Depressed interleukin-12 (IL-12), but not IL-18, production in response to a 30- or 32-kilodalton mycobacterial antigen in patients with active pulmonary tuberculosis

The secreted 30-kDa antigen (Ag) of Mycobacterium tuberculosis directly stimulates Th1-type protective cytokine responses in healthy tuberculin reactors but not in patients with active tuberculosis (TB). To examine the cytokine profiles attributable to Th1 suppression associated with active TB, interleukin-12 (IL-12), IL-18, and IL-10 production in response to a 30- or 32-kDa Ag in 16 patients with active pulmonary TB and 24 healthy controls was investigated by enzyme-linked immunosorbent assay. In TB patients, production of IL-12 p40, as well as gamma interferon (IFN-gamma), by 30- or 32-kDa Ag-stimulated peripheral blood mononuclear cells (PBMC) was significantly decreased compared with that in healthy tuberculin reactors. There were no significant differences in IL-18 production between patients and controls early during stimulation (16 h). However, PBMC from patients showed significantly enhanced IL-18 proteins after 96 h of stimulation. Similarly, higher IL-10 production was observed in the TB patients than in healthy tuberculin reactors. After 2 months of anti-TB therapy, the mean IFN-gamma and IL-12 p40 production and the mean blastogenic responses were significantly increased in PBMC in the 10 TB patients who were followed up. Our findings provide evidence that depressed IL-12 in response to the 30- or 32-kDa Ag is involved in the immunopathogenesis of human active pulmonary TB.     

Cytokine responses to stimulation of whole blood from patients with Buruli ulcer disease in Ghana

Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, follows an indolent course of initial progression to ulceration accompanied by extensive tissue damage. It has been suggested that healing disease stages are accompanied by a protective immune response. We hypothesized that interleukin-4 (IL-4)- or IL-10-induced downregulation of Th-1 responses plays a key role in the progression of early BUD and that healing is accompanied by an augmented Th-1 response. Gamma interferon (IFN-gamma), IL-4, and IL-10 responses were measured after in vitro stimulation with phytohemagglutinin (PHA) and tuberculin purified protein derivative (PPD) of whole blood from 39 (23 early- and 16 late-stage) BUD patients and 39 healthy control subjects in Ghana. Additionally, 30 patients with active or treated tuberculosis (TB) serving as PPD-responsive positive controls were studied. Early-stage BUD patients produced significantly lower levels of IFN and IFN-gamma/IL-4 ratios compared to late-stage BUD patients after PHA stimulation. Compared to that of controls, IFN-gamma production after tuberculin stimulation was significantly higher in late-stage but not in early-stage BUD patients (P=0.009). IL-10 and IL-4 levels did not differ between BUD patients and controls, although active TB patients had significantly higher IL-10 production levels than did treated TB patients. Multivariate analysis showed no confounding factors. In conclusion, Th-1 down regulation in early BUD appears to reverse in later stages of BUD, although an association with IL-10 or IL-4 production does not emerge from our data. Here we show differences in Th-1-type cytokine production between early- and late-stage BUD that might reflect an improved immune defense over time.     

Molecular signatures distinguishing active from latent tuberculosis in peripheral blood mononuclear cells, after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD) or Candida: a preliminary report

Purified protein derivative (PPD) or tuberculin skin testing is used to identify infected individuals with Mycobacterium tuberculosis (Mtb) and to assess cell-mediated immunity to Mtb. In the present study, we compared PBMC cultures in the presence of tuberculin or Candida antigens using cytokine bead arrays and RNA microarrays. Measurements of different cytokines and chemokines in supernatants of PMBC cultures in the presence of PPD showed increased levels of interferon (IFN)-gamma in active tuberculosis infection (ATBI) and latent TB infected (LTBI) compared to controls, and increased levels of TNF-alpha in ATBI compared with LTBI. Also, we found increase of IL-6 in cultures of PPD positive and controls but not in the cultures with Candida. We also report the molecular signature of tuberculosis infection, in ATBI patients, the following genes were found to be up-regulated and absent in LTBI individuals: two kinases (JAK3 and p38MAPK), four interleukins (IL-7, IL-2, IL-6, and IFNbeta1), a chemokine (HCC-4) a chemokine receptor (CxCR5), two interleukin receptors (IL-1R2 and IL-18R1), and three additional ones (TRAF5, Smad2, CIITA, and NOS2A). By contrast, IL-17 and IGFBP3 were significantly up-regulated in LTBI. And, STAT4, GATA3, Fra-1, and ICOS were down-regulated in ATBI but absent in LTBI. Conversely, TLR-10, IL-15, DORA, and IKK-beta were down-regulated in LTBI but not in ATBI. Interestingly, the majority of the up-regulated genes found in ATBI were found in cultures stimulated with tuberculin (PPD) or Candida antigens, suggesting that these pathogens stimulate similar immunological pathways. We believe that the molecular signature distinguishing active from latent tuberculosis infection may require using cytokine bead arrays along with RNA microarrays testing cell cultures at different times following in vitro proliferation assays using several bacterial antigens and PPD.     

Polarization of PPD-specific T-cell response of patients with tuberculosis from Th0 to Th1 profile after successful antimycobacterial therapy or in vitro conditioning with interferon-alpha or interleukin-12

The T helper (Th) 1/Th2 balance in the T-lymphocyte response to purified protein derivative (PPD) was evaluated at the clonal level in six Italian and five Gambian patients with pulmonary tuberculosis (TB) before and after antimycobacterial therapy, as well as in five Gambian and four Italian healthy immune control subjects. In untreated patients, most PPD-specific clones derived from either peripheral blood or pleural effusions showed a Th0 cytokine profile (production of both interferon [IFN]-gamma and interleukin [IL]-4/IL-5). After 6 mo of therapy and clinical healing, most PPD-specific clones showed a polarized Th1 profile (production of IFN-gamma but not IL-4/IL-5) in both Italian and Gambian patients. The Th1 polarization was less marked in Gambian than in Italian patients and failed to occur in another group of four Italian patients who experienced treatment failure. The cytokine profile observed after successful therapy in patients with TB was similar to that found in healthy control subjects. T-cell clones of undefined specificity generated from PPD-stimulated cultures showed a similar Th0/Th2 bias in Gambian individuals and Italian patients with treatment failure. The Th0/Th2-biased responses in Gambian patients before therapy could be modulated in vitro by IFN-alpha or IL-12, which induced a Th1 polarization of both PPD-specific and bystander T cells. Our data show that active TB associates with a predominant Th0 response to mycobacterial antigens that could play a role in the pathogenesis of the disease. Adjunctive immunotherapy using Th1-polarizing cytokines could increase host defense against mycobacteria and accelerate healing.     

Cytokine gene expression in human peripheral blood mononuclear cells stimulated by mannoprotein constituents from Candida albicans.

The expression of cytokine genes in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with mannoprotein constituents (MP) of Candida albicans has been studied by means of S1 nuclease mapping analysis, polymerase chain reaction, and enzyme-linked immunosorbent assay. MP induced early, consistent, and long-lasting production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, and IL-6 mRNAs. Similar results were obtained when the same PBMC cultures were stimulated with the purified protein derivative (PPD) from Mycobacterium tuberculosis or with IL-2, although lower levels of IL-6 mRNA were detected in IL-2-stimulated cells than in MP- or PPD-stimulated cells. MP, PPD, and IL-2 induced appreciable levels of granulocyte-macrophage colony-stimulating factor and gamma interferon, but only MP and PPD were able to induce IL-2 mRNA. MP were unable to stimulate a consistent expression of the genes encoding for IL-4, IL-5, and IL-10, while low, sometimes barely detectable levels of these cytokine mRNAs were observed in PPD- or IL-2-stimulated PBMC cultures. When protein synthesis of MP-stimulated PBMC was inhibited by cycloheximide, a superinduction of mRNAs for IL-4 and IL-10 and, more markedly, gamma interferon was observed. Overall, these results highlight the powerful, selective induction of cytokine gene expression by MP constituents of C. albicans in human PBMC cultures, thus providing some functional clues to explain the efficient state of the anticandidal response in normal human subjects.     

Eje interleucina 12/interferón gamma en pacientes de tuberculosis en una región europea con alta incidencia de enfermedad

The integrity of the interleukin 12/interferon gamma (IL-12/INF-γ) axis has been shown to be essential in the control of the Mycobacterium tuberculosis infection. The aim of this study was to assess whether the high incidence of tuberculosis (TB) in Galicia, Spain, could be related to an altered response in the IL-12/INF-γ axis in patients with TB. We studied 20 TB patients and 21 healthy controls: 7 with positive Tuberculin Skin test (TST+) and another 14 with TST-. The study of the IL-12/INF-γ axis was conducted by the analysis of INF-γ levels (in serum and supernatants of non-activated and activated cells) and by the cellular expression of IL-12Rβ1 and INF-γR1 in lymphocytes and monocytes, respectively. The assays were performed at baseline levels and after in vitro stimulation of peripheral blood mononuclear cells with phytohaemagglutinin (PHA) and purified protein derivative (PPD). Responses in patients and in healthy controls were analysed and compared. PHA and PPD in vitro stimulation significantly increased INF-gamma levels and the cellular expression of IL-12Rβ1 and INF-γR1 receptors compared to baseline levels in both TB patients and healthy controls (either with positive or negative TST). Our results suggest that there is an adequate immune response in all groups. Although the IL-12/INF-γ axis may have other abnormalities not analysed in this work, the functionality of the IL-12/INF-γ axis is preserved in the patients analysed with TB in Galicia. The high rates of disease historically observed in this community does not seem to be caused by a malfunction of the immune response at this level.     

Study of immune response in Warao children from communities with high tuberculosis prevalence

The present study was carried out in a Warao childhood population with extremely high tuberculosis (TB) rate of 3190/100,000 in 0-15 years old children. One hundred seven serum and saliva samples were tested, 32 from patients with active TB (27 positive and 5 negative for the tuberculin skin test, TST) and 75 apparently healthy contact children (45 positive and 30 negative for the TST). The innate, immunoglobulin and cellular responses were studied. The results showed that both, patients and controls, had a high percentage of children with increased levels of complement C3 and C4 components. A high percentage of children with increased total serum IgA and IgG (13.8% and 79.3% respectively) was observed in children with TB in comparison to control children (0% and 69.2%). A high percentage of control children had increased levels of IgM and sIgA (69.2% and 56.16%, respectively) in comparison to patients (48.3% and 31.25%). Both groups showed children with increased levels of IgE. The results concerning to the cellular immune response to PPD and the BCG vaccination status showed that there was a correlation between an increase in PPD reactivity and age. The PPD reactivity in children less than 7 years old was similar and also independent of the BCG vaccination status. A significant number of children without or with scars (46.8% and 27.6%, respectively) showed induration values of 0 mm to tuberculin skin test. The Candida reactivity showed a high percentage of children (80%) with anergy status. In conclusion, an increase in the levels of complement components C3 and C4 and hypergammaglobulinemia was observed in Warao children, and these results were independent from PPD reactivity and BCG vaccination. The isotype results showed that the decrease in sIgA could be and active disease marker, while the increase in IgM levels could represent a marker of recent disease.     

Cytokine Responses to Stimulation of Whole Blood from Patients with Buruli Ulcer Disease in Ghana

Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, follows an indolent course of initial progression to ulceration accompanied by extensive tissue damage. It has been suggested that healing disease stages are accompanied by a protective immune response. We hypothesized that interleukin-4 (IL-4)- or IL-10-induced downregulation of Th-1 responses plays a key role in the progression of early BUD and that healing is accompanied by an augmented Th-1 response. Gamma interferon (IFN-γ), IL-4, and IL-10 responses were measured after in vitro stimulation with phytohemagglutinin (PHA) and tuberculin purified protein derivative (PPD) of whole blood from 39 (23 early- and 16 late-stage) BUD patients and 39 healthy control subjects in Ghana. Additionally, 30 patients with active or treated tuberculosis (TB) serving as PPD-responsive positive controls were studied. Early-stage BUD patients produced significantly lower levels of IFN and IFN-γ/IL-4 ratios compared to late-stage BUD patients after PHA stimulation. Compared to that of controls, IFN-γ production after tuberculin stimulation was significantly higher in late-stage but not in early-stage BUD patients (P = 0.009). IL-10 and IL-4 levels did not differ between BUD patients and controls, although active TB patients had significantly higher IL-10 production levels than did treated TB patients. Multivariate analysis showed no confounding factors. In conclusion, Th-1 down regulation in early BUD appears to reverse in later stages of BUD, although an association with IL-10 or IL-4 production does not emerge from our data. Here we show differences in Th-1-type cytokine production between early- and late-stage BUD that might reflect an improved immune defense over time.     

Cytokine profile of t lymphocytes from peripheral blood and bronchoalveolar lavage fluid in patients with active pulmonary tuberculosis

The possible immunological relationship between the pattern of Th1/Th2 cytokine production and tuberculin reactivity was assessed in patients with active Mycobacterium tuberculosis infection. The production of the intracellular cytokines interferon (IFN)-gamma and interleukin-4 (IL-4) was measured in CD4(+) and CD8(+) T cells obtained from peripheral blood and bronchoalveolar lavage fluid (BALF) of 20 tuberculin skin-positive patients and compared with the findings recorded in nine tuberculin skin-negative patients with active pulmonary tuberculosis. Upon stimulation with phorbol 12-myristate acetate/ionomycin for 6 h, tuberculin-negative patients had a significantly higher proportion of IFN-gamma-producing CD4(+) T lymphocytes in BALF than in peripheral blood, while both CD4(+) and CD8(+) T-lymphocyte subsets in BALF of tuberculin-positive patients secreted more IFN-gamma than their peripheral blood counterparts. Tuberculin-negative patients had a significantly higher proportion of IFN-gamma-producing CD4(+) T lymphocytes in peripheral blood than tuberculin-positive patients. There was no significant difference in the production of IFN-gamma by BALF CD4(+) T lymphocytes, or by either peripheral blood or BALF CD8(+) T lymphocytes. In two tuberculin-negative patients, peripheral blood CD4(+) T lymphocytes produced IL-4. Study results suggested a higher immune activity in the blood of tuberculin-negative patients, with an increased lymphocyte activity in BALF versus peripheral blood in both patient groups.     

Increase of gamma/delta T cells in hospital workers who are in close contact with tuberculosis patients.

gamma/delta T cells are likely to participate in the immune response to tuberculous infection in humans. In this study, we carried out an investigation to characterize the responsiveness of gamma/delta T cells from tuberculous patients and healthy individuals to mycobacterial stimulation in vitro. Healthy subjects were assigned to the following two groups: those who had been exposed to tuberculosis (contacts) and those who had not been exposed (noncontacts). The percent gamma/delta T cells in fresh peripheral blood obtained from health care workers who were tuberculin skin test positive and who had constant contact with patients with active tuberculosis (healthy contacts) was significantly higher, whereas healthy noncontacts showed the normal range of gamma/delta T cells. Patients with active pulmonary tuberculosis also had low levels of gamma/delta T cells. HLA-DR antigen-bearing activated gamma/delta T cells were observed in higher percentages among healthy contacts than among healthy noncontacts or patients with pulmonary tuberculosis. In healthy contacts, gamma/delta T cells increased as a percentage of peripheral blood mononuclear cells after in vitro stimulation with purified protein derivative (PPD) tuberculin compared with the percentage of fresh peripheral blood mononuclear cells that they made up, whereas no such increase was observed in patients with tuberculosis or in healthy noncontacts. Phenotypic analysis of the gamma/delta T cells in healthy contacts, which increased in number in vitro in response to PPD, revealed the preferential outgrowth of CD4+ V gamma 2+ gamma/delta T cells. This expansion of gamma/delta T cells by PPD required accessory cells, and it was inhibited by the addition of an antibody against HLA-DR in culture. Proteolytic digestion of PPD showed that gamma/delta T cells increased in number in response to peptide, but not nonpeptide, components of PPD. These findings suggest that gamma/delta T cells, especially CD4+ V gamma 2+ gamma/delta T cells, may participate in the immune surveillance of tuberculous infections in humans.     

Tuberculin purified protein derivative-reactive T cells in cord blood lymphocytes.

Lymphocytes obtained from cord blood of newborn babies who were born of healthy mothers were studied in vitro for their responsiveness to purified protein derivative (PPD) of tuberculin. Cord blood lymphocytes proliferated in vitro by stimulation with PPD, despite wide variations in the results. Studies with fractionated lymphocytes revealed that PPD-responding cells belonged to E-rosetting, nylon wool-nonadherent T lymphocytes. Non-E-rosetting B lymphocytes alone did not proliferate at all after stimulation with PPD. In addition, bromodeoxyuridine and light treatment of in vitro PPD-stimulated lymphocytes eliminated the responsiveness to PPD. These results suggest that T lymphocytes do exist in cord blood and respond in vitro to stimulation with PPD. A possible role for PPD-reactive T lymphocytes in cell-mediated protective immunity in newborns is discussed.     

Development of a Human Gamma Interferon Enzyme Immunoassay and Comparison with Tuberculin Skin Testing for Detection of Mycobacterium tuberculosis Infection

A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-γ) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-γ per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-γ. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-γs. Cross-reactions with other human cytokines or with IFN-γs derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-γ in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-γ were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-γ responsiveness in vitro. These results support the further study of the blood culture–IFN-γ EIA system as an alternative to skin testing for the detection of human TB infection.     

Specific Lymphoproliferation, Gamma Interferon Production, and Serum Immunoglobulin G Directed against a Purified 32 kDa Mycobacterial Protein Antigen (P32) in Patients with Active Tuberculosis

Twenty-one patients treated for active tuberculosis were examined for immune reactivity to purified protein derivative (PPD) and to a purified 32-kDa protein antigen (P32) from Mycobacterium bovis , strain BCG. Lymphoproliferation of peripheral blood leucocytes to PPD and P32 was positive in 95% and 71% of the patients respectively. A positive IFN-γ response was detected in 62% against PPD and in 48% against P32. Low blastogenesis and IFN-γ production were observed, especially in patients with poor general health and advanced tuberculous lesions. Twelve out of twelve (100%) of the tuberculin-positive healthy volunteers responded to PPD and P32 with mean lymphoproliferation and IFN-γ values that were higher than in the patient group. Twelve tuberculin-negative control subjects were completely unreactive to PPD and P32 antigen. On the other hand, IgG antibodies in the serum were detected in 95% of the patients against PPD, in 77% of the patients against P32 but in none of the tuberculin-positive or negative healthy volunteers. The highest IgG levels against PPD were found in those patients with the lowest in vitro lymphoproliferation and IFN-γ production (r=-0.54; P <0.05). Nonspecific interferon production following induction with Newcastle disease virus, Corynebacterium parvum , or phytohaemagglutinin was comparable in the control and patient groups. Finally, low IFN-α titres were detected in the serum of about 50% of the patients.     

Defective gamma-interferon production in peripheral blood leukocytes of patients with acute tuberculosis

Production of interferon (IFN)-gamma by peripheral blood leukocytes (PBL) was examined in cultures of unseparated fresh whole blood exposed to phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). The yield of IFN-gamma was measured by a newly developed immunoradiometric assay. Nine of 14 patients with acute pulmonary tuberculosis (TB) showed a depressed IFN-gamma response to Con A and/or PWM. Only four of these TB patients also showed a depressed IFN-gamma response to PHA. Stimulation of the patients' PBL cultures with PHA in the presence of exogenous interleukin 2 (IL 2) produced normal IFN-gamma yields in all but the most severely depressed patients. PBL cultures of TB patients with defective IFN-gamma production in response to mitogenic lectins also produced less IFN-gamma after stimulation with tuberculin PPD. Although some patients showed a moderate degree of lymphopenia, their OKT4/T8 lymphocyte ratios were mostly normal or close to normal, with the notable exception of one TB patient who has been diagnosed to have the acquired immune deficiency syndrome (AIDS).     

In vitro kinetics of allergen- and microbe-induced IL-4 and IFN-gamma mRNA expression in PBMC of pollen-allergic patients

BACKGROUND: According to a hypothesis allergens induce Th2 responses in allergic patients, and microbes induce Th1 responses. We studied the kinetics of in vitro allergen-, tuberculin (PPD)- and tetanus toxin (TT)-induced IFN-gamma and IL-4 mRNA expression in peripheral blood mononuclear cell (PBMC) cultures of pollen-allergic patients and healthy controls. METHODS: PBMC of 10 birch or timothy pollen-allergic patients and of 13 healthy controls were stimulated in vitro with allergen (birch or timothy), PPD or TT. Pellets and supernatants were collected at 24, 48, 72 and 96 h after stimulation. IFN-gamma and IL-4 production was measured by enzyme linked immunosorbent assay and mRNA expression using RT-PCR and time-resolved fluorometry. RESULTS: Allergen induced IFN-gamma production and mRNA expression in PBMC more in allergic patients than in healthy controls. Also allergen induced IL-4 mRNA expression more in allergic patients than in healthy controls. PPD induced IFN-gamma mRNA expression both in allergic patients and healthy controls, whereas IFN-gamma production was induced only in healthy controls and IL-4 was not induced at all. TT induced IFN-gamma mRNA expression in both groups, IFN-gamma production in allergic patients, and IL-4 mRNA expression in both allergic patients and healthy controls. CONCLUSIONS: In vitro stimulation with allergen induced both IFN-gamma and IL-4 mRNA expression of PBMC in allergic patients. These observations challenge the clearcut division of microbe-specific Th1 and allergen-specific Th2 responses in peripheral blood.     

Association of strong immune responses to PPE protein Rv1168c with active tuberculosis

Accurate diagnosis of tuberculosis (TB) infection is critical for the treatment, prevention, and control of TB. Conventional diagnostic tests based on purified protein derivative (PPD) do not achieve the required diagnostic sensitivity. Therefore, in this study, we have evaluated the immunogenic properties of Rv1168c, a member of the PPE family, in comparison with PPD, which is routinely used in the tuberculin test, and Hsp60 and ESAT-6, well-known immunodominant antigens of Mycobacterium tuberculosis. In a conventional enzyme immunoassay, the recombinant Rv1168c protein displayed stronger immunoreactivity against the sera obtained from patients with clinically active TB than did PPD, Hsp60, or ESAT-6 and could distinguish TB patients from Mycobacterium bovis BCG-vaccinated controls. Interestingly, Rv1168c antigen permits diagnosis of smear-negative pulmonary TB as well as extrapulmonary TB cases, which are often difficult to diagnose by conventional tests. The immunodominant nature of Rv1168c makes it a promising candidate to use in serodiagnosis of TB. In addition, our studies also show that Rv1168c is a potent T-cell antigen which elicits a strong gamma interferon response in sensitized peripheral blood mononuclear cells obtained from TB patients.     

Depressed interleukin-12 production by peripheral blood mononuclear cells after in vitro stimulation with the 30-kDa antigen in recurrent pulmonary tuberculosis patients

Some patients develop recurrent tuberculosis (R-TB), even after successfully completing initial anti-tubercular treatment. Although R-TB may be caused by relapse or exogenous reinfection, little is known about the underlying host responses associated with R-TB. This study investigated the profile of cytokines [interferon (IFN)-gamma, interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, IL-6, and IL-10] present in peripheral blood mononuclear cells (PBMCs) of 17 R-TB patients after stimulation with the 30-kDa antigen (Ag) or purified protein derivative (PPD) Ag of Mycobacterium tuberculosis. These data were compared with data obtained from 15 patients with newly diagnosed pulmonary TB (N-TB), 22 patients with treatment failure (TF-TB), and 19 healthy tuberculin reactors (HTR). N-TB and R-TB patients were enrolled in this study within 1 month of beginning anti-tubercular chemotherapy. ELISA results showed that IFN-gamma production following stimulation with the 30-kDa Ag was significantly lower in each group of TB patients than in the HTR controls. In addition, patients with R-TB showed the most significant IL-12 depression among the subject groups after in vitro stimulation with either Ag. Furthermore, a significant decrease in TNF-alpha and IL-10 levels was observed in R-TB patients relative to N-TB patients. However, there was no statistical difference in TNF-alpha and IL-10 production between R-TB patients, TF-TB patients, and HTR controls. Our findings suggest that the underlying mechanisms of cytokine regulation might differ between N-TB and R-TB patients, and that decreased IL-12 production in response to the 30-kDa or PPD Ag might be involved in the immunopathogenesis of human R-TB.     

Gamma/delta T cell subsets in patients with active Mycobacterium tuberculosis infection and tuberculin anergy

Earlier data suggest that gamma/delta T cells may play an important role in the immune response to Mycobacterium tuberculosis. The aim of this study was to determine the percentage of different gamma/delta subsets in peripheral blood of active tuberculosis patients with a positive or negative tuberculin reaction. Thirty-eight patients infected with M. tuberculosis and 22 healthy controls were included in the study. Venous blood was taken before starting antimycobacterial treatment. Lymphocytes were reacted with monoclonal antibodies specific for different gamma/delta V chains (Vdelta1, Vdelta2, Vgamma9 and Vgamma4). The results were analysed in the context of tuberculin reactivity and X-ray findings. Our results revealed a selective loss of Vgamma9/Vdelta2 T cells in the peripheral blood of tuberculin-negative patients with active tuberculosis compared to healthy controls, while the ratio of Vgamma9/Vdelta2 T cells in the peripheral blood of patients with a positive skin test did not differ from that of healthy controls. These findings demonstrate a relationship between the loss of the major M. tuberculosis-reactive subset of gammadelta T cells and the absence of tuberculin reactivity. The data are consistent with the hypothesis that gammadelta T cells play a role in the protective immune response to M. tuberculosis infection.     

Incubation of whole blood at 39°C augments gamma interferon (IFN-γ)-induced protein 10 and IFN-γ responses to Mycobacterium tuberculosis antigens

A rarely challenged dogma in cell-mediated immune (CMI) assays is the incubation temperature, 37°C. Fever augments proinflammatory immune responses in vivo, and the aim of this study was to explore whether incubation at fever-range temperature could increase antigen-specific biomarker responses. We compared CMI responses following incubation of whole blood at 37°C and 39°C. Whole blood was obtained from (i) 34 healthy subjects whose blood was incubated with TB10.4 antigen, present in the Mycobacterium bovis bacillus Calmette-Guérin vaccine and many environmental mycobacteria; (ii) 8 TB patients and 8 controls incubated with Mycobacterium tuberculosis-specific antigens in the QuantiFERON-TB Gold test (QFT-IT); and (iii) from both groups incubated with a T cell mitogen. T cell responses (gamma interferon [IFN-γ]) and responses from antigen-presenting cells (IFN-γ-induced protein 10 [IP-10]) were determined. We further evaluated the effect of adding interleukin-7 (IL-7) and blocking IL-10 during incubation. In TB patients, IFN-γ and IP-10 levels were increased 4.1- and 3.4-fold, respectively, at 39°C incubation (P < 0.001). Similar results were seen after mitogen stimulation. In subjects responding to TB10.4, the effects were less pronounced and significant only for IP-10. Incubation at 39°C increased IP-10 and IFN-γ responsiveness to both antigens and mitogen in persons with baseline or initial low responses. Adding IL-7 and blocking IL-10 augmented the effects in synergy with fever-range temperature. Incubation at fever-range temperature vividly increases CMI responsiveness to antigen stimulation in vitro in tuberculosis patients and may increase the sensitivity of CMI assays.     

Cytokine profiles using whole-blood assays can discriminate between tuberculosis patients and healthy endemic controls in a BCG-vaccinated population

Whole-blood assays (WB) provide a simple tool for assessing immune cytokine profiles which may be useful laboratory predictors of early disease, aiding the evaluation of new tuberculosis (TB) vaccines and offering insights into disease pathogenesis. Although BCG does not provide protection against pulmonary disease in TB endemic areas, it does modulate immune responses to mycobacterial antigens. It is important, therefore, to evaluate any new tool in an endemic setting in both BCG vaccinees and patients with tuberculosis. We have assessed the optimal conditions in terms of dose and kinetics of those cytokines which are released early (TNF-alpha, IL6 and TGF-beta, IL10) or (interferon [IFN]-gamma and IL5) in WB cultures stimulated with mitogens and mycobacterial antigens. Responses were studied in parallel in untreated TB patients and endemic control groups. Optimal responses to LPS (predominantly monocyte-derived) occurred on days 1-2, whereas for PHA (predominantly T-cell-derived), they were on days 3-5. Secreted Mycobacterium tuberculosis culture filtrate proteins (CFP) provided a stronger stimulus for monocyte-derived cytokines compared to PPD, but both antigens were comparable for induction of T-cell cytokines. Using unpaired Student's t-tests, pulmonary tuberculosis patients (P.TB; n=11), in response to CFP, showed higher monocyte-derived IL6 (p=0.023) and IL10 (p=0.042) compared to endemic controls (EC; n=13), and significantly suppressed T-cell-derived IFN-gamma (p=0.028) and IL5 (p=0.012) secretion but increased IL10 (p=0.047) on day 5, indicating that CFP is a strong stimulus for IL10 secretion in pulmonary TB patients. Extrapulmonary TB patients (E.TB; n=6) showed no elevation of early monocyte-derived cytokines to either PPD or CFP, but showed a marked suppression of the T-cell-derived cytokines IFN-gamma (PPD, p=0.015; CFP, p=0.05) and IL5 (PPD, p=0.05; CFP, p=0.015). Cytokine analysis in WB cultures is, therefore, able to discriminate between active tuberculosis infection and nondiseased healthy controls.