Enhancement of human antigen-specific memory T-cell responses by interleukin-7 may improve accuracy in diagnosing tuberculosis

Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-γ) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-γ. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-γ message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-γ responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-γ responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R² = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-γ by 39% compared to the level with antigen alone. Increased production of IFN-γ induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-γ-based assays might improve TB diagnosis in those populations at highest risk for TB.     

Intranasal interleukin-12 therapy inhibits Mycoplasma pneumoniae clearance and sustains airway obstruction in murine pneumonia

Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentrations in respiratory secretions. We evaluated the effects of IL-12 therapy on microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in mice. BALB/c mice were inoculated with M. pneumoniae or SP4 broth. Mice were treated with intranasal IL-12 or placebo daily for 8 days, starting on day 1 after inoculation. Mice were evaluated at baseline and on days 1, 3, 6, and 8 after therapy. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic score (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and granulocyte-macrophage colony-stimulating factor), and plethysmography, both before and after methacholine treatment. M. pneumoniae-infected mice treated with IL-12 (MpIL12 mice) were found to have significantly higher BAL M. pneumoniae concentrations than those of M. pneumoniae-infected mice treated with placebo (MpP mice) (P < 0.001). MpIL12 mice had higher BAL concentrations of IL-12, IFN-γ, TNF-α, and IL-6, with differences in IL-12 and IFN-γ concentrations reaching statistical significance (P < 0.001). Airway obstruction was statistically elevated in MpIL12 mice compared to that in MpP mice (P = 0.048), while airway hyperreactivity was also elevated in MpIL12 mice but did not reach statistical significance (P = 0.081). Lung parenchymal pneumonia subscores were significantly higher in MpIL12 mice (P < 0.001), but no difference was found for overall HPS, even though a strong trend was noticed (P = 0.051). Treatment of experimental M. pneumoniae pneumonia with intranasal IL-12 was associated with more severe pulmonary disease and less rapid microbiologic and histological resolution.     

Changes in Antigen-Specific Cytokine and Chemokine Responses to Plasmodium falciparum Antigens in a Highland Area of Kenya after a Prolonged Absence of Malaria Exposure

Individuals naturally exposed to Plasmodium falciparum lose clinical immunity after a prolonged lack of exposure. P. falciparum antigen-specific cytokine responses have been associated with protection from clinical malaria, but the longevity of P. falciparum antigen-specific cytokine responses in the absence of exposure is not well characterized. A highland area of Kenya with low and unstable malaria transmission provided an opportunity to study this question. The levels of antigen-specific cytokines and chemokines associated in previous studies with protection from clinical malaria (gamma interferon [IFN-γ], interleukin-10 [IL-10], and tumor necrosis factor alpha [TNF-α]), with increased risk of clinical malaria (IL-6), or with pathogenesis of severe disease in malaria (IL-5 and RANTES) were assessed by cytometric bead assay in April 2008, October 2008, and April 2009 in 100 children and adults. During the 1-year study period, none had an episode of clinical P. falciparum malaria. Two patterns of cytokine responses emerged, with some variation by antigen: a decrease at 6 months (IFN-γ and IL-5) or at both 6 and 12 months (IL-10 and TNF-α) or no change over time (IL-6 and RANTES). These findings document that P. falciparum antigen-specific cytokine responses associated in prior studies with protection from malaria (IFN-γ, TNF-α, and IL-10) decrease significantly in the absence of P. falciparum exposure, whereas those associated with increased risk of malaria (IL-6) do not. The study findings provide a strong rationale for future studies of antigen-specific IFN-γ, TNF-α, and IL-10 responses as biomarkers of increased population-level susceptibility to malaria after prolonged lack of P. falciparum exposure.     

Cytokines as Mediators for or Effectors against Rotavirus Disease in Children

Rotavirus is the most common cause of severe gastroenteritis in young children, but the pathogenesis and immunity of this disease are not completely understood. To examine the host response to acute infection, we collected paired serum specimens from 30 children with rotavirus diarrhea and measured the levels of nine cytokines (interleukin-1β [IL-1β], IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) using a microsphere-based Luminex Flowmetrix system. Patients with acute rotavirus infection had elevated median levels of seven cytokines in serum, and of these, the levels of three (IL-6, IL-10, and IFN-γ) were significantly (P < 0.05) higher than those in serum from control children without diarrhea. Patients with fever had significantly (P < 0.05) higher levels of IL-6 in serum than control children, and those with fever and more episodes of diarrhea had significantly (P < 0.05) higher levels of TNF-α than those without fever and with fewer episodes of diarrhea. We further demonstrated a negative association (P < 0.05) between the levels of IL-2 and the number of stools on the day on which the first blood sample was collected. Finally, patients with vomiting had significantly (P < 0.05) lower levels of IFN-γ than those without vomiting. Our pilot study provides evidence that the types and magnitudes of cytokine responses to rotavirus infection in children influence or reflect the clinical outcome of disease. These findings suggest that certain cytokines may play an important role in the pathogenesis of and the protection against rotavirus disease in children and, consequently, may provide directions and insights that could prove critical to the prevention or treatment of this important disease.     

Measurement of Phenotype and Absolute Number of Circulating Heparin-Binding Hemagglutinin,ESAT-6 and CFP-10, and Purified Protein Derivative Antigen-Specific CD4 T Cells Can Discriminate Active from Latent Tuberculosis Infection

The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154⁺ TNF-α⁺ cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277; P < 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154⁺ TNF-α⁺ IFN-γ⁺ IL-2⁺ and CD154⁺ TNF-α⁺ CXCR3⁺. Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB.     

Analysis of Immune Responses against a Wide Range of Mycobacterium tuberculosis Antigens in Patients with Active Pulmonary Tuberculosis

Characterizing host immune responses to molecular targets of Mycobacterium tuberculosis is essential to develop effective immunodiagnostics and better vaccines. We investigated the immune response against a large series of M. tuberculosis antigens, including 5 classical and 64 nonclassical (39 DosR regulon-encoded, 4 resuscitation-promoting factor [RPF], and 21 reactivation-associated) antigens in active-pulmonary-tuberculosis (TB) patients. Whole blood from TB patients (n = 34) was stimulated in vitro with M. tuberculosis antigens. Gamma interferon (IFN-γ) was measured after 7 days of stimulation, using an enzyme-linked immunosorbent assay (ELISA). The majority of the study participants responded to the classical M. tuberculosis antigens TB10.4 (84.8%), early secreted antigenic target-6 kDa (ESAT-6)/CFP-10 (70.6%), and purified protein derivative (PPD) (55.9%). However, only 26.5% and 24.2% responded to HSP65 and Ag85A/B, respectively. Of the 64 nonclassical antigens, 23 (33.3%) were immunogenic (IFN-γ levels, >62 pg/ml) and 8 were strong inducers of IFN-γ (IFN-γ levels, ≥100 pg/ml). The RPF antigens were the most immunogenic. In addition, we observed distinct cytokine expression profiles in response to several M. tuberculosis antigens by multiplex immunoassay. Tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10), and IL-6 were commonly detected at high levels after stimulation with 4/15 latency antigens (Rv0081, Rv2006, Rv2629, and Rv1733c) and were found especially in supernatants of the three strong IFN-γ inducers (Rv2629, Rv1009, and Rv2389c). IL-8, IL-6, and IL-17 were exclusively detected after stimulation with Rv0574c, Rv2630, Rv1998, Rv054c, and Rv2028c. In conclusion, in active-pulmonary-TB patients, we identified 23 new immunogenic M. tuberculosis antigens. The distinct expression levels of IFN-γ, TNF-α, IL-6, and IL-10 in response to specific subsets of M. tuberculosis antigens may be promising for the development of immunodiagnostics.     

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.     

Discrimination between Active and Latent Tuberculosis Based on Ratio of Antigen-Specific to Mitogen-Induced IP-10 Production

Mycobacterium tuberculosis is the major causative agent of tuberculosis (TB). The gamma interferon (IFN-γ) release assay (IGRA) has been widely used to diagnose TB by testing cell-mediated immune responses but has no capacity for distinguishing between active TB and latent TB infection (LTBI). This study aims to identify a parameter that will help to discriminate active TB and LTBI. Whole-blood samples from 33 active TB patients, 20 individuals with LTBI, and 26 non-TB controls were applied to the commercial IFN-γ release assay, QuantiFERON-TB Gold In-Tube, and plasma samples were analyzed for interleukin-2 (IL-2), IL-6, IL-8, IL-10, IL-13, tumor necrosis factor-alpha (TNF-α), IFN-γ, monokine induced by IFN-γ (MIG), interferon gamma inducible protein 10 (IP-10), interferon-inducible T cell alpha chemoattractant (I-TAC), and monocyte chemoattractant protein 1 (MCP-1) by using a commercial cytometric bead array. The Mycobacterium tuberculosis antigen-specific production of most of the assayed cytokines and chemokines was higher in the active TB than in the LTBI group. The mitogen-induced responses were lower in the active TB than in the LTBI group. When the ratio of TB-specific to mitogen-induced responses was calculated, IL-2, IL-6, IL-10, IL-13, TNF-α, IFN-γ, MIG, and IP-10 were more useful in discriminating active TB from LTBI. In particular, most patients showed higher IP-10 production to Mycobacterium tuberculosis antigens than to mitogen at the individual level, and the ratio for IP-10 was the strongest indicator of active infection versus LTBI with 93.9% sensitivity and 90% specificity. In conclusion, the ratio of the TB-specific to the mitogen-induced IP-10 responses showed the most promising accuracy for discriminating active TB versus LTBI and should be further studied to determine whether it can serve as a biomarker that might help clinicians administer appropriate treatments.     

Production of interleukins 10 and 12 by peripheral blood mononuclear cells (PBMC) in chronic hepatitis C virus (HCV) infection

We previously reported that interferon-gamma (IFN-γ) production by PBMC in response to HCV core protein was increased in patients with type C chronic liver disease. To understand better the pathophysiology of this disease, we evaluated production of IL-10 and IL-12 by PBMC from 41 patients with chronic HCV infection, including asymptomatic HCV carriers with persistently normal serum ALT values. IL-10 is known to inhibit many effector functions of the immune system, suppressing Th1-type cell development, while IL-12 stimulates differentiation of Th1-type cells, facilitating cell-mediated immunity. IL-10 production was determined by culturing lymphocytes with concanavalin A (Con A), while IL-12 was produced by monocytes in the presence of Staphylococcus aureusCowan 1 (SAC) with or without recombinant HCV core protein, respectively. The cytokine levels in culture supernatants were measured by ELISA. Spontaneous IL-10 production was greater in patients with chronic hepatitis (CH) (229±119 pg/ml, P<0.01) and liver cirrhosis (LC) (185±88 pg/ml, P<0.05) than in controls (119±27 pg/ml), while it was decreased during IFN treatment (70±25 pg/ml). Both HCV core protein and Con A enhanced IL-10 production by cells from HCV-infected patients. IL-12 was not detectable in medium alone cultures, and SAC-induced IL-12 production did not differ between various patient groups and controls. Simultaneous addition of HCV protein resulted in an increase of IL-12 production in chronic liver disease compared with SAC-alone cultures. Addition of IL-10 to the cultures equally suppressed IFN-γ production for both controls and patient groups, but the enhancing effect of IL-12 on IFN-γ production was significantly less in LC than in controls and other patient groups. The findings suggest that secretion of IL-10/IL-12 by cells from control individuals and various patient groups may be different, and that the cytokines might show different effects on IFN-γ production by some cells.     

Identification of Immunological Biomarkers Which May Differentiate Latent Tuberculosis from Exposure to Environmental Nontuberculous Mycobacteria in Children

A positive gamma interferon (IFN-γ) response to Mycobacterium tuberculosis early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) has been taken to indicate latent tuberculosis (TB) infection, but it may also be due to exposure to environmental nontuberculous mycobacteria in which ESAT-6 homologues are present. We assessed the immune responses to M. tuberculosis ESAT-6 and cross-reactive responses to ESAT-6 homologues of Mycobacterium avium and Mycobacterium kansasii. Archived culture supernatant samples from children at 3 years post-BCG vaccination were tested for cytokine/chemokine responses to M. tuberculosis antigens. Furthermore, the IFN-γ responses to M. tuberculosis antigens were followed up for 40 children at 8 years post-BCG vaccination, and 15 TB patients were recruited as a control group for the M. tuberculosis ESAT-6 response in Malawi. IFN-γ enzyme-linked immunosorbent assays (ELISAs) on supernatants from diluted whole-blood assays, IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, QuantiFERON TB Gold-In Tube tests, and multiplex bead assays were performed. More than 45% of the responders to M. tuberculosis ESAT-6 showed IFN-γ responses to M. avium and M. kansasii ESAT-6. In response to M. tuberculosis ESAT-6/CFP-10, interleukin 5 (IL-5), IL-9, IL-13, and IL-17 differentiated the stronger IFN-γ responders to M. tuberculosis ESAT-6 from those who preferentially responded to M. kansasii and M. avium ESAT-6. A cytokine/chemokine signature of IL-5, IL-9, IL-13, and IL-17 was identified as a putative immunological biosignature to differentiate latent TB infection from exposure to M. avium and M. kansasii in Malawian children, indicating that this signature might be particularly informative in areas where both TB and exposure to environmental nontuberculous mycobacteria are endemic.     

Coexistent Malnutrition Is Associated with Perturbations in Systemic and Antigen-Specific Cytokine Responses in Latent Tuberculosis Infection

Malnutrition, as defined by low body mass index (BMI), is a major risk factor for the development of active tuberculosis (TB), although the biological basis underlying this susceptibility remains poorly characterized. To verify whether malnutrition affects the systemic and antigen-specific cytokine levels in individuals with latent TB (LTB), we examined circulating and TB antigen-stimulated levels of cytokines in individuals with LTB and low BMI (LBMI) and compared them with those in individuals with LTB and normal BMI (NBMI). Coexistent LBMI with LTB was characterized by diminished circulating levels of type 1 (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), type 2 (interleukin-4 [IL-4]), type 17 (IL-22), and other proinflammatory (IL-1α, IL-1β, and IL-6) cytokines but elevated levels of other type 2 (IL-5 and IL-13) and regulatory (IL-10 and transforming growth factor beta [TGF-β]) cytokines. In addition, LBMI with LTB was associated with diminished TB antigen-induced IFN-γ, TNF-α, IL-6, IL-1α, and IL-1β levels. Finally, there was a significant positive correlation between BMI values and TNF-α and IL-1β levels and a significant negative correlation between BMI values and IL-2, IL-10, and TGF-β levels in individuals with LTB. Therefore, our data reveal that latent TB with a coexistent low BMI is characterized by diminished protective cytokine responses and heightened regulatory cytokine responses, providing a potential biological mechanism for the increased risk of developing active TB.     

Monitoring of Peptide-Specific and Gamma Interferon-Productive T Cells in Patients with Active and Convalescent Tuberculosis Using an Enzyme-Linked Immunosorbent Spot Assay

To establish a high-efficiency gamma interferon-specific enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT assay) for detection of tuberculosis (TB), peptides (E6, E7, and C14) and peptide mixtures (E6 plus E7 and E6 plus E7 plus C14) were used to monitor peripheral blood (PBL) samples from patients with pulmonary TB (PTB), as well as control samples. The positive detection rates of the five IFN-γ ELISPOT assays were 78.38%, 74.86%, 55.83%, 90.43%, and 91.51%, respectively, and there were similar detection rates between the two combined peptide mixture IFN-γ ELISPOT assays and the tuberculin skin test (TST) (90.62% versus 95.59%). No significant difference was found between the detection rates of the two combined peptide mixture IFN-γ ELISPOT assays and the T-SPOT.TB assay for 86 patients with PTB (P > 0.05), but the median number of spot-forming cells/10⁶ cells (SFP value) for positive results was higher by the former than by the latter assay (P < 0.05). In contrast, the 29.93% positive detection rate and median SFP value of 482 by the two combined peptide mixture IFN-γ ELISPOT assays were significantly higher than the corresponding values of 14.29% and 152 by T-SPOT.TB assay for the same 147 community donors (P < 0.05). For nine PTB patients tracked, the SFP value of 7 for the two peptide mixture IFN-γ ELISPOT assays began to decrease from the second month after regular treatment. A relatively low, almost normal, SFP level was reached and maintained after the third or fourth month. Two in-house IFN-γ ELISPOT assays and the T-SPOT.TB assay could reduce the false-positive and false-negative detection rates of TST and sputum acid-fast staining. Therefore, these two combined peptide mixture IFN-γ ELISPOT assays have a potential advantage, beyond their greater specificity and sensitivity, for use in screening and detection of active TB infection (TBI) and latent TB infection (LTBI) in China.     

Depressed Type 1 Cytokine Synthesis by Superantigen-Activated CD4+ T Cells of Women with Human Papillomavirus-Related High-Grade Squamous Intraepithelial Lesions

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4⁺ T and CD8⁺ T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4⁺ T cells that produced IL-2 (P = 0.045), IFN-γ (P = 0.040), and TNF-α (P = 0.015) and a significantly lower percentage of activated CD8⁺ T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4⁺ T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8⁺ T cells.     

An assessment of peripheral immunity in patients with sarcoidosis using measurements of serum vitamin D3, cytokines and soluble CD23

The aetiology of the peripheral anergy in sarcoidosis is unclear. To investigate this further we measured the serum levels of several factors important in different aspects of immune regulation to obtain a profile of those factors which promote and inhibit immune activation in sarcoidosis. Thirty-seven patients with sarcoidosis and 20 healthy controls of similar sex and age comprised the study group. Serum IL-10, interferon-gamma (IFN-γ), soluble CD23 (sCD23), IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1β and tumour necrosis factor-alpha (TNF-α) were measured using in-house ELISAs. Vitamin D3 was measured using a radioreceptor assay. Serum levels of sCD23 and IL-10 were significantly elevated in patients with sarcoidosis relative to controls (median 13.9 versus 9.5 arbitrary units/ml, P < 0.01 for sCD23, and 9.6 versus 5.0 pg/ml, P < 0.04 for IL-10). Regardless of steroid therapy or disease activity, serum levels of IFN-γ, TNF-α, IL-1β, GM-CSF and IL-8 were no different in patients with sarcoidosis and controls. Vitamin D3 levels were significantly higher in patients with sarcoidosis versus normal controls (medians 78.0 versus 56.0, P < 0.001), active sarcoidosis (n = 20) versus inactive disease (n = 17) (medians 81.5 versus 66.0, P < 0.03) and active sarcoidosis versus controls (medians 81.5 versus 56.0, P < 0.0002). The levels were no different between patients with inactive sarcoidosis and controls. We suggest that IL-10 and vitamin D3 may contribute to the peripheral anergy in sarcoidosis. The elevated serum sCD23 suggests an increase in peripheral humoral immunity. Consistent with a quiescent peripheral immune system, factors capable of monocyte/macrophage activation (TNF-α, IFN-γ, GM-CSF and IL-8) were not elevated in the peripheral circulation.     

Frequencies of Region of Difference 1 Antigen-Specific but Not Purified Protein Derivative-Specific Gamma Interferon-Secreting T Cells Correlate with the Presence of Tuberculosis Disease but Do Not Distinguish Recent from Remote Latent Infections

The majority of individuals infected with Mycobacterium tuberculosis achieve lifelong immune containment of the bacillus. What constitutes this effective host immune response is poorly understood. We compared the frequencies of gamma interferon (IFN-γ)-secreting T cells specific for five region of difference 1 (RD1)-encoded antigens and one DosR-encoded antigen in 205 individuals either with active disease (n = 167), whose immune responses had failed to contain the bacillus, or with remotely acquired latent infection (n = 38), who had successfully achieved immune control, and a further 149 individuals with recently acquired asymptomatic infection. When subjects with an IFN-γ enzyme-linked immunospot (ELISpot) assay response to one or more RD1-encoded antigens were analyzed, T cells from subjects with active disease recognized more pools of peptides from these antigens than T cells from subjects with nonrecent latent infection (P = 0.002). The T-cell frequencies for peptide pools were greater for subjects with active infection than for subjects with nonrecent latent infection for summed RD1 peptide pools (P ≤ 0.006) and culture filtrate protein 10 (CFP-10) antigen (P = 0.029). Individuals with recently acquired (<6 months) versus remotely acquired (>6 months) latent infection did not differ in numbers of peptide pools recognized, proportions recognizing any individual antigen or peptide pool, or antigen-specific T-cell frequencies (P ≥ 0.11). The hierarchy of immunodominance for different antigens was purified protein derivative (PPD) > CFP-10 > early secretory antigenic target 6 > Rv3879c > Rv3878 > Rv3873 > Acr1, and the hierarchies were very similar for active and remotely acquired latent infections. Responses to the DosR antigen α-crystallin were not associated with latency (P = 0.373). In contrast to the RD1-specific responses, the responses to PPD were not associated with clinical status (P > 0.17) but were strongly associated with positive tuberculin skin test results (≥15-mm induration; P ≤ 0.01). Our results suggest that RD1-specific IFN-γ-secreting T-cell frequencies correlate with the presence of disease rather than with protective immunity in M. tuberculosis-infected individuals and do not distinguish recently acquired asymptomatic infection from remotely acquired latent infection.The immune response is responsible for both bacillary containment in latent tuberculosis infection (LTBI) and immunopathology in active tuberculosis (TB). Comparing key immune responses for these two states may therefore help us dissect which responses mediate or correlate with disease and protection. It is therefore of particular interest to compare the immune responses in individuals with latent infection, who have successfully achieved immune control, and individuals with active disease, in whom the immune response has failed to contain the bacterium but immunopathology is present.Gamma interferon (IFN-γ) is essential for controlling Mycobacterium tuberculosis infection (11, 17), and CD4⁺ IFN-γ-secreting T cells specific for mycobacterial antigens play a pivotal role (16, 31). Responses to region of difference 1 (RD1)-encoded antigens are of special interest because of the unique features of early secretory antigenic target 6 (ESAT-6) (1, 30) and culture filtrate protein 10 (CFP-10) (3, 5), which appear to be both virulence factors and putative targets of protective immunity.In a primary bovine TB infection, ESAT-6-specific IFN-γ production, although not a proliferative response, correlated with the severity of disease pathology (39). Likewise, increased ESAT-6 expression and CFP-10 expression in Mycobacterium bovis bacillus Calmette-Guerin (BCG) or Mycobacterium microti infections were associated with increased pathogenicity in susceptible mice and correlated with increased RD1-specific T-cell responses (9). However, RD1-specific responses induced by vaccination are associated with protective immunity against subsequent challenge with virulent organisms. Thus, mice infected with M. tuberculosis were resistant to reinfection (2), and protection correlated with accelerated accumulation of IFN-γ-secreting effector T cells responding to Ag85 and ESAT-6 (1). Mice (27) and guinea pigs (8) vaccinated with BCG::RD1 developed strong CD4⁺ IFN-γ and proliferative responses to ESAT-6. When challenged, these animals had superior protection compared with BCG-vaccinated or unvaccinated mice, as well as less severe pathology and reduced dissemination of the pathogen (32).However, the relationship between the magnitude of ESAT-6 responses and disease in humans is unclear; little is known about CFP-10, and almost nothing is known about the other RD1-encoded antigens. The chaperone protein α-crystallin (“Acr1,” “Rv2031c,” “HspX,” “16-kDa antigen”) is upregulated under oxidative stress conditions (41) and is important for growth in macrophages (12). Encoded by the M. tuberculosis “dormancy regulon” expressed during natural infection (25), this protein is upregulated during conditions of in vitro stress (35). For these reasons, it has been postulated that Acr1-specific T-cell responses may correlate with latency (12, 41).We recently recruited a cohort of 389 individuals suspected to have TB as part of a prospective study of the diagnostic utility of enzyme-linked immunospot (ELISpot) responses to RD1 antigens. (13). A total of 205 patients were given a definitive diagnosis of active or latent TB and had not received antituberculous chemotherapy. We enumerated IFN-γ-secreting T cells specific for ESAT-6, CFP-10, Rv3879c, Rv3878, Rv3873 (10), and purified protein derivative (PPD) and, in a subset, IFN-γ-secreting T cells specific for Acr1. We compared responses to these antigens in patients with active TB and patients with LTBI in a blinded, prospective manner to address the following questions. Are the frequencies of IFN-γ-secreting T cells specific for RD1 antigens, PPD, and Acr-1 different in patients with active TB and patients with LTBI? Do the breadth of the response to RD1-derived peptides in patients with active disease and the breadth of the response to RD1-derived peptides in patients with latent infection differ? Are the hierarchies of immunodominance similar? Do these responses vary with the tuberculin skin test (TST) results? Do responses differ between recently and remotely acquired latent infections?(Part of this work was presented at the winter meeting of the Acid Fast Club, United Kingdom, on 12 January 2007.)     

Protective effects of bifidobacterial adhesin on intestinal mucosa of stressed male rats via modulation of inflammation

This study aimed to assess BA impact on inflammation markers and repair of intestinal mucosa. Forty-eight rats were randomly divided into stress (n = 24) and BA (n = 24) groups. Stress was induced by fettering in all animals, fed enterally with 125.4 kJ/kg/d and 0.2 g/kg/d nitrogen. Then, rats were treated for 8 days with 5 mg/kg/d BA (BA group) or 5 mg/kg/d saline (Stress group). Levels of NF-κB, IL-10, TNF-α, and IFN-γ were measured at different time points, in plasma and intestinal mucosa samples. Changes in intestinal mucosa morphology were observed by electron microscopy. Plasma and/or mucosal levels of NF-κB, TNF-α, and IFN-γ were significantly higher in both groups after stress induction (P < 0.05). These high levels persisted in control animals throughout the experiment, and were significantly reduced in the BA group, 3 and 8 days after stress induction (P < 0.05). Interestingly, IL-10 levels were increased after BA treatment (P < 0.05). At day 8, ileal mucosal villi and crypt structure were significantly restored in the BA group. Bifidobacterial adhesin plays a role in repairing intestinal mucosa injury after stress by regulating the release of inflammatory mediators in the intestinal mucosa.     

Differential Live Mycobacterium tuberculosis-, M. bovis BCG-, Recombinant ESAT6-, and Culture Filtrate Protein 10-Induced Immunity in Tuberculosis

The high prevalence of Mycobacterium tuberculosis makes it imperative that immune responses to evaluate could be predictive of infection. We investigated live Mycobacterium- and recombinant antigen-induced cytokine and chemokine responses in patients with active tuberculosis (TB) compared with those of healthy controls from an area where TB is endemic (ECs). M. tuberculosis-, M. bovis BCG-, ESAT6-, and culture filtrate protein 10 (CFP10)-induced responses were determined in peripheral blood mononuclear cells from patients with pulmonary TB (n = 38) and ECs (n = 39). The levels of the cytokines gamma interferon (IFN-γ) and interleukin-10 (IL-10) and the chemokines CCL2, CCL3, and CXCL9 were measured. The levels of M. tuberculosis- and BCG-induced IFN-γ secretion were significantly reduced (P = 0.002 and P < 0.01, respectively), while the amount of IL-10 induced by both virulent (P < 0.01) and avirulent (P = 0.002) mycobacteria was increased in patients with TB. The ESAT6-induced IFN-γ responses were increased in the patients with TB (P = 0.013) compared with those in the EC group. When tuberculin skin test (TST)-negative (TST⁻; induration, <10 mm) and TST-positive (TST⁺) donors were studied separately, both TST⁻ and TST⁺ individuals showed increased IFN-γ responses to M. tuberculosis compared with the responses of the patients with TB (P = 0.037 and P = 0.006, respectively). However, only TST⁺ ECs showed reduced IFN-γ responses to ESAT6 (P = 0.008) compared with the responses of the patients with TB. The levels of M. tuberculosis-induced CCL2 (P = 0.006) and CXCL9 (P = 0.017) were greater in the patients with TB. The levels of CCL3 secretion in response to Mycobacterium and antigen stimulation were comparable between the two groups. While the levels of ESAT6-induced chemokines did not differ between the patients with TB and the ECs, the levels of CFP10-induced CCL2 (P = 0.01) and CXCL9 (P = 0.001) were increased in the patients. These data indicate differential host IFN-γ, CXCL9, and CCL2 responses to live mycobacteria and mycobacterial antigens and have implications for the identification of potential biomarkers of infection which could be used for the diagnosis of TB.Tuberculosis (TB) remains an important cause of mortality and morbidity worldwide, causing 2 million deaths annually. Vaccination with Mycobacterium bovis BCG provides protection mostly during childhood and primarily against the extrapulmonary forms of the disease (28, 45, 49). It is important to identify immune markers that may be reflective of natural infection in the host and that may therefore be useful in predicting the outcome of the disease.Host immune responses determine the outcome of M. tuberculosis infection. The balance between the key T-cell cytokines gamma interferon (IFN-γ) and interleukin-10 (IL-10), which are produced by macrophages and subsets of T cells, is key to the maintenance of effector responses against the mycobacterium (13, 34). This is further reflected by the relationship between IFN-γ and IL-10, which differs according to the severity of the disease, especially in patients with extrapulmonary M. tuberculosis infections (24).The formation and maintenance of granulomas are essential for the control of mycobacterial infections. Tumor necrosis factor alpha (TNF-α) is critical to an effective response against M. tuberculosis and for efficient granuloma formation (38). TNF^−/− mice show increased pathology and a more rapid progression of disease during infection with M. tuberculosis (6).IFN-γ- and macrophage-induced TNF-α activates and regulates the C-C chemokines CCL2, CCL3, CCL4, and CCL5 and the C-X-C chemokines CXCL8, CXCL9, CXCL10, and CXCL11 (2) (42). These chemokines have been shown to play an important role in the recruitment and activation of leukocytes at the site of granuloma formation.CCL2 is the most potent activator of monocytes and is essential for recruitment and migration to the lung. Studies with CCL2^−/− mice have investigated the pathology of M. tuberculosis infection (26) and have shown that CCCR2^−/− mice exhibit faulty granuloma formation and increased susceptibility to disease caused by M. tuberculosis (44). IFN-γ-activated chemokines, such as CXCL9 and CXCL10, are induced by M. tuberculosis antigens (1, 40).The circulating levels of cytokines such as IFN-γ, IL-10, transforming growth factor-β, and chemokine CXCL9 are found to be raised in patients with TB (17, 33). Patients with pulmonary TB also display increases in the level of monocyte-activating chemokine CCL2 (20). An increase in the CCL2 level is associated with monocyte migration to the lung and the reduced progression of disease (26, 43). Recent studies have identified molecular mechanisms which are deficient in patients with pulmonary TB. These include reduced IFN-γ responses at the disease site (21) and a reduced monocyte maturation ability concomitant with a reduction in the levels of markers of antigen presentation, such as major histocompatibility complex class II expression (36). In addition, it has been shown that granulomas differ in cavitary and noncavitary TB (48). This further emphasizes the need to understand the pattern of recognition of mycobacterial antigens in different forms of TB and in transitions from latent to active infection.Six-kilodalton early secreted antigenic target 6 (ESAT6) and culture filtrate protein 10 (CFP10) are both encoded by region of difference 1 (RD1), which is present in M. tuberculosis and M. bovis but which is absent from M. bovis BCG and most environmental mycobacteria (8, 16). ESAT6 is an immunodominant T-cell-stimulatory antigen and is recognized by specific IFN-γ-secreting T cells that are present in greater numbers in patients with active disease than in those who are uninfected (37, 47). The importance of these recombinant antigens is in their immune recognition by T cells specific to infected individuals. They are a primary component of new diagnostic assays and are meant to detect latent infections in individuals because of their lack of cross-reactivity with M. bovis BCG, which is used for vaccination (27). Commercial tests that use IFN-γ-release assays are increasingly available as diagnostic and predictive tests for newer-generation vaccines. ESAT6- and CFP10-induced IFN-γ responses have been shown to be useful in discriminating infected individuals from healthy controls (1, 9, 46). Demissie et al. have shown that immune responses to ESAT6 are more likely to be associated with active infection and that a dormancy-related antigen (HspX or α-crystallin) might be a better marker of latency in patients with TB, while cross-reactive antigens, such as the antigen 85A complex, do not distinguish between active and latent TB (11).Antigen recognition and binding to cell surface receptors result in cytokine activation and intracellular signaling cascades within the host cell (14, 39). Virulent M. tuberculosis organisms are efficient at evading macrophage host defenses, such as phagolysosome fusion; downregulation of the activation of proinflammatory cytokines, such as TNF-α; inhibition of antigen presentation; and also host apoptotic responses (19, 25). In contrast, avirulent mycobacteria, such as the M. bovis BCG strain, are processed along the endosomal pathway, activating stronger responses of proinflammatory cytokines, such as TNF-α, IL-2, and IL-6, than the responses activated by virulent mycobacteria (7). In addition, the uptake and processing of live and dead mycobacteria differ in host cells (18). Therefore, although both mycobacteria and mycobacterial antigens can stimulate host immunity, it is likely that their immunogenicities may differ due to processing and presentation of antigens.There is as yet a limited understanding of how host cell responses differ when the host is challenged with live mycobacteria as opposed to mycobacterial antigens. We investigated the differential cytokine responses to live mycobacteria (virulent M. tuberculosis and avirulent M. bovis BCG strains) and compared them with those to the M. tuberculosis antigens ESAT6 and CFP10. BCG vaccination is widespread in Pakistan and since 1981 has routinely been administered at birth through the Expanded Program for Immunization (51). Therefore, the levels of cytokine and chemokine secretion by peripheral blood mononuclear cells (PBMCs) in response to different stimuli were analyzed in patients with pulmonary TB and were compared with those in healthy vaccinated controls from areas where TB is endemic (ECs). Our results highlight the variability of host responses to mycobacteria and mycobacterial antigens.     

Haemophilus influenzae and Streptococcus pneumoniae induce different intracerebral mRNA cytokine patterns during the course of experimental bacterial meningitis

Using in situ hybridization with radiolabelled oligonucleotide probes, we studied the mRNA expression of IL-1β, IL-4, IL-6, IL-10, IL-12, tumour necrosis factor-alpha (TNF-α), TNF-β, interferon-gamma (IFN-γ), and transforming growth factor-beta (TGF-β) in the brain during the lethal course of experimental meningitis in a rat model inoculated intracisternally with Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae and in uninfected control rats inoculated with the same volume of PBS. The production of IL-1β, IL-4, IL-6 and IFN-γ was also evaluated by immunohistochemistry. In the brain of Hib-inoculated rats, there was marked mRNA expression of IL-1β, IL-6, TNF-α, IL-12 and IFN-γ. IL-1β, IL-6 and TNF-α were up-regulated throughout the observation period at 2, 8 and 18 h post-inoculation (p.i.), with similar patterns of induction. The Th1 cytokines IFN-γ and TNF-β were up-regulated within 8 h p.i. IL-10 and TGF-β were down-regulated at 18 h p.i., while IL-4 was not detected. In contrast, the brain of S. pneumoniae-inoculated rats showed lower levels of IL-1β, IL-6 and TNF-α, but higher levels of TNF-β and detectable mRNA expression of IL-4 when compared with Hib-inoculated rats. IL-12, IFN-γ, IL-10 and TGF-β exhibited similar patterns of induction in the brains of Hib- and S. pneumoniae-inoculated rats. At 18 h p.i., immunohistochemistry showed similar patterns of IL-1β, IL-4, IL-6 and IFN-γ as mRNA expression in the brains of Hib- and S. pneumoniae-inoculated rats. The differences of cytokine profiles induced by the two bacterial strains may imply that different immunomodulating approaches should be considered, depending on etiology.     

Temporal regulation of interleukin-12p70 (IL-12p70) and IL-12-related cytokines in splenic dendritic cell subsets during Leishmania donovani infection

Dendritic cells (DC) play an essential role in initiating and directing T-cell responses, in part by production of interleukin-12p70 (IL-12p70), IL-23, and IL-27. However, comparative studies on the capacity for cytokine production of DC subsets are rare. Here, we compare splenic CD8α⁺, CD4⁺, and double-negative (DN) DC, isolated 5 h to 28 days after Leishmania donovani infection, for (i) production of IL-12p70, (ii) accumulation of IL-12/23p40, IL-12p35, IL-23p19, and IL-27p28 mRNAs, and (iii) their capacity to direct CD4⁺ T-cell differentiation. At 5 h, conventional DC (cDC) accumulated mRNA for IL-12/23p40 (CD8α>CD4>DN), IL-23p19 (CD4>CD8α>DN), and IL-27p28 (CD8α>CD4>DN), in an infection dose-dependent manner. IL-12p70 was restricted to CD8α⁺ cDC, reflecting the subset-specific accumulation of IL-12p35 mRNA. In contrast, cDC from mice infected for 14 to 28 days accumulated little mRNA for IL-12p40 and IL-12p19, though IL-27p28 mRNA remained detectable (CD8α>DN>CD4). IL-12p70 secretion by CD8α⁺ cDC was also absent, reflecting deficient IL-12/23p40, rather than IL-12p35, mRNA accumulation. The capacity of CD8α⁺ cDC isolated early after infection to direct Th1 cell differentiation was mediated through IL-12/23p40, whereas this ability in CD4⁺ and DN cDC was independent of IL-12/23p40 and did not result from overexpression of Delta 4 Notch-like ligand. However, DN cDC produced gamma interferon (IFN-γ) and also contained a rare population of CD11c^hi DX5⁺ IFN-γ-producing cells. Our data illustrate the extensive diversity in, and temporal regulation of, splenic cDC subsets during infection and suggest caution in interpreting data obtained with unfractionated or minimally purified DC.