Background

The European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) is the instrument most frequently used to measure quality of life in cancer patients, whereas the EQ-5D is widely used to measure and evaluate general health status. Although the EORTC QLQ-C30 has been mapped to EQ-5D utilities, those studies were limited to patients with a single type of cancer. The present study aimed to develop a mapping relationship between the EORTC QLQ-C30 and EQ-5D-based utility values at the individual level.

Methods

The model was derived using patients with different types of cancer who were receiving chemotherapy. The external validation set comprised outpatients with colon cancer. Ordinary least squares regression was used to estimate the EQ-5D index from the EORTC QLQ-C30 results. The predictability, goodness of fit, and signs of the estimated coefficients of the model were assessed. Predictive ability was determined by calculating the mean absolute error, the estimated proportions with absolute errors > 0.05 and > 0.1, and the root-mean-squared error (RMSE).

Results

A model that included global health, physical, role, emotional functions, and pain was optimal, with a mean absolute error of 0.069 and an RMSE of 0.095 (normalized RMSE, 8.1%). The explanatory power of this model was 51.6%. The mean absolute error was higher for modeled patients in poor health.

Conclusions

This mapping algorithm enabled the EORTC QLQ-C30 to be converted to the EQ-5D utility index to assess cancer patients in Korea.

Identification and characterization of serologically active mycobacterial antigens are prerequisites for the development of diagnostic reagents. We examined the humoral immune responses of active tuberculosis (TB) patients against Triton-soluble proteins extracted from Mycobacterium tuberculosis by immunoblotting. A 29-kDa protein reacted with immunoglobulin M (IgM) in the pooled sera of the patients, and its N-terminal amino acid sequence matched that of the heparin-binding hemagglutinin (HBHA). Recombinant full-length HBHA was expressed in Escherichia coli (rEC-HBHA) and M. smegmatis (rMS-HBHA). In immunoblot analysis, the IgM antibodies of the TB patients reacted strongly with rMS-HBHA but not with rEC-HBHA, whereas the IgG antibodies of these patients reacted weakly with both recombinant HBHA proteins. In enzyme-linked immunosorbent assay analysis using rMS-HBHA and 85B as antigens, the mean levels and sensitivities of the anti-HBHA IgM antibodies of the TB patients were significantly higher than those of the anti-antigen 85B IgM antibodies, while the IgG antibodies showed the opposite results. Of interest in this respect, the pooled sera from the TB patients that contained anti-HBHA IgM antibodies neutralized the entry of M. tuberculosis into epithelial cells. These findings suggest that IgM antibody to HBHA may play a role in protection against extrapulmonary dissemination.

Background

Chronic heart failure accounts for a great deal of the morbidity and mortality in the aging population. Evidence-based treatments include angiotensin-2 receptor blockers (ARBs), angiotensin-converting enzyme inhibitors (ACE-I), beta-blockers, and aldosterone antagonists. Underutilization of these treatments in heart failure patients were frequently reported, which could lead to increase morbidity and mortality. The aim of this study was to evaluate the utilization of evidence-based treatments and their related factors for elderly patients with chronic heart failure.

Methods

This is retrospective observational study using the Korean National Health Insurance claims database. We identified prescription of evidence based treatment to elderly patients who had been hospitalized for chronic heart failure between January 1, 2005, and June 30, 2006.

Results

Among the 28,922 elderly patients with chronic heart failure, beta-blockers were prescribed to 31.5%, and ACE-I or ARBs were prescribed to 54.7% of the total population. Multivariable logistic regression analyses revealed that the prescription from outpatient clinic (prevalent ratio, 4.02, 95% CI 3.31–4.72), specialty of the healthcare providers (prevalent ratio, 1.26, 95% CI, 1.12–1.54), residence in urban (prevalent ratio, 1.37, 95% CI, 1.23–1.52) and admission to tertiary hospital (prevalent ratio, 2.07, 95% CI, 1.85–2.31) were important factors associated with treatment underutilization. Patients not given evidence-based treatment were more likely to experience dementia, reside in rural areas, and have less-specialized healthcare providers and were less likely to have coexisting cardiovascular diseases or concomitant medications than patients in the evidence-based treatment group.

Conclusions

Healthcare system factors, such as hospital type, healthcare provider factors, such as specialty, and patient factors, such as comorbid cardiovascular disease, systemic disease with concomitant medications, together influence the underutilization of evidence-based pharmacologic treatment for patients with heart failure.

Purpose

Live/killed mycobacteria and culture supernatants can suppress asthmatic reactions. This study investigated whether mycobacterial secretory proteins have therapeutic effects on asthma.

Methods

Mycobacterium bovis bacille Calmette-Guérin (BCG; 2×105 CFUs) and mycobacterial secretory proteins (Ag85 complex, 38-kDa protein or MPB70; 4 or 20 µg) were administered intraperitoneally to female BALB/c mice with established airway hyperresponsiveness. One week after treatment, the mice underwent a methacholine challenge test, and then inflammatory cell numbers in bronchoalveolar lavage fluid (BAL) and around bronchi (<500 µm), and cytokine levels in splenocyte supernatants, were assessed.

Results

BCG and all of the tested secretory proteins significantly improved airway sensitivity compared to baseline values (P<0.05). The secretory protein Ag85 complex significantly suppressed airway reactivity also (P<0.05), while 38-kDa protein significantly suppressed reactivity and maximal narrowing (P<0.05). The number of eosinophils in BAL and around bronchi, and the goblet cell proportion, were also significantly reduced in mice in both the BCG and secretory protein groups compared to the asthma control group. IFN-γ/IL-5 ratios were significantly higher in mice treated with BCG, 4 µg MPB70 or 4 µg 38-kDa protein than in asthma control mice (P<0.05), and were negatively associated with airway hyperresponsiveness, peribronchial eosinophil numbers and goblet cell proportion (all P<0.05). IL-17A was positively correlated with IL-5 (r=0.379, P<0.001), maximal airway narrowing, peribronchial eosinophil numbers and goblet cell proportion (all P<0.05).

Conclusions

Secretory proteins from BCG and M. tuberculosis and live BCG were effective against established asthma, their effects being accompanied by increased IFN-γ/IL-5 ratios. Thus, allergic asthma could be effectively treated with mycobacterial secretory proteins.

We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg.

Background

Apoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages.

Methodology/Principal Findings

Here, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2α was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2α phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2α. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response.

Conclusion/Significance

These findings indicate that eIF2α/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria.

Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA), a virulence factor involved in extrapulmonary dissemination and a strong diagnostic antigen against tuberculosis, is both surface-associated and secreted. The role of HBHA in macrophages during M. tuberculosis infection, however, is less well known. Here, we show that recombinant HBHA produced by Mycobacterium smegmatis effectively induces apoptosis in murine macrophages. DNA fragmentation, nuclear condensation, caspase activation, and poly (ADP-ribose) polymerase cleavage were observed in apoptotic macrophages treated with HBHA. Enhanced reactive oxygen species (ROS) production and Bax activation were essential for HBHA-induced apoptosis, as evidenced by a restoration of the viability of macrophages pretreated with N-acetylcysteine, a potent ROS scavenger, or transfected with Bax siRNA. HBHA is targeted to the mitochondrial compartment of HBHA-treated and M. tuberculosis-infected macrophages. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in both macrophages and isolated mitochondria treated with HBHA. Disruption of HBHA gene led to the restoration of ΔΨm impairment in infected macrophages, resulting in reduced apoptosis. Taken together, our data suggest that HBHA may act as a strong pathogenic factor to cause apoptosis of professional phagocytes infected with M. tuberculosis.

Author Summary

Cell death is a common outcome during infection with a number of pathogenic microorganisms. Therefore, defining the factors responsible for killing of host cells is important to uncovering mechanisms of pathogenesis. World-wide, two billon people are latently infected with Mycobacterium tuberculosis, which is still killing 2–3 million people each year. Heparin-binding hemagglutinin (HBHA) protein of M. tuberculosis is known to interact specifically with non-phagocytic cells and to be involved in dissemination from lungs to other tissues. Nevertheless, the role of HBHA in phagocytic cells such as macrophages, which are the first cells of the immune system to encounter inhaled pathogens, has been unknown. In the present study, we suggest HBHA as a critical bacterial protein for macrophage cell death. After M. tuberculosis infection or HBHA treatment of macrophages, HBHA targeted to mitochondria and then caused mitochondrial damage and oxidative stress, which eventually lead to apoptosis. A mutant of M. tuberculosis lacking HBHA induced less apoptosis with moderated mitochondrial damage. These experiments provide a candidate virulence factor which may be a novel target for tuberculosis treatment.

Objective

To investigate the usefulness of bioimpedance measurement for predicting the treatment outcome in breast cancer related lymphedema (BCRL) patients.

Method

Unilateral BCRL patients who received complex decongestive therapy (CDT) for 2 weeks (5 days per week) were enrolled in this study. We measured the ratio of extracellular fluid (ECF) volume by using bioelectrical impedance spectroscopy (BIS), and single frequency bioimpedance analysis (SFBIA) at a 5 kHz frequency before treatment. Arm circumferences were measured at 10 cm above and below the elbow before and after treatment. We also investigated whether there is correlation between ECF ratio and SFBIA ratio with the change of arm circumference after CDT.

Results

A total of 73 patients were enrolled in this study. The higher ECF ratio was significantly correlated with higher reduction of arm circumference at both above and below the elbow after treatment, but the higher SFBIA ratio was correlated only with the higher reduction of arm circumference below the elbow.

Conclusion

These results show that ECF volume measurements and SFBIA before treatment are useful tools for predicting the outcome of patients with lymphedema. We concluded that ECF volume measure can be used as a screening tool for predicting treatment outcome of BCRL patients.

A novel duplex PCR method based on variable-number tandem-repeat targets to discriminate among Mycobacterium abscessus complex isolates was developed and evaluated in 85 clinical isolates. The assay accuracy was confirmed by a multiple-target sequence analysis. The duplex PCR assay is a one-step, reliable, and accurate assay for discriminating M. abscessus species.

We evaluated high-resolution melting (HRM) curve analysis as a tool for detecting rifampin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis in an accurate, affordable, and rapid manner. Two hundred seventeen M. tuberculosis clinical isolates of known resistance phenotype were used. Twenty-nine known rpoB mutant DNAs, including rare mutations, were also included. Four pairs of primers were designed: rpoB-F/R (for codons 516 to 539 of rpoB), rpoB-516F/R (for codons 508 to 536 of rpoB), katG-F/R (for the codon 315 region of katG), and inhA-F/R (for the nucleotide substitution of C to T at position −15 of inhA). An HRM curve was generated for each isolate after real-time PCR differentiated the mutant from the wild-type strains. DNA sequencing of the target regions was performed to confirm the results of the HRM curve analysis. All but one of the 73 RIF-resistant (RIF-R) strains and all 124 RIF-susceptible (RIF-S) isolates were correctly identified by HRM curve analysis of rpoB. Twenty-seven of 29 known rpoB mutants were detected. In HRM curve analysis of katG and inhA, 90 INH-R strains that harbored katG or inhA mutations, or both, and all INH-S strains were correctly identified. Ten phenotypically INH-R strains not harboring katG or inhA mutations were not detected. The HRM curve analysis will be a useful method for detection of RIF and INH resistance in M. tuberculosis in a rapid, accurate, simple, and cost-effective manner.

Background

Mycobacterium tuberculosis (Mtb) heparin binding hemagglutinin (HBHA) is an Ag known to evoke effective host immune responses during tuberculosis infection. However, the molecular basis of the host immune response to HBHA has not been fully characterized. In this study, we examined the molecular mechanisms by which HBHA can induce the expression of proinflammatory cytokines in macrophages.

Methods

HBHA-induced mRNA and protein levels of proinflammatory cytokines were determined in bone marrow-derived macrophages (BMDMs) using RT-PCR and ELISA analysis. The roles of intracellular signaling pathways for NF-κB, PI3-K/Akt, and MAPKs were investigated in macrophage proinflammatory responses after stimulation with HBHA.

Results

HBHA robustly activated the expression of mRNA and protein of both TNF-α and IL-6, and induced phosphorylation of NF-κB, Akt, and MAPKs in BMDMs. Both TNF-α and IL-6 production by HBHA was regulated by the NF-κB, PI3-K, and MAPK pathways. Furthermore, PI3-K activity was required for the HBHA-induced activation of ERK1/2 and p38 MAPK, but not JNK, pathways.

Conclusion

These data suggest that mycobacterial HBHA significantly induces proinflammatory responses through crosstalk between the PI3-K and MAPK pathways in macrophages.

Background/Aims

Fabry disease is an X-linked recessive and progressive disease caused by α-galactosidase A (α-GaL A) deficiency. We sought to assess the prevalence of unrecognized Fabry disease in dialysis-dependent patients and the efficacy of serum globotriaosylceramide (GL3) screening.

Methods

A total of 480 patients of 1,230 patients among 17 clinics were enrolled. Serum GL3 levels were measured by tandem mass spectrometry. Additionally, we studied the association between increased GL3 levels and cardiovascular disease, cerebrovascular disease, or left ventricular hypertrophy.

Results

Twenty-nine patients had elevated serum GL3 levels. The α-GaL A activity was determined for the 26 patients with high GL3 levels. The mean α-GaL A activity was 64.6 nmol/hr/mg (reference range, 45 to 85), and no patient was identified with decreased α-GaL A activity. Among the group with high GL3 levels, 15 women had a α-GaL A genetics analysis. No point mutations were discovered among the women with high GL3 levels. No correlation was observed between serum GL3 levels and α-GaL A activity; the Pearson correlation coefficient was 0.01352 (p = 0.9478). No significant correlation was observed between increased GL3 levels and the frequency of cardiovascular disease or cerebrovascular disease.

Conclusions

Fabry disease is very rare disease in patients with end-stage renal disease. Serum GL3 measurements as a screening method for Fabry disease showed a high false-positive rate. Thus, serum GL3 levels determined by tandem mass spectrometry may not be useful as a screening method for Fabry disease in patients with end stage renal disease.

Purpose

Live Mycobacterium bovis Bacille Calmette-Guérin (BCG) has a suppressive effect on asthma, but its use in clinical practice may be limited due to adverse reactions. To develop a product that is effective for suppressing asthma with minimal adverse reactions, we investigated whether the heat-killed body or culture supernatants of mycobacteria could also prevent asthma development.

Methods

Female BALB/c mice were treated with live BCG, the heat-killed body, or culture supernatants of BCG or Mycobacterium tuberculosis intraperitoneally, while sensitizing and provoking with ovalbumin. Then they underwent a methacholine bronchoprovocation test, and the peribronchial inflammatory cell numbers and cytokine levels in splenocyte culture supernatants were assessed.

Results

The airway sensitivity to methacholine decreased significantly after treatment with not only live BCG (30.8 versus 10.0 mg/mL, P<0.001) but also with the culture supernatant (BCG, 23.0 mg/mL, P<0.05; M. tuberculosis, 20.5 mg/mL, P<0.05). In contrast, heat-killed mycobacteria did not effectively decrease airway sensitivity. The peribronchial eosinophil counts and the goblet cell proportions in total epithelial cells decreased significantly in most of the groups. The interferon-γ/interleukin-5 ratios increased significantly in most of the treatment groups except for the heat-killed groups, and were significantly related to airway sensitivity (r=0.312, P<0.01) and peribronchial eosinophil counts (r=-0.416, P<0.001). Interleukin-17A level was inversely related to airway sensitivity (r=-0.212, P<0.05) and was significantly lower in the live BCG group than in the control (137±20 versus 308±57 pg/mL, P<0.05).

Conclusions

BCG and mycobacteria culture supernatants may effectively prevent the development of asthma associated with altered Th1/Th2 cytokines and interleukin-17A levels.

Tuberculosis (TB) is the leading cause of death from a single infectious agent in Korea. In this study, we compared the proteins present in culture filtrates from Mycobacterium tuberculosis strain K, which is the dominant clinical isolate in Korea, with those present in culture filtrates from M. tuberculosis H37Rv. Several differences in expression were detected between the two strains for those proteins with a molecular mass of <20 kDa. ESAT-6, HSP-X, and CFP-10 were found to be abundantly expressed in the strain K culture filtrates by liquid chromatography-electrospray ionization-time of flight mass spectrometry. The serodiagnostic potentials of recombinant antigens rESAT-6, rHSP-X, and rCFP-10 and two native antigens (Ag85 and PstS1) were evaluated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera collected from 46 TB patients with active disease and 46 healthy controls. As for our ELISA results, HSP-X was superior to the other antigens in terms of sensitivity when a single antigen was employed. The results of a receiver operator characteristic analysis revealed that a cocktail ELISA using all five antigens was significantly more sensitive (77.8%) than the use of a single antigen and offered equivalent specificity; moreover, it produced the largest area under the curve (0.91 versus 0.55 to 0.87). Therefore, a cocktail ELISA containing abundantly expressed antigens enhances the sensitivity of a single antigen and can be a useful diagnostic tool for the detection of active TB.

Background

In recent years T cell based interferon gamma release assays (IGRA) have been developed for immunodiagnosis of M. tuberculosis infection. At present these assays do not discriminate between disease and latency. Therefore, more promising antigens and diagnostic tools are continuously being searched for tuberculosis immunodiagnostics. The heparin binding hemagglutinin (HBHA) is a surface protein of M. tuberculosis which promotes bacterial aggregation and adhesion to non-phagocytic cells. It has been previously assumed that native, methylated form of this protein would be a promising antigen to discriminate latent from active infection.

Methodology and Principal Findings

We performed a pilot investigation to study humoral and T-cell mediated immunological responses to recombinant HBHA produced in M. smegmatis or to synthetic peptides in patients with recent or past tuberculosis, with atypical mycobacteriosis, or in healthy vaccinated individuals. The T cell reactivities to HBHA were compared to the respective reactivities towards Purified Protein Derivative (PPD) and two surface secreted proteins, ie. Early Secretory Antigen Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10). Our pilot results indicate that methylated recombinant HBHA induced a strong T cell mediated immune response and the production of IgG and IgM-class antibodies in all patient groups, most surprisingly in young Finnish vaccinees, as well. We observed a positive correlation between the reactivities to HBHA and non-specific PPD among all studied subjects. As expected, ESAT-6 and CFP-10 were the most powerful antigens to discriminate disease from immunity caused by vaccination.

Conclusions

On the basis of results of this exploratory investigation we raise concerns that in countries like Finland, where BCG vaccination was routinely used, HBHA utility might not be sufficient for diagnostics because of inability to explicitly discriminate tuberculosis infection from immunoreactivity caused by previous BCG vaccination.

Mycobacterium tuberculosis produces a large number of structurally diverse lipids generated from the carboxylation products of acetyl-CoA and propionyl-CoA. A biotin-dependent acyl-CoA carboxylase was purified from M. tuberculosis H37Rv by avidin affinity chromatography, and the three major protein components were determined by N-terminal sequencing to be the 63-kDa α3-subunit (AccA3, Rv3285), the 59-kDa β5-subunit (AccD5, Rv3280), and the 56-kDa β4-subunit (AccD4, Rv3799). A minor protein of about 24 kDa that co-purified with the above subunits was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to be the product of Rv3281 that is located immediately downstream of the open reading frame encoding the β5-subunit. This protein displays identity over a short stretch of amino acids with the recently discovered ε-subunits of Streptomyces coelicolor, suggesting that it might be an ε-subunit of the mycobacterial acyl-CoA carboxylase. To test this hypothesis, the carboxylase subunits were expressed in Escherichia coli and purified. Acyl-CoA carboxylase activity was successfully reconstituted for the first time from purified subunits of the acyl-CoA carboxylase of M. tuberculosis. The reconstituted α3-β5 showed higher activity with propionyl-CoA than with acetyl-CoA, and the addition of the ε-subunit stimulated the carboxylation by 3.2- and 6.3-fold, respectively. The α3-β4 showed very low activity with the above substrates but carboxylated long chain acyl-CoA. This ε-subunit contains five sets of tandem repeats at the N terminus that are required for maximal enhancement of carboxylase activity. The Rv3281 open reading frame is co-transcribed with Rv3280 in the mycobacterial cell, and the level of ε-protein was highest during the log phase and decreased during the stationary phase.

Although the 38-kDa glycolipoprotein of Mycobacterium tuberculosis H37Rv is known to evoke prominent cellular and humoral immune responses in human tuberculosis (TB), little information is known about intracellular regulatory mechanisms involved in 38-kDa antigen (Ag)-induced host responses. In this study, we found that purified 38-kDa glycolipoprotein activates mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38) and induces tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in human monocytes. When the 38-kDa Ag was applied to monocytes from TB patients and healthy controls, the activation of ERK1/2 and p38 MAPK and the subsequent cytokine secretion were greater in the monocytes from the active pulmonary TB patients than in monocytes from the healthy controls. Additionally, neutralizing antibodies for Toll-like receptor 2 (TLR2) or TLR4 significantly reduced the ERK1/2 and p38 activation induced by the 38-kDa protein when the antibodies were applied to HEK293 cells overexpressing TLR2 or TLR4 as well as human primary monocytes. Furthermore, the inhibition of TLR2 significantly, and that of TLR4 partially, decreased the 38-kDa Ag-induced secretion of TNF-α and IL-6 in human monocytes. The intact protein moieties of the 38-kDa protein were responsible for biologic activities by this Ag. These data collectively demonstrate that the 38-kDa glycolipoprotein, acting through both TLR2 and TLR4, induces the activation of the ERK1/2 and p38 MAPK pathways, which in turn play an essential role in TNF-α and IL-6 expression during mycobacterial infection.

Although Mycobacterium marinum is closely related to Mycobacterium tuberculosis H37Rv genomically, the clinical outcome in humans is quite different for M. marinum and M. tuberculosis infections. We investigated possible factors in the host macrophages for determining differential pathological responses to M. tuberculosis and M. marinum using an in vitro model of mycobacterial infection. Using suppression-subtractive hybridization, we identified 12 differentially expressed genes in the human monocytic cell line U937 infected with M. tuberculosis and M. marinum. Of those genes, the most frequently recovered transcript encoded interleukin-8 (IL-8). Northern hybridization revealed that IL-8 mRNA was highly upregulated in M. tuberculosis-infected U937 cells compared with M. marinum-infected cells. In addition, enzyme-linked immunosorbent assay showed that IL-8 protein secretion was significantly elevated in M. tuberculosis-infected U937 cells, human primary monocytes, and monocyte-derived macrophages compared with that in M. marinum-infected cells. The depressed IL-8 expression was unique in M. marinum-infected cells compared with cells infected with other strains of mycobacteria, including M. tuberculosis H37Ra, Mycobacterium bovis BCG, or Mycobacterium smegmatis. When the expression of NF-κB was assessed in mycobacterium-infected U937 cells, IκBα proteins were significantly degraded in M. tuberculosis-infected cells compared with M. marinum-infected cells. Collectively, these results suggest that differential IL-8 expression in human macrophages infected with M. tuberculosis and M. marinum may be critically associated with distinct host responses in tuberculosis. Additionally, our data indicate that differential signal transduction pathways may underlie the distinct patterns of IL-8 secretion in cells infected by the two mycobacteria.

The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-14C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

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-γ), 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-γ 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.

The three proteins of the antigen 85 complex (85A, 85B, and 85C), which are major secretory products of Mycobacterium tuberculosis, were purified to homogeneity in large amounts by a combination of chromatography on hydroxylapatite, DEAE-Sepharose, and DEAE-Sephacel and gel filtration from M. tuberculosis culture filtrate. Then we examined the immunological reactivity of the three proteins in tuberculosis patients and healthy controls. Antibody responses to the 85B and 85A proteins in patients were significantly greater than responses to the 85C protein. In contrast, all three antigens induced significant lymphoproliferation and gamma interferon production in peripheral blood mononuclear cells from healthy tuberculin reactors.