Improved diagnostic evaluation of suspected tuberculosis.

BACKGROUND: The role of new T-cell-based blood tests for tuberculosis in the diagnosis of active tuberculosis is unclear. OBJECTIVE: To compare the performance of 2 interferon-gamma assays and tuberculin skin testing in adults with suspected tuberculosis. DESIGN: Prospective study conducted in routine practice. SETTING: 2 urban hospitals in the United Kingdom. PATIENTS: 389 adults, predominantly of South Asian and black ethnicity, with moderate to high clinical suspicion of active tuberculosis. INTERVENTION: Tuberculin skin testing, the enzyme-linked immunospot assay (ELISpot) incorporating early secretory antigenic target-6 and culture filtrate protein-10 (standard ELISpot), and ELISpot incorporating a novel antigen, Rv3879c (ELISpot(PLUS)) were performed during diagnostic assessment by independent persons who were blinded to results of the other test. MEASUREMENTS: Sensitivity, specificity, predictive values, and likelihood ratios. RESULTS: 194 patients had a final diagnosis of active tuberculosis, of which 79% were culture-confirmed. Sensitivity for culture confirmed and highly probable tuberculosis was 89% (95% CI, 84% to 93%) with ELISpot(PLUS), 85% (CI, 79% to 90%) with standard ELISpot, 79% (CI, 72% to 85%) with 15-mm threshold tuberculin skin testing, and 83% (CI, 77% to 89%) with stratified thresholds of 15 and 10 mm in vaccinated and unvaccinated patients, respectively. The ELISpot(PLUS) assay was more sensitive than tuberculin skin testing with 15-mm cutoff points (P = 0.01) but not with stratified cutoff points (P = 0.10). The ELISpot(PLUS) assay had 4% higher diagnostic sensitivity than standard ELISpot (P = 0.02). Combined sensitivity of ELISpot(PLUS) and tuberculin skin testing was 99% (CI, 95% to 100%), conferring a negative likelihood ratio of 0.02 (CI, 0 to 0.06) when both test results were negative. LIMITATIONS: Local standards for tuberculin skin testing differed from others used internationally. The study sample included few immunosuppressed patients. CONCLUSION: The ELISpot(PLUS) assay is more sensitive than standard ELISpot and, when used in combination with tuberculin skin testing, enables rapid exclusion of active infection in patients with moderate to high pretest probability of tuberculosis.

Dynamic relationship between IFN-gamma and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load.

Distinct IFN-gamma and IL-2 profiles of Ag-specific CD4(+) T cells have recently been associated with different clinical disease states and Ag loads in viral infections. We assessed the kinetics and functional profile of Mycobacterium tuberculosis Ag-specific T cells secreting IFN-gamma and IL-2 in 23 patients with untreated active tuberculosis when bacterial and Ag loads are high and after curative treatment, when Ag load is reduced. The frequencies of M. tuberculosis Ag-specific IFN-gamma-secreting T cells declined during 28 mo of follow-up with an average percentage decline of 5.8% per year (p = 0.005), while the frequencies of Ag-specific IL-2-secreting T cells increased during treatment (p = 0.02). These contrasting dynamics for the two cytokines led to a progressive convergence of the frequencies of IFN-gamma- and IL-2-secreting cells over 28 mo. Simultaneous measurement of IFN-gamma and IL-2 secretion at the single-cell level revealed a codominance of IFN-gamma-only secreting and IFN-gamma/IL-2 dual secreting CD4(+) T cells in active disease that shifted to dominance of IFN-gamma/IL-2-secreting CD4(+) T cells and newly detectable IL-2-only secreting CD4(+) T cells during and after treatment. These distinct T cell functional signatures before and after treatment suggest a novel immunological marker of mycobacterial load and clinical status in tuberculosis that now requires validation in larger prospective studies.

Regulatory T cells are expanded in blood and disease sites in patients with tuberculosis.

RATIONALE: T-cell responses during tuberculosis (TB) help contain Mycobacterium tuberculosis in vivo but also cause collateral damage to host tissues. Immune regulatory mechanisms may limit this immunopathology, and suppressed cellular immune responses in patients with TB suggest the presence of regulatory activity. CD4+CD25(high) regulatory T cells mediate suppressed cellular immunity in several chronic infections but have not been described in TB. OBJECTIVE: To determine whether regulatory T cells are increased in patients with TB and whether they suppress cellular immune responses. METHODS: We compared the frequency of circulating regulatory T cells in 27 untreated patients with TB and 23 healthy control subjects using two specific markers: cell-surface CD25 expression and FoxP3 mRNA expression in peripheral blood mononuclear cells. MEASUREMENTS AND MAIN RESULTS: We detected a threefold increase in the frequency of CD4 + CD25(high) T cells (p < 0.001) and a 2.2-fold increase in FoxP3 expression (p = 0.006) in patients with TB, and there was a positive correlation between these markers (r = 0.58, p < 0.001). Increased expression of interleukin-10 and transforming growth factor-beta1 mRNA was also detected in patients with TB but did not correlate with regulatory T-cell markers. Ex vivo depletion of CD4 + CD25(high) cells from peripheral blood mononuclear cells resulted in increased numbers of M. tuberculosis antigen-specific IFN-gamma-producing T cells in seven of eight patients with TB (p = 0.005). Finally, FoxP3 expression was increased 2.3-fold in patients with extrapulmonary TB compared with patients with purely pulmonary TB (p = 0.01) and was amplified 2.6-fold at disease sites relative to blood (p = 0.043). CONCLUSIONS: Regulatory T cells are expanded in patients with TB and may contribute to suppression of Th1-type immune responses.

Determination of minimum herpes simplex virus type 1 components necessary to localize transcriptionally active DNA to ND10.

DNA viruses such as herpes simplex virus type 1 (HSV-1) appear to start their replicative processes at specific nuclear domains known as ND10. In analyses to determine the minimum viral components needed for transcript accumulation at ND10, we find that a specific viral DNA sequence, OriS, and the viral immediate-early proteins ICP4 and ICP27 are sufficient for a reporter gene placed in cis to the OriS sequence to transcribe at ND10. A chromatin immunoprecipitation assay demonstrated expected critical intermediates in retaining the minimal genome at ND10 for the HSV-1 replication origin through direct or indirect binding to the host protein Daxx. Coimmunoprecipitation assays with antibodies to Daxx and ICP4, ICP27, and ICP8 showed that the respective proteins interact, possibly forming a complex. A potential complex between the origin, early viral DNA-binding protein ICP8 and Daxx did not result in transcription at ND10. Thus, the deposition of transcriptionally active HSV-1 genomes at ND10 is most likely a consequence of retention at ND10 through the interaction of viral genome-bound ICP4 and ICP27 with Daxx. Such a complex might be more likely immobilized at the outside of ND10 by the PML-interacting Daxx than at other nuclear sites.

Interaction of PRMT1 with BTG/TOB proteins in cell signalling: molecular analysis and functional aspects.

BACKGROUND: Several recent reports have connected protein methylation with differentiation. Furthermore, the BTG/TOB proteins have also been implicated in such control. BTG1 and 2 have been shown to interact with PRMT1 (predominant cellular arginine N-methyltransferase of type I). RESULTS: First, we have studied the interaction between PRMT1 and the proteins of the BTG/TOB family. We show that boxC, a sequence present only in BTG1 and BTG2, is essential for this association. Using boxC peptide, we have investigated the importance of PRMT1/BTG protein association during type I protein methylation reactions. Finally, we show that the addition of boxC fused to penetratin interferes with the neuronal differentiation of PC12 cells and ES cell-derived neurones. CONCLUSIONS: Taken together, these results indicate that PRMT1/BTG proteins could play a key role in the arginine methylation-mediated signalling pathway as well as in neuronal differentiation.