Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.

Streptococcus pneumoniae (S. pneumoniae) causes high early mortality in pneumococcal pneumonia, which is characterized by acute lung injury (ALI). The molecular mechanisms underlying ALI and the high early mortality remain unknown. Despite recent studies that identify deubiquitinating enzyme cylindromatosis (CYLD) as a key regulator for T cell development, tumor cell proliferation, and NF-kappaB transcription factor signaling, its role in regulating bacteria-induced lethality, however, is unknown. Here, we showed that CYLD deficiency protected mice from S. pneumoniae pneumolysin (PLY)-induced ALI and lethality. CYLD was highly induced by PLY, and it inhibited MKK3-p38 kinase-dependent expression of plasminogen activator inhibitor-1 (PAI-1) in lung, thereby potentiating ALI and mortality. Thus, CYLD is detrimental for host survival, thereby indicating a mechanism underlying the high early mortality of pneumococcal pneumonia.

HBsAg and anti-HBs with an unmatched specificity coexisted in 4.9% of patients. The presence of anti-HBs was not associated with the appearance of specific HBV mutants in patients with chronic infection. Apparently, the presence of anti-HBs in patients with chronic HBV infection did not lead to a selection of HBV escape mutants.

Information remains sparse about non-HIV patients with cryptococcal meningitis in the era of triazole therapy. Particularly of interest are the clinical manifestations and prognosis of the infection in these previously healthy patients. We retrospectively reviewed 154 non-HIV-infected patients with cryptococcal meningitis who presented in our hospital from 1997 to 2007. We compared the clinical features and outcomes between predisposed and otherwise healthy hosts. The number of cases per year showed a steady increase over time. The majority of patients were otherwise apparently healthy (103 patients, 66.9%) and predisposing factors were identified in only 51 (33.1%) patients. Corticosteroid medication accounted for the most common underlying factor in these cases (n = 21). Morbidity was appallingly high, with seizures in 28.6%, cranial nerves palsies in 51.5% and cerebral herniation in 19.5%. Despite these complications, overall mortality during 1 year was 28.7% (41/143), close to that reported from other centers with non-HIV patients. Death attributed to cryptococcosis occurred in 19.6% (28/143) patients with most receiving amphotericin B as a component of their initial therapy. Among surviving patients who had lumbar punctures at weeks 2 and 10, those given amphotericin B for initial therapy achieved higher rates of overall response than those receiving initial fluconazole therapy at either week 2 (84.4% of 96 patients vs. 33.3% of 24 patients, P <0.001) or week 10 (85.0% of 93 patients vs. 66.7% of 24 patients, P = 0.041). In multivariate analysis, coma, cerebral herniation, and initial antifungal therapy without amphotericin B were independently correlated with both increased overall and attributable mortality, while advanced age (>/= 60 years) was correlated with increased overall mortality only. Patients with apparently normal immune status were overall younger than those who were immunocompromised. In addition, previously healthy patients for whom diagnosis was delayed had more severe disease, experiencing more brain herniation, coma, seizures, hydrocephalus and more surgical shunt procedures. On the other hand, immunocompromised patients were more commonly found to have high fever and brain parenchymal involvement. However, both groups had a similar treatment response and 1-year survival.

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BackgroundHumans infected with Mycobacterium tuberculosis (MTB) can delete the pathogen or otherwise become latent infection or active disease. However, the factors influencing the pathogen clearance and disease progression from latent infection are poorly understood. This study attempted to use a genome-wide transcriptome approach to identify immune factors associated with MTB infection and novel biomarkers that can distinguish active disease from latent infection.Methodology/Principal FindingsUsing microarray analysis, we comprehensively determined the transcriptional difference in purified protein derivative (PPD) stimulated peripheral blood mononuclear cells (PBMCs) in 12 individuals divided into three groups: TB patients (TB), latent TB infection individuals (LTBI) and healthy controls (HC) (n = 4 per group). A transcriptional profiling of 506 differentially expressed genes could correctly group study individuals into three clusters. Moreover, 55- and 229-transcript signatures for tuberculosis infection (TB<BI) and active disease (TB) were identified, respectively. The validation study by quantitative real-time PCR (qPCR) performed in 83 individuals confirmed the expression patterns of 81% of the microarray identified genes. Decision tree analysis indicated that three genes of CXCL10, ATP10A and TLR6 could differentiate TB from LTBI subjects. Additional validation was performed to assess the diagnostic ability of the three biomarkers within 36 subjects, which yielded a sensitivity of 71% and specificity of 89%.Conclusions/SignificanceThe transcription profiles of PBMCs induced by PPD identified distinctive gene expression patterns associated with different infectious status and provided new insights into human immune responses to MTB. Furthermore, this study indicated that a combination of CXCL10, ATP10A and TLR6 could be used as novel biomarkers for the discrimination of TB from LTBI.

BackgroundThe Mycobacterium tuberculosis (Mtb)-specific T-cell interferon gamma release assays (IGRAs) are useful in detecting Mtb infection but perform poorly at distinguishing active tuberculosis disease (ATB) and latent tuberculosis infection (LTBI). This study is aimed at evaluating additional cytokines as biomarkers besides interferon-gamma (IFN-γ) to improve the identification of ATB and LTBI.Methodology/Principal FindingsSixty-six patients with ATB, 73 household contacts (HHC) of ATB patients and 76 healthy controls (HC) were recruited to undergo QuantiFERON TB GOLD in-tube assay (QFT) and the enzyme-linked immunosorbent assay (ELISA) where the release of IFN-γ, IFN-γ inducible protein 10 (IP-10), Interleukin 2 (IL-2) and Tumor Necrosis Factor-α (TNF-α) was determined in the whole blood with or without antigen-stimulation. The positive rates of the QFT, IP-10 and IL-2 tests were 86.4%, 89.4% and 86.4% for the ATB group with no difference between them (p>0.05). However, QFT in combination with IP-10 and IL-2 significantly increased the detection rate to 95.5% in the ATB group (p = 0.03) and the indeterminate rate of all samples decreased from 2.3% (5/215) to 0.4% (1/215). The un-stimulated level of IP-10 was significantly higher in the HHC than the ATB and HC groups. The IP-10 responses were strongly associated with extended Mtb exposure time and the degree of smear-positivity of the index cases. The IL-2/IFN-γ ratio in the antigen-stimulated plasma could discriminate LTBI from ATB with a sensitivity of 77.2% and a specificity of 87.2%.ConclusionThe increased Mtb-specific antigen-stimulated expression of IP-10 and IL-2 may be useful for detecting both ATB and LTBI. Combining the QFT with IP-10 and IL-2 could increase the detection accuracy of active TB over the QFT alone.

The aim of this study was to establish reference ranges for lymphocyte subsets in Chinese adults. Venous blood specimens were obtained from 614 healthy, human immunodeficiency virus (HIV)-seronegative adults in Shanghai. Flow cytometry was used to determine percentages and absolute numbers of CD4 and CD8 T lymphocytes. Mean values for CD4 and CD8 lymphocytes were 727 and 540 cells/l, respectively, yielding a CD4/CD8 ratio of 1.49. While CD8 lymphocyte values varied with age and gender, no significant differences in CD4 lymphocyte values were observed. Shanghai adults had approximately 100 fewer CD4 lymphocytes/l on average than Caucasians, suggesting that lower CD4 lymphocyte cutoffs for classifying and monitoring HIV infection may be needed in China. Measurements of CD4ϩ lymphocytes are essential for assessing human immunodeficiency virus (HIV) disease course, clinical staging, epidemiological studies, and decisions regarding prophylactic therapies against opportunistic infections (2,3,8,9,21). Only in industrialized countries has it been feasible to routinely monitor CD4 lymphocyte subsets during routine HIV clinical care. In China, HIV has spread to all 31 provinces, regions, and municipalities and is currently moving into new segments of the populace (22). It is estimated that more than 1 million people are infected in China, and this number may reach 10 million by 2010 (11). Information is generally lacking on the normal range of lymphocyte subpopulations, including CD4 and CD8 lymphocytes, in China.To provide normal ranges for CD4 and CD8 lymphocyte subsets, and for CD4/CD8 ratios in normal Chinese adults, blood specimens were collected from healthy native adult residents of Shanghai who received routine annual health evaluations at Huashan Hospital between August 2000 and February 2001. Subjects were excluded if they were diagnosed with HIV type 1 (HIV-1) infection or other recent viral or bacterial infections or chronic organ diseases, were immunocompromised, or were recently exposed to toxic chemicals. Wholeblood samples were collected using sterile EDTA Vacutainer tubes. The Science and Research Bureau of Fudan University approved the study.Flow cytometry of lymphocyte subsets was carried out using a lamp-based flow cytometer (Bryte-HS, Bio-Rad, Hercules, Calif.) according to the manufacturer's instructions. Briefly, white blood cell counting and differentiation were performed using a Symex-SF3000 Coulter counter (Coulter Electronic, Luton, London). Blood samples were then stained using OptiClone CD4/CD8, immunoglobulin G1-fluorescein isothiocyanate, and immunoglobulin G1-phycoerythrin monoclonal antibodies (Coulter-Immunotech, Miami, Florida). The monoclonal antibodies, 13B8.2 and B9.11, were used to bind specifically to CD4 and CD8 subsets of peripheral blood T lymphocytes, respectively (7, 17). The determination of positive and negative cells for any combination of reagents was set with directly conjugated antibodies of irrelevant specificity as negative controls. Positive and negative controls were...