smokers. Most AM recovered by bronchoalveolar lavage from normal lung segments also strongly expressed B7-1, B7-2 and CD40 molecules. In comparison, resting blood monocytes were B7-1- and only moderately positive for B7-2. Activation of monocytes with lipopolysaccharide (LPS) induced expression of these costimulatory molecules to levels similar to that of AM from the control subjects. EC in granulomatous lesions also expressed easily detectable levels of B7-1, B7-2 and CD40. T lymphocytes within and surrounding the granulomas expressed CD28, the counter-receptor for B7, and many of these T cells also expressed B7-1 and B7-2. These findings suggest that both AM and EC can deliver costimulatory signals through B7-1, B7-2 and CD40 molecules, and indicate that the impairment in accessory cell activity observed for normal AM cannot be attributed to the absence of expression of these costimulatory molecules.
Subject(s)
Antigens, CD/analysis , B7-1 Antigen/analysis , CD40 Antigens/analysis , Granuloma/immunology , Macrophages, Alveolar/chemistry , Membrane Glycoproteins/analysis , Tuberculosis/immunology , Adult , Aged , B7-2 Antigen , Epithelial Cells/chemistry , Female , Flow Cytometry , Humans , Male , Middle AgedABSTRACT
The mechanisms through which immune and inflammatory responses stimulate the expression of antimycobacterial activity by human macrophages remain poorly defined. To study this question, we developed a method permitting the rapid quantification of viable mycobacteria, based on the detection of luciferase activity expressed by a Mycobacterium bovis Bacillus Calmette-Guerin (BCG) reporter strain, and used this approach to evaluate mycobacterial survival in human monocyte-derived macrophages following stimulation with cytokines and through crosslinking of costimulatory molecules expressed on the cell surface. Modest proliferation, followed by persistence of mycobacteria, was observed in unpretreated macrophages as assessed both by measurement of luciferase activity and by the evaluation of colony forming units. Of the 19 cytokines tested, only granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to improve the mycobactericidal activity of monocyte-derived macrophages. In both cases, this effect was observed only when macrophages were pretreated with the cytokines prior to infection. In contrast, pretreatment of human macrophages with interferon-gamma, either alone or in combination with other mediators (including tumor necrosis factor-alpha and 1,25[OH]2-vitamin D3), did not improve mycobacterial killing. The stimulation of macrophages through several different costimulatory molecules known to participate in macrophage-lymphocyte interactions (CD4, CD40, CD45, CD86, CD95 [Fas/Apo-1]) also failed to improve mycobactericidal activity. This study shows that GM-CSF and IL-3, cytokines whose receptors are known to share a common subunit and to use common second messengers, may contribute to the stimulation of mycobactericidal activity in humans. The ability to rapidly screen the effects of different macrophage stimuli on mycobacterial survival through the detection of luciferase activity should help define additional signals required for optimal antimycobacterial responses.
Subject(s)
Intracellular Membranes/microbiology , Macrophages/microbiology , Mycobacterium bovis/physiology , Colony Count, Microbial , Cytokines/pharmacology , Genes, Reporter/physiology , Humans , Interferon-gamma/pharmacology , Luciferases/metabolism , Macrophages/drug effects , Mycobacterium bovis/drug effects , Mycobacterium bovis/enzymology , Mycobacterium bovis/genetics , Stimulation, ChemicalABSTRACT
Pulmonary Langerhans cell histiocytosis (LCH) is an uncommon disorder occurring most often in young smokers. Histologically, the disease is characterized by granulomatous lesions containing LC that destroy distal bronchioles. The etiology of the disease remains unknown, but progress has been made in understanding its pathogenesis. Modifications in the bronchiolar epithelium induced by smoking, such as the increased secretion of GM-CSF by these cells, are probably responsible for the initial accumulation of large numbers of LC. However, given the rarity of pulmonary LCH compared with the frequency of smoking, an as yet unidentified genetic predisposition may also be necessary for the development of the disease. Although LC in LCH granulomas may be clonal in origin, several observations argue against the idea that the disease, which can regress spontaneously, is a malignant process. Cells of dendritic cell lineage (including LC), are potent antigen presenting cells, suggesting that pulmonary LCH results from an uncontrolled immune response initiated by LC. Consistent with this idea, LC and T-cells are the predominant cell populations found in the early lesions of pulmonary LCH, and unlike LC in the normal bronchial mucosa and those accumulating in other pathologic situations, LC in pulmonary LCH granulomas express surface molecules important for the activation of T-lymphocytes. A number of mediators are produced in the microenvironment of granulomas that probably influence the outcome of the local immune and inflammatory reaction. Ultimately, precise knowledge of the pathogenesis of this disorder should permit the development of specific treatment. In the interim, therapies aimed at modifying the state of activation of LC in the granulomatous lesions may prove useful.
Subject(s)
Histiocytosis, Langerhans-Cell/etiology , Smoking/adverse effects , Adult , Cytokines/biosynthesis , Cytokines/immunology , Cytokines/pharmacology , Histiocytosis, Langerhans-Cell/immunology , Histiocytosis, Langerhans-Cell/pathology , Humans , Lung/pathology , T-Lymphocytes/immunologyABSTRACT
Receptors belonging to the tumour necrosis factor receptor (TNF-R) superfamily are implicated in a variety of important biological processes. Although messenger ribonucleic acid coding for many of these receptors has been detected in lung, little is known about their expression in this organ. In this study, immunohistochemical techniques were used to evaluate the expression of three receptors of this family (4-1BB, lymphotoxin-beta receptor (LTbeta-R), and Fas) in normal human lung, lung carcinomas and by tuberculous and sarcoid granulomas. The 4-1BB receptor was uniformly expressed by endothelial cells of small vessels and by basal epithelial cells within pseudostratified bronchial epithelium. LTbeta-R expression by parenchymal cells was limited to those of epithelial origin (bronchial and bronchiolar epithelium, hyperplastic alveolar epithelium and lung carcinomas). Fas was present on both fibroblasts and epithelial cells (bronchial and alveolar epithelium and most, but not all, carcinomas), but not on endothelial cells. A subpopulation of T-lymphocytes expressed these receptors, but only Fas was detected on normal alveolar macrophages and epithelioid cells. Thus, 4-1BB, LTbeta-R, and Fas have characteristic and only partially overlapping patterns of expression in the lung. The findings should facilitate further evaluation of their role in lung homeostasis and pathology.
Subject(s)
Lung/chemistry , Receptors, Tumor Necrosis Factor/analysis , fas Receptor/analysis , Adenocarcinoma/chemistry , Adult , Aged , Antigens, CD , Biomarkers/analysis , Biopsy, Needle , Carcinoma, Squamous Cell/chemistry , Culture Techniques , Female , Humans , Immunohistochemistry , Lung Neoplasms/chemistry , Lymphotoxin beta Receptor , Male , Membrane Glycoproteins/analysis , Middle Aged , Receptors, Nerve Growth Factor/analysis , Reference Values , Sarcoidosis, Pulmonary/pathology , Sensitivity and Specificity , Tuberculosis, Pulmonary/pathology , Tumor Necrosis Factor Receptor Superfamily, Member 9ABSTRACT
The pathogenesis of sarcoidosis has intrigued clinicians since the first descriptions of the disease, and a number of causes have been proposed. Among the candidate agents, the possible role of mycobacterial infection in the pathogenesis of sarcoidosis has attracted by far the most attention. The purpose of this review is to summarize current evidence concerning the role of mycobacterial infection in sarcoidosis. First, the similarities in clinical and histological features of tuberculosis and sarcoidosis that have raised suspicion that mycobacterial infection could be involved in the pathogenesis of sarcoidosis are discussed. In addition, experimental evidence for and against a role of mycobacterial infection is summarized, including recent attempts to detect mycobacterial DNA in clinical samples from patients with sarcoidosis by polymerase chain reaction.
Subject(s)
Sarcoidosis, Pulmonary/microbiology , Tuberculosis, Pulmonary/complications , Animals , DNA, Bacterial/analysis , Humans , Mycobacterium tuberculosis/isolation & purification , Polymerase Chain ReactionABSTRACT
A luminescence-based procedure that permits the rapid evaluation of the survival of mycobacteria within mononuclear phagocytes was developed and used to screen insertional mutants of Mycobacterium smegmatis for their ability to survive in human monocyte-derived macrophages. Among the 5000 mutants tested, eight mutants were identified that demonstrated impaired intracellular survival in human macrophages but that grew normally in the absence of cells. For each mutant, a portion of the gene interrupted by the transposition event was amplified by ligand-mediated PCR and sequenced. In all cases, the existence of homologous genes of as yet unknown function were identified in the Mycobacterium tuberculosis genome. Complementation of the mutant mycobacterial strains with cosmids containing the homologous loci from M. tuberculosis restored normal intracellular growth in three of the four mutants tested, supporting the idea that these loci contain genes that are important for intracellular survival. This study demonstrates the feasibility of directly screening mutant mycobacterial strains to identify genes coding for activities necessary for the intracellular survival in human mononuclear phagocytes, an important initial step in the identification of potential targets for new therapeutic agents.
Subject(s)
Mutation , Mycobacterium smegmatis/genetics , Phagocytes/microbiology , Base Sequence , DNA Transposable Elements , Genes, Bacterial , Genetic Complementation Test , Humans , Luminescent Measurements , Macrophages/microbiology , Microbiological Techniques , Molecular Sequence Data , Mycobacterium smegmatis/pathogenicity , Mycobacterium tuberculosis/geneticsABSTRACT
In most patients with pulmonary Langerhans cell histiocytosis (LCH), clinical and radiological abnormalities initially either stabilize or regress, often without treatment. Little information is available, however, concerning the subsequent evolution of disease in patients who initially follow a benign course. We describe four patients with biopsy-confirmed pulmonary LCH whose initial course was characterized by regression of parenchymal nodular lesions, but who subsequently developed one or more episodes of active disease 7 mo to 7.5 yr after their initial presentation. In each case, the subsequent episodes of active disease were characterized by the reappearance or marked increase in nodular radiographic abnormalities, whose presence was confirmed by high-resolution computed tomography (HRCT). Thus, initial regression of nodular lesions in pulmonary LCH does not preclude the reappearance of one or more episodes of active disease, and may have important consequences on the long-term prognosis of these patients.
Subject(s)
Histiocytosis, Langerhans-Cell/diagnostic imaging , Lung/diagnostic imaging , Adult , Female , Histiocytosis, Langerhans-Cell/pathology , Histiocytosis, Langerhans-Cell/therapy , Humans , Lung/pathology , Radiography , RecurrenceABSTRACT
Pulmonary Langerhans cell granulomatosis (LCG), also called histiocytosis X, is a disorder of unknown etiology characterized by the presence of destructive granulomas containing numerous Langerhans cells (LCs). The process may be localized or multifocal, and it remains unclear whether the same pathogenic mechanism is involved in all forms of the disease. It is often assumed that the massive accumulation of LCs at the sites of the lesions results from the abnormal proliferation of these cells, although it has been suggested that LCG in adults, at least in the lung, could be a reactive disorder initiated by activated LCs. Little is known, however, concerning the mechanisms responsible for the accumulation of large numbers of LCs in the course of the disease, and the relative contribution of recruitment and local proliferation of these cells remains to be established. To investigate this question, the proportion of replicating LCs was evaluated in biopsied granulomas from patients with localized or diffuse form of LCG by means of several histopathological techniques currently used in assessment of cell proliferation. The findings demonstrate that, except for proliferating cell nuclear antigen (PCNA), all parameters measured are low in all forms of the disease. They are similar to those of renewing epithelial cells and clearly less than those of neoplastic cells. These data strongly suggest that LCs in LCG granulomas are not a rapidly dividing cell population and that local LC replication makes only a minimal contribution to granuloma maintenance. Caution appears to be necessary in the use of PCNA as a marker of growth fraction.
Subject(s)
Histiocytosis, Langerhans-Cell/pathology , Langerhans Cells/pathology , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adult , Aged , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Division , Child , Child, Preschool , DNA/analysis , DNA, Neoplasm/analysis , Female , Histiocytosis, Langerhans-Cell/metabolism , Humans , Immunohistochemistry , Infant , Ki-67 Antigen/metabolism , Langerhans Cells/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Middle Aged , Mitotic Index , Nucleolus Organizer Region/metabolism , Nucleolus Organizer Region/pathology , Proliferating Cell Nuclear Antigen/metabolismABSTRACT
A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.
Subject(s)
DNA Transposable Elements/genetics , DNA, Bacterial/analysis , Mutagenesis, Insertional/methods , Mycobacterium tuberculosis/genetics , Cloning, Molecular/methods , DNA Primers , Genome, Bacterial , Polymerase Chain ReactionABSTRACT
To evaluate the role of mycobacterial infection in the pathogenesis of sarcoidosis, several groups have attempted to identify mycobacterial DNA in clinical samples from these patients by polymerase chain reaction (PCR), but widely divergent results have been obtained. It has been suggested that differences in the sensitivity of the procedures used may explain these discrepant results. To test this possibility, the presence of mycobacterial DNA was sought in biopsies from patients with sarcoidosis using sequence capture-PCR, a procedure that is 100-fold more sensitive in detecting mycobacterial DNA in paucibacillary samples than standard PCR protocols. Using this approach, DNA corresponding to two different sequences specific for organisms of the Mycobacterium tuberculosis complex (the 1S6110 insertion element and the DR region) could not be detected in any of the 15 biopsies from patients with sarcoidosis, whereas a high proportion of positive results was obtained for tissue biopsies and other clinical samples from patients with active tuberculosis, including samples that were smear-negative/culture-positive and smear-negative/culture-negative. These results support prior studies suggesting that M. tuberculosis does not play a pathogenic role in sarcoidosis in most patients.
Subject(s)
DNA, Bacterial/analysis , Mycobacterium tuberculosis/genetics , Polymerase Chain Reaction/methods , Sarcoidosis/microbiology , Tuberculosis/complications , Biopsy , Case-Control Studies , Humans , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
The reasons why severe allergic reactions to bee and wasp stings develop in only a small portion of exposed individuals are incompletely understood, but differences in T cell responses to venom antigens comparing allergic and non-allergic individuals are likely to be important. To identify such differences, venom-induced proliferative responses and cytokine mRNA production by blood mononuclear cells from Vespula venom-allergic patients and non-allergic individuals were compared. Mononuclear cells from most venom-allergic patients proliferated in response to alkylated Vespula venom (7275 +/- 8387 ct/min, n = 19), and the extent of proliferation was greater for patients with a history of multiple prior stings and those with high levels of venom-specific IgE. Although mononuclear cells from non-allergic subjects showed little or no proliferation in response to venom (926 +/- 711 ct/min, n = 8), production of mRNAs coding for IL-2, IL-4, IL-5, IL-10 and interferon-gamma (IFN-gamma) in response to Vespula venom by cells from non-allergic subjects was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), indicating that these individuals had been previously sensitized to venom antigens. In contrast to the Th0 cytokine mRNA profile observed for non-allergic individuals, venom-allergic patients released a more restricted profile of cytokines following stimulation with venom. Only IFN-gamma mRNA expression was detected in all individuals evaluated, whereas IL-2 mRNA was not detected during the first 48 h of stimulation, and T cells from only one of three venom-allergic individuals produced detectable IL-4 or IL-5 mRNA. The difference in cytokine profiles observed comparing venom-allergic patients and non-allergic controls could not be attributed to intrinsic differences in T cells from these individuals, because polyclonal stimulation with phorbol myristate acetate (PMA) + ionophore induced similar cytokine mRNA profiles in the two groups. These studies demonstrate clear differences in the T cell responses of venom-allergic subjects, that may contribute to the development of severe allergic reactions in these individuals.
Subject(s)
Anaphylaxis/immunology , Cytokines/biosynthesis , Insect Bites and Stings/immunology , Lymphocyte Activation , RNA, Messenger/biosynthesis , Wasp Venoms/immunology , Wasps , Adult , Allergens/immunology , Anaphylaxis/etiology , Animals , Cytokines/genetics , DNA Primers/chemistry , DNA Probes/chemistry , Humans , Insect Bites and Stings/complications , Polymerase Chain Reaction , T-Lymphocytes/immunologyABSTRACT
Techniques based on the polymerase chain reaction (PCR) can be used to rapidly identify DNA from Mycobacterium tuberculosis in clinical samples from patients with tuberculosis, but prior studies evaluating this approach in the diagnosis of paucibacillary forms of pulmonary tuberculosis have reported poor sensitivity and/or specificity. We have developed a procedure in which mycobacterial DNA in crude samples is specifically captured prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other inhibitors of the amplification reaction (sequence capture PCR). To evaluate the usefulness of this approach in the diagnosis of paucibacillary forms of pulmonary tuberculosis, sequence capture PCR was performed prospectively on samples of bronchoalveolar lavage fluid from consecutive patients suspected of having pulmonary tuberculosis but for whom three consecutive samples of respiratory secretions were smear negative. Of the 27 patients evaluated, active tuberculosis was diagnosed in nine; sequence capture PCR was positive for all of these patients, including the three for whom all specimens submitted for culture were negative. No positive results were obtained for lavage fluid from the 18 patients for whom the diagnosis of active tuberculosis was subsequently excluded or 25 additional patients undergoing bronchoalveolar lavage for evaluation of other pulmonary problems, even though many of these patients had a history of prior tuberculosis or radiographic evidence of prior tuberculous infection. Paucibacillary forms of pulmonary tuberculosis can be rapidly identified with high sensitivity and specificity using sequence capture PCR performed on samples obtained by bronchoalveolar lavage.