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1.  Association of visceral obesity and early colorectal neoplasia 
AIM: To examine whether visceral adipose tissue (VAT) serves as a risk factor for colorectal adenoma-early colorectal cancer (CRC) sequence.
METHODS: A retrospective case-control study was conducted with 153 patients with stage I CRC, age/sex-matched 554 patients with colorectal adenoma and 557 normal controls. All subjects underwent various laboratory tests, abdominal fat computed tomography (CT), and colonoscopy. VAT was defined as an intra-abdominal adipose tissue area measured by CT scan. Adipose tissue area was measured at the level of the umbilicus from CT scan. We used the lowest quartile of VAT and subcutaneous adipose tissue area as a reference group.
RESULTS: The body mass index (BMI), total cholesterol, fasting glucose and VAT areas were significantly different among normal, adenoma and CRC groups. The VAT area was 120.6 ± 49.0 cm2 in normal controls, 130.6 ± 58.4 cm2 in adenoma group and 117.6 ± 51.6 cm2 in CRC group (P = 0.002). In univariate analysis, increased BMI was a risk factor for CRC compared to control (P = 0.025). However, VAT area was not a risk factor for CRC compared to control. In multivariate analysis that adjusted for smoking, alcohol consumption and subcutaneous adipose tissue area, VAT area was inversely related to CRC, compared to the adenoma (OR = 0.53, 95%CI: 0.31-0.92, highest quartile vs lowest quartile).
CONCLUSION: Our study shows that visceral obesity is not a risk factor for early CRC. Visceral obesity might influence the normal-adenoma sequence but not the adenoma-early carcinoma sequence.
doi:10.3748/wjg.v19.i45.8349
PMCID: PMC3857459  PMID: 24363527
Adipose tissue; Visceral fat; Obesity; Colorectal cancer; Colorectal adenoma; Abdominal computed tomography
2.  Serodiagnostic Potential of Mycobacterium avium MAV2054 and MAV5183 Proteins 
The Mycobacterium avium-M. intracellulare complex (MAC) causes a pulmonary disease (PD) similar to tuberculosis (TB). Diagnosis of MAC-PD is complicated and time-consuming. In this study, the serodiagnostic potential of the newly identified MAV2054 and MAV5183 proteins was evaluated in subjects with MAC-PD, pulmonary TB, or latent TB and in noninfected healthy controls (HC), together with HspX and the 38-kDa antigen, well-known serodiagnostic M. tuberculosis antigens. All four antigens evoked significantly higher IgG responses in MAC-PD and active TB than in latent TB and HC subjects. Among the antigens, MAV2054 elicited the highest antibody responses in pulmonary TB and MAC-PD patients. IgG titers against MAV2054 and MAV5183 were significantly higher in MAC-PD than in pulmonary TB subjects. In addition, the levels of IgG against all antigens in the M. intracellulare and fibrocavitary forms were higher than those in the M. avium and nodular bronchiectatic forms, respectively. Based on sensitivity and receiver operator characteristic curve analysis, the best candidates for detection of MAC-PD and pulmonary TB were MAV2054 and the 38-kDa antigen, respectively. In total, 76.0% of MAC-PD and 65.0% of active TB patients were reactive to at least two antigens. In contrast, only 2.8% of HC subjects were reactive with two or more antigens. Our findings suggest that an enzyme-linked immunosorbent assay (ELISA) using the four antigens would be valuable for screening for mycobacterial lung disease, including MAC-PD and pulmonary TB, although it does not provide good discrimination of the disease-causing pathogens.
doi:10.1128/CVI.00649-12
PMCID: PMC3571277  PMID: 23269416
3.  Therapeutic Effects of Mycobacterial Secretory Proteins Against Established Asthma in BALB/c Mice 
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.
doi:10.4168/aair.2012.4.4.214
PMCID: PMC3378928  PMID: 22754715
Ag85 complex; asthma; MPB70; mycobacteria; 38-kDa protein
4.  Improved Sensitivity of Diagnosis of Tuberculosis in Patients in Korea via a Cocktail Enzyme-Linked Immunosorbent Assay Containing the Abundantly Expressed Antigens of the K Strain of Mycobacterium tuberculosis▿  
Clinical and Vaccine Immunology : CVI  2008;15(12):1788-1795.
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.
doi:10.1128/CVI.00231-08
PMCID: PMC2593165  PMID: 18945883
5.  Mycobacterium tuberculosis HBHA Protein Reacts Strongly with the Serum Immunoglobulin M of Tuberculosis Patients 
Clinical and Vaccine Immunology  2006;13(8):869-875.
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.
doi:10.1128/CVI.00103-06
PMCID: PMC1539112  PMID: 16893986
6.  Reliability of the Percent Density in Digital Mammography with a Semi-Automated Thresholding Method 
Journal of Breast Cancer  2014;17(2):174-179.
Purpose
The reliability of the quantitative measurement of breast density with a semi-automated thresholding method (Cumulus™) has mainly been investigated with film mammograms. This study aimed to evaluate the intrarater reproducibility of percent density (PD) by Cumulus™ with digital mammograms.
Methods
This study included 1,496 craniocaudal digital mammograms from the unaffected breast of breast cancer patients. One rater reviewed each mammogram and estimated the PD using the Cumulus™ method. All images were reassessed by the same rater 1 month later without reference to the previously assigned values. The repeatability of the PD was evaluated by an intraclass correlation coefficient (ICC). All patients were grouped based on their body mass index (BMI), age, family history of breast cancer, breastfeeding history and breast area (calculated with Cumulus™), and subgroup analysis for the ICC of each group was performed. All patients were categorized by their Breast Imaging Reporting and Data System (BI-RADS) density pattern, and the mean and standard deviation of the PD by each BI-RADS categories were compared.
Results
The ICC for the PD was 0.94, indicating excellent repeatability. The discrepancy between the paired PD values ranged from 0 to 23.93, with an average of 3.90 (standard deviation=3.39). The subgroup ICCs for the PD ranged from 0.88 to 0.96, indicating excellent reliability in all subgroups regardless of patient variables. The ICCs of the PD for the high-risk (BI-RADS 3 and 4) and low-risk (BI-RADS 1 and 2) groups were 0.90 and 0.88, respectively.
Conclusion
This study suggests that PD calculated with digital mammograms has an acceptable reliability regardless of patient age, BMI, family history of breast cancer, breastfeeding history, breast size, and BI-RADS density pattern.
doi:10.4048/jbc.2014.17.2.174
PMCID: PMC4090321  PMID: 25013440
Breast; Mammography; Observer variation
7.  Association between Non-invasive Fibrosis Markers and Mortality among Adults with Non-alcoholic Fatty Liver Disease in the United States 
Hepatology (Baltimore, Md.)  2013;57(4):1357-1365.
The clinical and public health significance of non-alcoholic fatty liver disease (NAFLD) is not well established. We investigate the long-term impact of NAFLD on mortality. This analysis utilizes the National Health and Nutrition Examination Survey conducted in 1988–1994 and subsequent follow-up data for mortality through December 31, 2006. NAFLD was defined by ultrasonographic detection of hepatic steatosis in the absence of other known liver diseases. The presence and severity of hepatic fibrosis in subjects with NAFLD was determined by the NAFLD fibrosis score (NFS), the AST-platelet ratio index (APRI), and the FIB-4 score. Out of 11,154 participants, 34.0% had NAFLD - the majority (71.7%) had NFS consistent with lack of significant fibrosis (NFS < −1.455), whereas 3.2% had a score indicative of advanced fibrosis (NFS > 0.676). After a median follow-up of 14.5 years, NAFLD was not associated with higher mortality (age- and sex-adjusted hazard ratio (HR) 1.05, 95% confidence interval (CI) 0.93–1.19). In contrast, there was a progressive increase in mortality with advancing fibrosis scores. Compared to subjects without fibrosis, those with a high probability of advanced fibrosis had a 69% increase in mortality (HR 1.69 (95% CI 1.09–2.63) for NFS, 1.85 (1.02–3.37) for APRI, 1.66 (0.98–2.82) for FIB-4) after adjustment for other known predictors of mortality. These increases in mortality were almost entirely from cardiovascular causes (HR 3.46 (95% CI 1.91–6.25) for NFS, 2.53 (1.33–4.83) for APRI, 2.68 (1.44–4.99) for FIB-4).
Conclusions
Ultrasonography-diagnosed NAFLD is not associated with increased mortality. However, advanced fibrosis as determined by non-invasive fibrosis marker panels is a significant predictor of mortality, mainly from cardiovascular causes, independent of other known factors.
doi:10.1002/hep.26156
PMCID: PMC3622816  PMID: 23175136
Hepatic steatosis; Fatty liver; National Health and Nutrition Examination Survey; Survival; Mortality
8.  Effect Modification of Hormonal Therapy by p53 Status in Invasive Breast Cancer 
Journal of Breast Cancer  2013;16(4):386-394.
Purpose
We aimed to confirm the prognostic and predictive value of p53 expression, particularly in invasive breast cancer patients, according to immunohistochemical hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status.
Methods
Immunohistochemical data for p53, estrogen receptor, progesterone receptor, and HER2 expression from a total of 15,598 patients were retrospectively retrieved from the web-based database of the Korean Breast Cancer Society. Overall survival (OS) and breast cancer-specific survival (BCSS) were calculated and compared using the Kaplan-Meier method and log-rank test, respectively. Multivariate analyses were performed using a stratified Cox proportional hazard regression model. A model evaluating interactions between p53 expression and both hormonal therapy and chemotherapy was used to determine the treatment benefit from both modalities.
Results
The prognostic value of p53 for OS and BCSS was most significant in the HR+/HER2- subgroup, with hazard ratios of 1.44 (95% confidence interval [CI], 1.08-1.93) and 1.47 (95% CI, 1.09-1.99), respectively. The p53 overexpression hazard ratios were of borderline significance for the HR+/HER2+ subgroup and were not significant for the HR-/HER2+ and HR-/HER2- subgroups. The model with interaction terms revealed that hormonal therapy significantly interacts with p53 status (p=0.002 and p=0.007 for OS and BCSS, respectively), suggesting an insignificant prognostic value for p53 status (p=0.268 and p=0.296 for OS and BCSS, respectively). An interaction between chemotherapy and p53 status was not found in this model.
Conclusion
p53 overexpression has independent prognostic value, particularly in cases of HR+/HER2- invasive breast cancer, which may be due to effect modification of hormonal therapy dependent on p53 status.
doi:10.4048/jbc.2013.16.4.386
PMCID: PMC3893340  PMID: 24454460
Breast neoplasms; Drug resistance; Tumor suppressor protein p53
9.  Inhalation Toxicity of Humidifier Disinfectants as a Risk Factor of Children’s Interstitial Lung Disease in Korea: A Case-Control Study 
PLoS ONE  2013;8(6):e64430.
Background
The occurrence of numerous cases of interstitial lung disease in children (chILD) every spring in Korea starting in 2006 raised suspicion about a causal relationship with the use of humidifier disinfectants (HDs). The aim of this study was to evaluate the association between HD use and the risk of chILD.
Methods
This retrospective, 1∶3 matched case-control study consisted of 16 cases of chILD that had developed between 2010 and 2011. The three groups of parallel controls (patients with acute lobar pneumonia, asthma, and healthy children) were matched by age, gender, and index date. Indoor/outdoor environmental risk factors, including HD use, were investigated by asking the guardians to complete a questionnaire.
Results
The median age of the affected children (43.8% male) was 26 months (18.25–36.25). The chILD group did not differ significantly from the control groups with respect to socio-demographic and clinical variables. Indoor and outdoor environmental factors were not associated with a risk of chILD. However, the previous use of HDs (OR; 2.73. 95% CI; 1.41–5.90, P = 0.00) were independently associated with an increased risk.
Conclusions
This study showed that HDs, which are widely used in South Korea in the winter season, independently increased the risk of chILD in spring. Therefore, continuous monitoring and, if needed, changes in policy are essential to prevent and control pediatric diseases caused by toxic chemicals.
doi:10.1371/journal.pone.0064430
PMCID: PMC3673956  PMID: 23755124
10.  Mapping EORTC QLQ-C30 onto EQ-5D for the assessment of cancer patients 
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.
doi:10.1186/1477-7525-10-151
PMCID: PMC3542092  PMID: 23244763
EQ-5D; EORTC QLQ-C30; Cancer; Mapping; Quality of life
11.  Utilization of evidence-based treatment in elderly patients with chronic heart failure: using Korean Health Insurance claims database 
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.
doi:10.1186/1471-2261-12-60
PMCID: PMC3468388  PMID: 22849621
Congestive heart failure; Drug utilization evaluation; Elderly; Type 2 angiotensin receptor antagonists; Angiotensin-converting enzyme antagonists; Beta-adrenergic blockers
12.  Label-Free Electrochemical Diagnosis of Viral Antigens with Genetically Engineered Fusion Protein 
Sensors (Basel, Switzerland)  2012;12(8):10097-10108.
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.
doi:10.3390/s120810097
PMCID: PMC3472818  PMID: 23112590
hepatitis B virus; Gold-binding polypeptide; fusion protein; electrochemical analysis
13.  Endoplasmic Reticulum Stress Pathway-Mediated Apoptosis in Macrophages Contributes to the Survival of Mycobacterium tuberculosis 
PLoS ONE  2011;6(12):e28531.
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.
doi:10.1371/journal.pone.0028531
PMCID: PMC3237454  PMID: 22194844
14.  Targeting of Mycobacterium tuberculosis Heparin-Binding Hemagglutinin to Mitochondria in Macrophages 
PLoS Pathogens  2011;7(12):e1002435.
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.
doi:10.1371/journal.ppat.1002435
PMCID: PMC3234249  PMID: 22174691
15.  Prediction of Treatment Outcome with Bioimpedance Measurements in Breast Cancer Related Lymphedema Patients 
Annals of Rehabilitation Medicine  2011;35(5):687-693.
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.
doi:10.5535/arm.2011.35.5.687
PMCID: PMC3309267  PMID: 22506192
Breast cancer related lymphedema; Bioelectrical impedance spectroscopy; Single frequency bioimpedance analysis
16.  Efficient Differentiation of Mycobacterium abscessus Complex Isolates to the Species Level by a Novel PCR-Based Variable-Number Tandem-Repeat Assay▿ 
Journal of Clinical Microbiology  2011;49(3):1107-1109.
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.
doi:10.1128/JCM.02318-10
PMCID: PMC3067748  PMID: 21177894
17.  High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Isolates ▿  
Journal of Clinical Microbiology  2010;48(11):3893-3898.
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.
doi:10.1128/JCM.00396-10
PMCID: PMC3020879  PMID: 20844231
18.  Mycobacterial Heparin-binding Hemagglutinin Antigen Activates Inflammatory Responses through PI3-K/Akt, NF-κB, and MAPK Pathways 
Immune Network  2011;11(2):123-133.
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.
doi:10.4110/in.2011.11.2.123
PMCID: PMC3100523  PMID: 21637390
Mycobacterium tuberculosis; Heparin-binding hemagglutinin (HBHA); Macrophages; MAPK; PI3-K; NF-κB
19.  Serum Globotriaosylceramide Assay as a Screening Test for Fabry Disease in Patients with ESRD on Maintenance Dialysis in Korea 
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.
doi:10.3904/kjim.2010.25.4.415
PMCID: PMC2997971  PMID: 21179280
Fabry disease; Globotriaosylceramide; End-stage renal disease
20.  Preventive effects of mycobacteria and their culture supernatants against asthma development in BALB/c mice 
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.
doi:10.4168/aair.2010.2.1.34
PMCID: PMC2831609  PMID: 20224676
Asthma; BCG vaccine; interferons; interleukins; mycobacterium
21.  Pilot Study of Diagnostic Potential of the Mycobacterium tuberculosis Recombinant HBHA Protein in a Vaccinated Population in Finland 
PLoS ONE  2008;3(9):e3272.
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.
doi:10.1371/journal.pone.0003272
PMCID: PMC2533402  PMID: 18815611
22.  Development of Amperometric Hydrogen Peroxide Sensor Based on Horseradish Peroxidase-Immobilized Poly(Thiophene-co-EpoxyThiophene) 
Sensors (Basel, Switzerland)  2008;8(7):4110-4118.
A modified electrode for hydrogen peroxide (H2O2) sensing was prepared via thiophene (Th) with epoxy group. Thiophene (EpoxyTh) with epoxy group was synthesized by reaction of 3-bromothiophene and glycidyl methacrylate (GMA) in acetonitrile according to Heck Reaction. The electrocopolymerization of Th and EpoxyTh was performed on the surface of indium tin oxide (ITO) electrode by cycling the potential between -1.0 and +2.5 V in mixture of thiophene (Th) and EpoxyTh. Poly(Th-co-EpoxyTh) grown onto the ITO electrode was successfully confirmed by SEM, AFM, and water contact angle analysis, respectively. Finally, the HRP was immobilized on the surface of poly(Th-co-EpoxyTh) electrode by covalent binding. The amperometric response of the HRP-immobilized poly(Th-co-EpoxyTh) electrode for H2O2 was examined by cyclic voltammetry (CV). The HRP-immobilized poly(Th-co-EpoxyTh) electrode showed linearity from 0.1 to 30 mM H2O2, good reproducibility, and long life time.
doi:10.3390/s8074110
PMCID: PMC3697164
Poly(Th-co-EpoxyTh) electrode; Horseradish peroxidase; Cyclic voltammetry; H2O2
23.  Identification and Characterization of Rv3281 as a Novel Subunit of a Biotin-dependent Acyl-CoA Carboxylase in Mycobacterium tuberculosis H37Rv* 
The Journal of biological chemistry  2005;281(7):3899-3908.
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.
doi:10.1074/jbc.M511761200
PMCID: PMC1523427  PMID: 16354663
24.  The Mycobacterial 38-Kilodalton Glycolipoprotein Antigen Activates the Mitogen-Activated Protein Kinase Pathway and Release of Proinflammatory Cytokines through Toll-Like Receptors 2 and 4 in Human Monocytes  
Infection and Immunity  2006;74(5):2686-2696.
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.
doi:10.1128/IAI.74.5.2686-2696.2006
PMCID: PMC1459749  PMID: 16622205
25.  Interleukin-8 Is Differentially Expressed by Human-Derived Monocytic Cell Line U937 Infected with Mycobacterium tuberculosis H37Rv and Mycobacterium marinum  
Infection and Immunity  2003;71(10):5480-5487.
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.
doi:10.1128/IAI.71.10.5480-5487.2003
PMCID: PMC201049  PMID: 14500465

Results 1-25 (28)