Biomedical subjects

P T Lukey

Publications and source records attributed to P T Lukey.

At least 19 recordsLinked to original sources

PubMed2006

Changes in leucocyte and lymphocyte subsets during tuberculosis treatment; prominence of CD3dimCD56+ natural killer T cells in fast treatment responders.

The immune responses against pulmonary tuberculosis are still poorly defined. This study describes changes in leucocyte and lymphocyte subsets during treatment to find reliable immunological markers for the disease and treatment response. Flow cytometric peripheral blood immune phenotyping, routine haematology and sputum microbiology were performed on 21 HIV-negative adult tuberculosis (TB) patients with positive sputum cultures during therapy in comparison with 14 healthy purified protein derivative (PPD)-positive volunteers. Patients at diagnosis showed high absolute neutrophil and monocyte counts which fell during treatment but low lymphocyte subset counts which increased [except natural killer (NK) and NK T cells]. High counts of a population of CD3(dim)/CD56+ NK T cells at diagnosis correlated significantly with negative sputum culture after 8 weeks of treatment. A multivariate classification technique showed improved correlation when NK cells were taken into account. In conclusion, peripheral blood white cell counts change significantly during treatment and counts at diagnosis, especially CD3(dim)/CD56+ NK T cells, hold promise in predictive models of TB treatment response.

Adolescent↗

PubMed2006

Immune markers measured before treatment predict outcome of intensive phase tuberculosis therapy.

The development of a statistical model based on simple immunological markers which could predict the response to tuberculosis treatment would facilitate clinical trials of new anti-tuberculosis drugs. We have examined the ability of immunological biomarkers, measured at diagnosis and after 4 weeks of treatment, to predict sputum smear status at week 8. Eighteen tuberculosis patients with positive Ziehl-Nielsen (ZN)-stained sputum smears 8 weeks after initiation of treatment (slow response) were matched for age, gender, sputum smear grade and extent of disease on chest radiograph to 18 patients with negative sputum smears at week 8 (fast response). In addition to total white blood cell (WBC) counts and absolute lymphocyte, monocyte and neutrophil numbers, concentrations of six serum markers were measured by enzyme-linked immunosorbent assay (ELISA) in all patients (soluble interleukin-2 receptor alpha (sIL-2Ralpha), granzyme B, soluble tumour necrosis factor alpha receptors 1 and 2 (sTNF-R1 and -2), nitrotyrosine and interferon-gamma (IFN-gamma). At diagnosis, 4 biomarkers (sTNF-R1, total WBC, absolute monocyte and absolute neutrophil numbers) were significantly higher in slow response patients. At week 4, total WBC count and absolute monocyte and neutrophil numbers remained significantly higher in slow responders. Discriminant analysis of the diagnosis and week 4 data provided models for classification of slow response patients with 67% and 83% predictive accuracy. We suggest that treatment response phenotypes can be determined before the start of treatment. Reliable predictive models would allow targeted interventions for patients at risk for slow treatment response to standard tuberculosis therapy.

Adult↗

PubMed2001

HLA-B*35-restricted CD8 T cell epitopes in the antigen 85 complex of Mycobacterium tuberculosis.

Few target epitopes have been described for human CD8 T lymphocytes in antigens of Mycobacterium tuberculosis. By use of a reverse immunogenetics approach, 23 motif-bearing peptides of the Ag85 complex were tested for binding to HLA-B*35, one of the common B-types in West Africa. Three 9-mer peptides bound with high affinity to HLA-B*3501 and displayed low dissociation rates of peptide-major histocompatibility complexes (MHCs). IC(50) and half-life values of peptide-MHC class I complexes were in the same range as reported earlier for other immunogenic peptides. Immune responses against peptide Ag85C (aa 204-212) WPTLIGLAM were characterized in detail. Peptide-stimulated effector cells were able to kill macrophages infected with M. tuberculosis or bacille Calmette-Guérin. Peptide-specific CD8 T cells could be visualized by using HLA-B*3501 tetramers and were shown to produce interferon-gamma and tumor necrosis factor-alpha. Together with other published epitopes, these peptides can be used to study more closely the role of CD8 T cells in mycobacterial infection and tuberculosis.

Acyltransferases↗

PubMed2001

Granule-dependent cytolysis of Mycobacterium tuberculosis-infected macrophages by human gammadelta+ T cells has no effect on intracellular mycobacterial viability.

One of the most important effector functions of activated gammadelta+ T cells in tuberculosis is their strong cytolytic activity against a variety of target cells, including M. tuberculosis-infected macrophages. In the present study, we investigated the relationship between the mechanism of cytolysis utilized by gammadelta+ CTL and intracellular M. tuberculosis survival using a panel of cytolytic human M. tuberculosis-specific gammadelta+ CTL clones. Cytolysis mediated by the gammadelta+ T-cell clones was found to be Ca2+-dependent, sensitive to Cyclosporin A, and was completely abrogated following Sr2+-induced de-granulation of the gammadelta+ T cell effectors. These data demonstrate that gammadelta+ T-cell-mediated cytoxicity was mediated via the granule exocytosis/perforin pathway. Despite significant cytolytic activity against mycobacteria infected U937 cells, the gammadelta+ CTL clones had no impact on the survival of intracellular M. tuberculosis.

Calcium↗

PubMed2001

The human macrophage cell line U937 as an in vitro model for selective evaluation of mycobacterial antigen-specific cytotoxic T-cell function.

Despite strong evidence for CD8+ T-cell function in murine mycobacterial infections, their corresponding role in human tuberculosis has proven more difficult to demonstrate. We have evaluated the human macrophage (Mphi) cell line U937 as an in vitro model for human leucocyte antigen (HLA) class I-restricted presentation of mycobacterial antigens, as HLA class I is constitutively expressed at high levels by U937 cells in the absence of detectable HLA class II or CD1 molecules. U937 cells were evaluated for their ability to phagocytose Mycobacterium tuberculosis and for their ability to present mycobacterial antigens to human HLA class I-matched cytotoxic T lymphocytes (CTLs). Differentiated U937 cells were capable of efficient phagocytosis of M. tuberculosis but did not generate a subsequent respiratory burst response, and were permissive for intracellular growth of both bacillus Calmette-Guérin (BCG) and the virulent M. tuberculosis H37Rv strain. CTL activity was restricted to live mycobacterial organisms and was shown to be mediated by M. tuberculosis-specific, HLA class I-matched, purified CD8+ CTL lines and CD8+ T-cell clones. Furthermore, M. tuberculosis-infected U937 targets were more rapidly and strongly lysed by CD8+ CTLs than were infected autologous Mphi. Finally, M. tuberculosis-infected U937 cells simultaneously provided a sensitive indicator for detection of mycobacterial-specific, HLA-unrestricted gammadelta+ CTL activity.

Antigen-Presenting Cells↗

PubMed2000

Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A.

The role of CD8(+) CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-gamma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P(48-56) and P(242-250), were revealed within the core sequences. CD8(+) T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8(+) T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.

Acyltransferases↗

PubMed2000

Monocyte-derived macrophage cytokine responses induced by M. bovis BCG.

SETTING: One important aspect of macrophage function is the production of inflammatory and anti-inflammatory cytokines, which in turn affect the survival of intracellular organisms such as mycobacteria. OBJECTIVE: To determine the relationship between phagocytosis of mycobacteria and expression of intracellular cytokines. DESIGN: Phagocytosis and cytokine production were studied simultaneously within human monocyte-derived macrophages (MDMs) from healthy donors using fluorescent labelling of M. bovis BCG and flow cytometry. RESULTS: At a range of infection ratios (5:1, 1:1, 0.2:1) TNF- alpha, IL-10, IL-6 and IL-12 were all produced in a dose-dependent manner. At an infection ratio representative of the in vivo situation (1:1), cytokine production was induced in both MDMs containing intracellular M. bovis BCG and in uninfected bystander MDMs. Phagocytosis increased over time, but there was considerable donor variation: the proportions of cells containing one or more mycobacterium were 15.4+/-14.8% (mean+/-SD) at 4 h and 32.7+/-21.1% at 24 h (n=19). Analysis of cytokine production by MDMs not containing mycobacteria (bystander cells) at 4 h revealed that these uninfected cells produced 79+/-6.6% of the TNF- alpha, 53.9+/-40.0% of the IL-10 and 64.2+/-12.4% of the IL-12. By 20 h these proportions had decreased to 57+/-13.5%, 30.9+/-7.4% and 45.5+/-13.3% respectively. CONCLUSION: Both infected and bystander MDMs can be stimulated to produce cytokines in response to M. bovis BCG, indicating that the ability of MDMs to produce cytokines is not necessarily dependent on the ability to phagocytose mycobacteria.

Adult↗

PubMed2000

In situ production of gamma interferon, interleukin-4, and tumor necrosis factor alpha mRNA in human lung tuberculous granulomas.

Human tuberculous granulomas from five adults undergoing surgery for hemoptysis were analyzed by nonradioactive in situ hybridization for tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), and interleukin-4 (IL-4) gene expression. All of the patients produced TNF-alpha mRNA. Three patients stained positive for both IFN-gamma and IL-4 mRNA; the other two stained positive for IFN-gamma but not IL-4 mRNA. Heterogeneity between the granulomas was observed in those patients staining positive for both IFN-gamma and IL-4 mRNA; these patients exhibited granulomas having IFN-gamma and not IL-4 mRNA as well as granulomas positive for both cytokine mRNAs. There was no evidence of caseation in these granulomas, and the cytokine patterns may represent events in the evolution of the granuloma. However, in those granulomas exhibiting caseous necrosis, very little IFN-gamma or IL-4 mRNA was observed, implying that progression of the granuloma is accompanied by a down regulation of T-cell responses. TNF-alpha mRNA expression was highest in patients with both IFN-gamma and IL-4 mRNA. Populations of CD68 positive macrophage-like cells within the granulomas produce mRNA for TNF-alpha, IFN-gamma, and IL-4. This implies that macrophages within the tuberculous granuloma may not be dependent on T-cell cytokines for modulation of their function but may be able to regulate their own activation state and that of the surrounding T cells. These findings have implications on the delivery of immunotherapies to patients with tuberculosis.

Adult↗

PubMed1998

Kinetics of purified protein derivative (PPD) proliferation reflects underlying suppressor mechanisms revealed by limiting dilution analysis (LDA) in patients with extrapulmonary tuberculosis (TB).

Mononuclear leucocytes from the blood (PBML) and effusion (EML) of patients undergoing pericardiocentesis were assayed for proliferative response to purified protein derivative of Mycobacterium tuberculosis (PPD). Of the 23 patients tested, 10 had culture-positive tuberculous effusions, while 13 had non-tuberculous aetiologies. Three different kinetic responses were identified: (i) accelerated responses (found in 70% of EML from patients with culture-positive tuberculous effusions); (ii) 'flat' responses (found in 10% of EML from patients with culture-positive tuberculous effusions); and (iii) normal kinetic responses. These differences in kinetic response may reflect underlying immune mechanisms important in the immunopathogenesis of TB. In order to address this possibility we performed LDA on a selection of patients with culture-positive extrapulmonary TB: three patients with accelerated responses, two with normal responses, and one with a 'flat' response. The results confirm the previously reported accumulation of PPD-specific responder cells in the effusion of patients with TB. Cell-mediated suppressor mechanisms (as shown by 'V'-shaped LDA curves) were found in the blood of one patient and the effusion of another. In both cases 'flat' PPD-proliferative responses were observed. However, the LDA data also suggested the presence of in vivo mechanisms limiting the clonal burst size. Thus it appears that immune responses in extrapulmonary TB are influenced by an array of inhibitory mechanisms, modulation of which may influence the outcome of infection.

Humans↗

PubMed1996

Memory lymphocytes from tuberculous effusions: purified protein derivative (PPD) stimulates accelerated activation marker expression and cell cycle progression.

Accelerated PPD-specific proliferation and generation of CD4+ cytotoxic effectors by mononuclear leucocytes (MNL) from tuberculous effusions (EMNL) has been previously reported by our laboratory. In order to explore the contribution of the state of activation of MNL to accelerated reactivity, EMNL and peripheral blood (PB)MNL from seven patients with tuberculosis were assessed both ex vivo and after PPD stimulation. Flow cytometry revealed no difference in the activation state (IL-2 receptor and HLA-DR expression) or cell cycle progression ex vivo. However, CD4+ CD29+ memory T cells were accumulated in EMNL compared with PBMNL. In vitro stimulation of EMNL with PPD resulted in accelerated expression of activation markers and progression through the cell cycle (peak after 4 days), whilst PBMNL exhibited normal activation kinetics (peak after 7 days). Accelerated reactivity could not be accounted for by quantitative differences in effusion CD4+ CD29+ memory T cells compared with blood, but may be due to a qualitative difference in effusion memory T cells, which are shown to be in a postactivation state of differentiation. T cells entering S and G2/M phases of the cell cycle were largely of the activated memory phenotype. Activation marker expression occurred in association with up-regulation of CD4 antigen expression on the surface of EMNL. Thus accelerated expression of activation markers and cell cycle progression by CD4+ CD29+ memory T cells may in part account for accelerated PPD reactivity in tuberculous effusions.

Adult↗

PubMed1995

Prostanoid modulation of synovial antigen-specific CD4+ T-cell cytotoxic function in rheumatoid arthritis.

The recent demonstration of cytolytic mediators within synovial CD4+ T-cells of patients with rheumatoid arthritis (RA) has suggested an additional role for these cells in the pathogenesis of the disease. In this study we have investigated the function and regulation of antigen-specific class II-restricted cytotoxic T-cells from the synovial fluid (SFMNC) and peripheral blood (PBMNC) of 20 seropositive RA patients, and correlated in vitro findings with clinical data. Regulatory factors including prostaglandin E2 (PGE2), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were measured in cell supernatants. A diversity in SFMNC antigen-specific cytotoxicity that correlated with therapy and PGE2 production was found, and shown to be mediated by synovial prostanoid (products of cyclooxygenase metabolism) inhibition of effector function. Our findings indicate that SFMNC cytotoxicity may be important in the pathogenesis and treatment of RA. Cyclooxygenase inhibition as the sole treatment early in RA may reduce the potentially beneficial inhibitory effect of synovial prostanoids on antigen-specific SFMNC cytotoxicity.

Adult↗

PubMed1994

Basic fibroblast growth factor antagonizes transforming growth factor beta-mediated erythroid differentiation in K562 cells.

Basic fibroblast growth factor (bFGF) and transforming growth factor-beta 1 (TGF-beta) have both been shown to act on hematopoietic progenitor cells. bFGF is a hematopoietic cytokine that acts on progenitor cells in concert with other cytokines to promote their proliferation. TGF-beta induces erythroid differentiation in K562 cells. To determine whether bFGF might act on progenitor cells by antagonizing the effects of cytokines that induce differentiation, we determined the effects of bFGF on the TGF-beta-mediated induction of hemoglobin synthesis in K562 cells. bFGF antagonized the TGF-beta-mediated induction of hemoglobin in a dose-dependent manner, with 0.1 ng/mL bFGF inhibiting hemoglobin induction by 40% and 10 ng/mL bFGF completely abrogating hemoglobin production. bFGF was most effective at antagonizing the TGF-beta-mediated induction of hemoglobin if it and TGF-beta were added simultaneously to K562 cells, but delayed addition of bFGF to TGF-beta-treated cultures still resulted in significant inhibition of hemoglobin synthesis. The inhibitory effects of bFGF on hemoglobin production were fully reversible, showing that bFGF did not permanently alter the phenotype of K562 cells. The hemin-mediated induction of hemoglobin synthesis in K562 cells was only partially negated by bFGF. bFGF also diminished the expression of glycophorin A on the surface of K562 cells. These results indicate that bFGF might increase progenitor/stem cell numbers by antagonizing the effects of cytokines that induce differentiation, thereby increasing the pool of proliferating progenitor/stem cells.

Cell Differentiation↗

PubMed1994

Reduced NK activity correlates with active disease in HIV- patients with multidrug-resistant pulmonary tuberculosis.

There has been a global increase in the incidence of multidrug-resistant pulmonary tuberculosis (TB). As there are no previous reports of immune function in HIV- patients with multidrug-resistant pulmonary TB, a comprehensive assessment of cellular immunity in this setting was undertaken. This involved a prospective, case-controlled study which included five patients with active multidrug-resistant pulmonary TB and five matched controls with active non-resistant infection, and documented the changes in immune parameters which occurred upon clinical resolution. Patients with multidrug-resistant TB had significantly lower fresh natural killer (NK) cell activity than matched controls with non-resistant pulmonary TB (P < 0.05). This was a specific abnormality, as there were no significant differences in antigen-specific cytotoxicity or lymphocyte proliferation in the case-controlled study. Follow-up assessment of the patients with multidrug-resistant infections indicated that clinical improvement correlated with a moderate increase in NK cell activity. Impaired NK cell function may be involved in the pathogenesis of multidrug-resistant TB.

Adult↗

PubMed1992

Reduced natural killer cell activity in multi-drug resistant pulmonary tuberculosis.

Cellular immune status in five patients with multi-drug resistant pulmonary tuberculosis was investigated and compared with five matched controls with non-resistant tuberculosis. A significant reduction in fresh natural killer (NK)-cell activity was found in the resistant group (P less than 0.005). There were no significant differences between the two groups in lymphocyte phenotype, proliferation or PPD-specific cytotoxicity. Reduced NK-cell function may play a role in the pathogenesis of multi-drug resistant pulmonary tuberculosis.

Cytotoxicity, Immunologic↗

PubMed1992

Evidence for in vivo generation of cytotoxic T cells. PPD-stimulated lymphocytes from tuberculous pleural effusions demonstrate enhanced cytotoxicity with accelerated kinetics of induction.

Mycobacterium tuberculosis is a bacterial pathogen capable of survival and replication within human macrophages. Cytotoxic T cells are thought to be important for the eradication of infected macrophages. To test this hypothesis, pleural effusion lymphocytes from patients with tuberculous pleuritis were stimulated in vitro with PPD, and proliferation and cytotoxicity were assessed by thymidine incorporation and chromium release, respectively. The level and kinetics of generation of antigen-specific cytotoxicity were measured and compared with those in autologous peripheral blood, control peripheral blood, and nontuberculous effusions. Both proliferation and cytotoxicity in tuberculous pleural effusions were augmented and accelerated in comparison to autologous or control peripheral blood. By contrast, low levels of cytotoxicity were observed in nontuberculous effusions, without evidence of accelerated kinetics. Cell subset fractionation by panning indicated that the cytotoxicity was mediated by CD4+ cells. The accelerated kinetics of induction of PPD-specific cytotoxic T cells demonstrated here suggests reactivation of in vivo generated cytotoxic T cells. These findings provide evidence that cytotoxic T cells are induced at the site of pathology in vivo and suggest that these cells play an important role in protection in vivo against infection with tuberculosis.

Cytotoxicity, Immunologic↗

PubMed1988

Benoxaprofen activates membrane-associated oxidative metabolism in human polymorphonuclear leucocytes by apparent modulation of protein kinase C.

1. The non-steroidal anti-inflammatory drug (NSAID) benoxaprofen at concentrations of 15, 30 and 60 micrograms ml-1 caused a dose-related activation of superoxide generation by human polymorphonuclear leucocytes (PMNL) in vitro. 2. The protein kinase C (PKC) inhibitor H-7 prevented benoxaprofen-mediated activation of superoxide generation by PMNL. 3. Benoxaprofen, by apparent substitution for phosphatidylserine, caused a dose-related activation of purified PKC from rat brain and in cytosolic extracts from human platelets. 4. Benoxaprofen-mediated stimulation of PMNL membrane-associated oxidative metabolism is due to apparent activation of PKC by this NSAID. These findings establish the molecular basis of the pro-oxidative properties of benoxaprofen.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1987

Ascorbate and cysteine-mediated selective neutralisation of extracellular oxidants during N-formyl peptide activation of human phagocytes.

The effects of sodium ascorbate and cysteine (2.5 X 10(-5) M-2.5 X 10(-4) M) on the intensity and profile of luminol-enhanced chemiluminescence, superoxide generation, extracellular myeloperoxidase (MPO) activity and auto-iodination were measured in purified human polymorphonuclear leukocytes activated by the leukoattractant FMLP in vitro. Chemiluminescence studies were also performed using a whole-blood method. Cysteine (10(-4) M-2.5 X 10(-4) M) and ascorbate (2.5 X 10(-5) M-2.5 X 10(-4) M) caused significant inhibition of the early extracellular peak of FMLP-activated chemiluminescence and increased the intensity of the later occurring intracellular peak in both PMNL and blood. At the same concentrations both agents scavenged superoxide released by FMLP-activated PMNL, inhibited oxidant generation by extracellular MPO and decreased FMLP-induced auto-oxidation of PMNL. Administration of a single 1 gram oral dose of ascorbate to adult human volunteers was associated with significant reduction and enhancement respectively of the extracellular and intracellular luminol-enhanced chemiluminescence responses of FMLP-activated blood. These results show that the water soluble anti-oxidants cysteine and especially ascorbate selectively neutralise the reactivity of harmful reactive oxidants released by phagocytes, whilst the intracellular generation of antimicrobial oxidants remains intact.

Adult↗

PubMed1987

An in-vitro comparison of the intraphagocytic bioactivity of erythromycin and roxithromycin.

The binding to human polymorphonuclear leucocytes and the intracellular bioactivity of the macrolide antibiotics erythromycin and roxithromycin on Legionella micdadei, Listeria monocytogenes and Staphylococcus aureus were investigated in vitro by the combination of a fluorochrome microassay and a radioassay. Polymorphs with intact or absent membrane-associated oxidative metabolism were used to investigate the interactions which may occur between the intrinsic oxygen-dependent antimicrobial systems of human polymorphs and the test antibiotics, in the elimination of intracellular microbial pathogens. Elimination of O2-dependent antimicrobial systems with retention of phagocytic activity was achieved by using polymorphs from children with chronic granulomatous disease NaF-pulsed normal polymorphs. Both antimicrobial agents were actively concentrated by polymorphs. Erythromycin was concentrated ten-fold and roxithromycin approximately thirty-fold above extra cellular levels. Both agents possessed intracellular bacteriostatic activity for all three test microbial pathogens. Depletion of polymorph O2-dependent intrinsic antimicrobial systems interfered with the intracellular bioactivity of both antibiotics. This emphasizes the importance of interactions between cell-associated antibiotics and phagocyte antimicrobial systems in the elimination of intracellular microbial pathogens. Like erythromycin, roxithromycin is concentrated by human phagocytes and is bioactive intracellularly.

Bacteria↗