Characterisation of dendritic cell subsets in lung cancer micro-environments.

The aim of the current study was to seek evidence for a correlation between mediators present in lung cancer micro-environments and subsets of dendritic cells (DCs) infiltrating these tumours. Immunohistochemistry and recently available antibodies were used to define the phenotype of DCs present in surgical biopsies from 12 patients with lung carcinomas, and the local expression of chemokines potentially involved in the recruitment of these cells was evaluated, both at mRNA and protein levels. Real-time PCR was used to analyse the expression of mRNA coding for cytokines known to influence the maturation of DCs in vitro. Different subsets of myeloid DCs were present in lung cancers, but no plasmocytoid DCs were identified. Both Langerhans cells and CD1a+/Langerin cells were interspersed among tumour cells, in numbers that were correlated to the amounts of CC chemokine ligand 20 produced in these tumours. In most specimens, DC-specific intercellular adhesion molecule-grabbing nonintegrin-positive DCs were also present at the periphery of the tumour beds. No DC-lysosomal associated membrane protein-positive DCs were identified and CD83+ DCs were rarely present in the tumour stroma. All tumours expressed interleukin (IL)-10, transforming growth factor-beta and vascular endothelial growth factor, whereas IL-12 was virtually absent. Thus, various types of dendritic cells infiltrate lung carcinomas and display an immature phenotype, presumably because of the inhibitory cytokine micro-environment.

Caspase-dependent and -independent T-cell death pathways in pathogenic simian immunodeficiency virus infection: relationship to disease progression.

Studies of human immunodeficiency virus (HIV) and nonhuman primate models of pathogenic and nonpathogenic simian immunodeficiency virus (SIV) infections have suggested that enhanced ex vivo CD4 T-cell death is a feature of pathogenic infection in vivo. However, the relative contributions of the extrinsic and intrinsic pathways to programmed T-cell death in SIV infection have not been studied. We report here that the spontaneous death rate of CD4+ T cells from pathogenic SIVmac251-infected rhesus macaques ex vivo is correlated with CD4 T-cell depletion and plasma viral load in vivo. CD4+ T cells from SIVmac251-infected macaques showed upregulation of the death ligand (CD95L) and of the proapoptotic proteins Bim and Bak, but not of Bax. Both CD4+ and CD8+ T cells from SIVmac251-infected macaques underwent caspase-dependent death following CD95 ligation. The spontaneous death of CD4+ and CD8+ T cells was not prevented by a decoy CD95 receptor or by a broad-spectrum caspase inhibitor (zVAD-fmk), suggesting that this form of cell death is independent of CD95/CD95L interaction and caspase activation. IL-2 and IL-15 prevented the spontaneous death of CD4+ and CD8+ T cells, whereas IL-10 prevented only CD8 T-cell death and IL-7 had no effect on T-cell death. Our results indicate that caspase-dependent and caspase-independent pathways are involved in the death of T cells in pathogenic SIVmac251-infected primates.

Changes in human immunodeficiency virus type 1 populations after treatment interruption in patients failing antiretroviral therapy.

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.

Discordant increases in CD4+ T cells in human immunodeficiency virus-infected patients experiencing virologic treatment failure: role of changes in thymic output and T cell death.

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.

Effect of stimulation of human macrophages on intracellular survival of Mycobacterium bovis Bacillus Calmette-Guerin. Evaluation with a mycobacterial reporter strain.

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.

Identification of genetic loci implicated in the survival of Mycobacterium smegmatis in human mononuclear phagocytes.

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.

Absence of DNA from mycobacteria of the M. tuberculosis complex in sarcoidosis.

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.

Cytokine patterns in tuberculous and sarcoid granulomas: correlations with histopathologic features of the granulomatous response.

Cytokines play an important role in granuloma formation, but the extent that cytokine profiles are similar in different granulomatous diseases and whether differences in the histopathologic features of the granulomatous response results from differences in cytokine production have not been evaluated. To investigate these questions, we used RT-PCR to quantify the expression of mRNAs coding for 16 cytokines in granulomatous lymph nodes from patients with tuberculosis and sarcoidosis and from control tissues, and we sought correlations between the level of expression of these cytokines and the histopathologic features of the granulomas. Expression of mRNAs coding for a number of cytokines (IL-1beta, IFN-gamma, TNF-alpha, granulocyte-macrophage (GM)-CSF, IL-12 (p40), and lymphotoxin-beta) was increased in tuberculous and sarcoid granulomas compared with that of control tissues. All sarcoid granulomas were shown to express a Th1 pattern of cytokine mRNAs, while tuberculous lymph nodes expressed either a Th1 or a Th0 profile. GM-CSF and lymphotoxin-beta mRNAs were more abundant in sarcoid than in tuberculous granulomas, whereas IL-8 mRNA was strongly expressed only in tuberculous lymph nodes. Strong expression of GM-CSF, TNF-alpha, and IL-8 by granulomas was shown to be correlated, respectively, with the presence of florid granulomatous lesions, the absence of central necrosis, and the presence of neutrophil infiltration. These results demonstrate that the formation of tuberculous and sarcoid granulomas in humans is associated with the expression of characteristic cytokine profiles and indicate that the expression of certain cytokines is associated with the development of specific pathologic features in the resulting granulomas.

Characterization of proliferative responses and cytokine mRNA profiles induced by Vespula venom in patients with severe reactions to wasp stings.

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.

Diagnosis of smear-negative pulmonary tuberculosis using sequence capture polymerase chain reaction.

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.

Activation of T-cells through an antigen-independent alternative pathway induces precocious sensitivity to Fas-induced apoptosis.

Autoreactive T-cells can be activated inadvertently during immune responses through antigen-independent pathways. It has been suggested that Fas/Fas ligand interactions may play a role in eliminating these cells, but the extent that cells activated through such alternative pathways are sensitive to Fas-induced apoptosis has not been extensively evaluated. Proliferation of peripheral blood T-cells from normal individuals activated for 4 days with PHA or PMA + ionophore was not influenced by the presence of anti-Fas antibody. When the same cells were activated with soluble factors produced by previously activated T-cells (lymphostimulatory activity), anti-Fas antibodies inhibited thymidine incorporation by 74+/-4%. The presence of typical morphological changes and oligonucleosomal fragmentation of DNA indicated that the reduced proliferation resulted from apoptotic death of the lymphoblasts. Fas-sensitivity of T-cells activated by lymphostimulatory activity was first detectable 4 days after activation, and at 5 days the majority of lymphoblasts had become sensitive to Fas, whereas no evidence of sensitivity to Fas was observed for lymphoblasts generated by PHA or PMA + ionophore during the first 5 days of culture. Incubation of cells activated with PHA or PMA+ ionophore in the presence of IL-2 at concentrations 10-fold higher than that present in lymphostimulatory activity did not induce early sensitivity to Fas, indicating that exposure to IL-2 could not explain the precocious development of sensitivity to Fas seen following activation by lymphostimulatory activity. These studies demonstrate that T-cells activated through an antigen-independent 'alternative' pathway develop precocious sensitivity to Fas-induced apoptosis, which may be important in permitting the elimination of autoreactive bystander cells activated in the course of immune responses.

Extensive apoptosis of lung T-lymphocytes maintained in vitro.

The phenotypic and functional properties of T cells recovered from the lung indicate that many of these cells have been recently activated. Because such recently activated cells are often more susceptible to death through apoptotic mechanisms, the viability of lung T cells recovered from bronchoalveolar lavage and those isolated from peripheral blood was compared. The progressive loss of viable cells following in vitro culture was considerably greater for lavage T cells than blood T cells, and was observed for cells from both patients with sarcoidosis and control subjects. Following 4 days of culture, 76 +/- 14% of blood cells, but only 31 +/- 13% of lavage cells from sarcoid patients were viable. The evaluation of morphologic features and flow cytometric profiles, as well as the demonstration of typical oligonucleosomal fragmentation of DNA extracted from these cells indicated that lavage T cells were dying by apoptotic mechanisms. CD4+ T cells appeared to be particularly sensitive to apoptosis. Most lavage T cells from controls and sarcoid patients expressed Fas (CD95) antigen. Although some lavage T Cells were sensitive to Fas-induced apoptosis, the viability of lavage T cells was not improved by incubation in the presence of a monoclonal antibody that inhibits Fas-induced apoptosis. Culture in the presence of interleukin 2 did prevent, at least in part, the progressive death of lavage T cells, suggesting that the viability of T cells in the lung may depend on the presence of locally delivered trophic signals. These studies emphasize that T cells on the alveolar surface are in a different state of activation and differentiation compared with that of circulating T cells, and offer a possible explanation for the impaired functional capacities observed for lavage T cells in some in vitro studies.

Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens.

The rapid identification of mycobacterial DNA in clinical samples by PCR can be useful in the diagnosis of tuberculous infections, but several large studies have found that the sensitivity of this approach is not better than that of culture. In order to improve the sensitivity of detection of mycobacterial DNA in clinical specimens from patients with paucibacillary forms of tuberculosis, we have developed a procedure permitting the specific capture of mycobacterial DNA in crude samples prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other potential inhibitors of the amplification reaction (sequence capture-PCR). By using this approach to capture and amplify two different sequences specific for organisms of the Mycobacterium tuberculosis complex (IS6110 and the direct repeat region), it was possible to detect as little as one genome of mycobacterial DNA in samples containing up to 750 micrograms of total DNA, representing a 10- to 100-fold increase in sensitivity compared with that obtained by purifying total DNA prior to amplification. Detection of the IS6110 sequence in pleural fluid samples from patients with tuberculous pleurisy by sequence capture-PCR gave positive results in 13 of 17 cases, including 3 of 3 culture-positive samples and 10 of 14 culture-negative samples. In contrast, when total DNA was purified from these samples by adsorption to a silica matrix prior to amplification, only the three culture-positive samples were positive by PCR. The sensitivity of detection of the direct repeat sequence in these samples by sequence capture-PCR was similar to that of IS6110 and, in addition, permitted immediate typing of the strains from some patients. We conclude that sequence capture-PCR improves the sensitivity of detection of mycobacterial DNA in paucibacillary samples. This approach should be useful in detecting rare target sequences from organisms implicated in other pathologic processes.

Expression and modulation of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in skin fibroblasts from patients with type Ib pseudohypoparathyroidism.

To explore the possibility that defects in the regulation of expression of the messenger ribonucleic acid (mRNA) coding for the PTH receptor could be involved in pseudohypoparathyroidism type Ib (PHP-Ib), PTH-induced cAMP production and PTH/PTH-related peptide (PTH-rp) receptor mRNA expression, measured using a ribonuclease protection assay, were compared in untreated and dexamethasone (dexa)-pretreated (5 x 10(-7) mol/L; 7 days) cultured skin fibroblasts from controls (n = 4) and patients with PHP-Ib (n = 6). In control fibroblasts, stimulation of cAMP production by PTH and expression of PTH/PTH-rp receptor mRNA were easily detectable and were not significantly affected by dexa pretreatment. In fibroblasts from three PHP-Ib patients demonstrating reduced PTH-induced cAMP production that was reversed by dexa, the level of basal PTH/PTH-rp receptor mRNA was also reduced, but increased to levels similar to those in control cells after dexa pretreatment. In fibroblasts from a patient with resistance to PTH not reversed by dexa, PTH/PTH-rp receptor mRNA expression was also significantly lower than that in control cells (18 +/- 13%; P < 0.001) and remained only 30 +/- 15% of that observed in control cells after dexa pretreatment (P < 0.001). In fibroblasts from two PHP-Ib patients expressing normal cAMP responsiveness to PTH before and after dexa treatment, the level of PTH/PTH-rp receptor mRNA was not different from that in control cells before or after dexa treatment. Thus, in all conditions where PTH-induced cAMP production by PHP-Ib fibroblasts was reduced, the abnormality could be explained by the reduced level of PTH/PTH-rp receptor mRNA in these cells. These results suggest that defects in the regulation of expression of the PTH/PTH-rp receptor mRNA, not structural defects in the receptor itself, explain the PTH resistance in PHP-Ib in the patients evaluated, but several different defects must exist.

Activation of T cells by previously activated T cells. HLA-unrestricted alternative pathway that modifies their proliferative potential.

When cultured in the presence of previously activated T cells, up to 30% of resting T cells are activated, as indicated by lymphoblastic morphology, formation of cell aggregates, expression of activation Ags (CD25 and HLA-DR), and a proliferative response. This activation occurred in the absence of accessory cells and was not HLA restricted, and the TCR repertoire of the responding cells was extremely diverse without evidence for preferential expansion of T cells expressing certain V beta families or clonal populations. The ability of activated T cells to stimulate resting T cells was a transient phenomenon, which was first detected 24 h after activation and which peaked between 48 and 96 h; the stimulation of previously resting T cells produced more lymphostimulatory activity than restimulation of recently activated cells. Both resting CD4+ and CD8+ T cells expressing TCR-alpha beta and -gamma delta responded to previously activated cells, including cells with both the naive and memory phenotypes. When T cells were activated by this pathway and recultured in the presence of Ag and accessory cells, the strong proliferative response observed at 5 to 7 days with use of fresh T cells was almost entirely absent, and this impaired Ag-induced proliferative response could not be explained by the generation of suppressor cells or the inability of these cells to respond to growth factors. These findings are compatible with the possibility that Ag-specific activation of T cells permits the subsequent Ag-independent activation of other T cells and could explain the broad TCR repertoire and impaired Ag-induced proliferation of activated T cells at sites of immune reactions.

T cell receptor repertoire of T lymphocytes recovered from the lung and blood of patients with sarcoidosis.

We evaluated the repertoire of V beta segments used in forming the T-cell receptor of lavage and blood T lymphocytes from 11 sarcoid patients and 10 normal subjects using procedures based on quantitative polymerase chain reaction, permitting analysis of both the abundance of transcripts using each of 20 different V beta families and the diversity of the VDJC beta rearrangements within each V beta family. Blood and lung T cells from sarcoid patients had a very diverse V beta repertoire. For all V beta families but one, the abundance of the V beta transcripts fell within the mean +/- 2 SD of that observed for normal blood lymphocytes; no difference in the overall abundance was observed comparing lavage and blood T cells, and the length of VDJC beta rearrangements for a given V beta family in samples from sarcoid patients was usually quite heterogeneous. Despite the overall polyclonality, evidence for selective expansion of T cells was found, in that an increased abundance of V beta 19 transcripts was observed for sarcoid blood and/or lung T cells in eight out of 11 patients studied, and rearrangements of a single predominant length using certain (e.g., V beta 19, V beta 14), but not all, V beta families were present. Sequencing confirmed the presence of a single predominant VDJC beta rearrangement in these cases. These findings suggest that the alveolitis in sarcoidosis results from two distinct processes, a local clonal expansion of T cells associated with an apparently nonspecific accumulation of T cells with an extremely diverse V beta repertoire.

Diagnosis of primary tuberculosis in children by amplification and detection of mycobacterial DNA.

Standard microbiologic techniques were compared with a rapid diagnostic method based on the amplification by polymerase chain reaction (PCR) of a fragment of the IS6110 insertion element (present in multiple copies in the Mycobacterium tuberculosis genome) for the detection of M. tuberculosis in specimens obtained from children diagnosed as having primary tuberculosis on clinical grounds. Two (n = 7) or three (n = 15) gastric aspirates were obtained from the 22 children with primary tuberculosis. All specimens were negative for mycobacteria by acid-fast staining and culture. When DNA was purified from the clinical specimens and aliquots of each sample were amplified in duplicate, 15 of 59 (25%) specimens gave at least one positive result. Increasing beyond two the number of times that samples were tested did not appreciably improve sensitivity. Testing multiple samples from the same individual increased the diagnostic yield. Thus, when three different samples from the same subject were tested two times each, two or more positive results were obtained from 9 of 15 children with primary tuberculosis but 0 of 17 control subjects. Samples from children with symptoms, recent contact with patients with active tuberculosis, vesicular tuberculin responses, or abnormal chest radiographs were more frequently positive than those from patients whose only manifestation of tuberculosis was a positive (but not vesicular) tuberculin response. Thus, M. tuberculosis DNA can be detected by PCR in gastric aspirates of many children with primary tuberculosis, despite that specimens from these patients are negative by culture. Multiple samples must be tested to optimize the diagnostic yield.

Restricted diversity of V gamma 9-JP rearrangements in unstimulated human gamma/delta T lymphocytes.

The diversity of human peripheral blood gamma/delta T cells is known to be limited by the preferential use of V genes coding for V gamma 9 (usually linked to JP) and V delta 2. We show that the diversity of these cells is further limited at the junctional region. First, an identical rearrangement is found in 10%-30% of all gamma/delta T cells which contain V gamma 9-JP rearrangements. Second, the vast majority of V gamma 9-JP rearrangements which are different from this predominant sequence have, nevertheless, the same length or code for variable regions whose length differs by only one amino acid (+/- 1). Overall, 30%-50% of V gamma 9-JP rearrangements have a junctional region which encodes for a peptide with the amino acid sequence E VX EL, in which EV is predominantly, but not exclusively, encoded by the germ-line V gamma 9 sequence and EL is encoded by JP. The X amino acid is variable, but a glutamine is over-represented. The diversity of the V gamma 9-JP repertoire is fairly constant in different individuals and at different ages, including before, during and after the post-natal expansion of peripheral blood gamma/delta T cells.

A search for mycobacterial DNA in granulomatous tissues from patients with sarcoidosis using the polymerase chain reaction.

We have used the polymerase chain reaction as a tool to detect the presence of mycobacterial DNA from organisms of the Mycobacterium tuberculosis complex and other species of mycobacteria in samples from patients with sarcoidosis. Using systems based on the amplification of a fragment of the gene coding for the 65-kD mycobacterial antigen, which were demonstrated to detect approximately 20 mycobacterial genomes/microgram total DNA, DNA from M. tuberculosis was reproducibly identified in DNA extracted from granulomatous tissues from two patients with sarcoidosis, but could not be detected in DNA extracted from tissue biopsies (n = 16) or cells recovered by lavage (n = 6) from most sarcoid patients or from control subjects (n = 22). Using a system based on the amplification of a fragment of the IS6110 insertion element, which could reliably detect two genomes of mycobacterial DNA/microgram total DNA, no additional positive results were observed. In an effort to identify another species of Mycobacterium present in granulomatous tissues from sarcoid patients but not control tissues, a fragment of the 65-kD mycobacterial antigen was amplified and then reamplified using "nested" primers recognizing sequences that are highly conserved among mycobacteria and closely related species, and the amplified DNA products were cloned and sequenced. Amplified DNA was observed in a minority of samples from patients and control subjects (32/84 and 34/77 attempts, respectively, p greater than 0.2), resulting from amplification of DNA from at least 17 different organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of mycobacterial DNA in pleural fluid from patients with tuberculous pleurisy by means of the polymerase chain reaction: comparison of two protocols.

BACKGROUND: The detection of mycobacterial DNA in clinical samples on the basis of the polymerase chain reaction is a promising approach for the rapid diagnosis of tuberculous infections. No consensus exists, however, about which protocols are most sensitive, and the usefulness of this approach in the diagnosis of tuberculous effusions has been assessed in few patients. METHODS: The sensitivity of two protocols was compared for the detection of DNA from Mycobacterium tuberculosis in samples containing known amounts of mycobacterial DNA and in DNA extracted from 15 tuberculous pleural effusions. The results obtained for pleural fluid have been compared with cytological findings and with results obtained by standard microbiological techniques. RESULTS: Mycobacteria could be detected by acid fast staining in none and by culture in three of the 15 pleural fluid samples. A protocol based on the detection of the IS6110 insertion element (which could detect one mycobacterial genome/sample reproducibly) gave a positive result in nine of the 15 tuberculous effusions, though some samples were only intermittently positive (p less than 0.05 compared with culture). In contrast, a protocol based on the detection of the gene coding for the 65 kD mycobacterial antigen (which could detect mycobacterial genomes only if there were at least 10/sample) gave a positive result in three of the 15 tuberculous effusions. Pleural fluid that was always positive with the amplification procedure detecting the IS6110 sequence contained more neutrophils (30% (SD 27%)) than samples that were intermittently positive or always negative (3% (3%)); mycobacterial DNA was never detected in the four samples containing less than 1% neutrophils. CONCLUSIONS: The amplification of the IS6110 insertion element represents a rapid and sensitive means of detecting M tuberculosis in tuberculous effusions. The enrichment of cells containing mycobacteria (possibly neutrophils) before DNA extraction may be required to improve the sensitivity of this approach.