Apoptosis is one way of controlling immune responses, and a variety of immunosuppressive drugs suppress harmful immune responses by inducing apoptosis of lymphocytes. In this study we observed that rosmarinic acid, a secondary metabolite of herbal plants, induced apoptosis in an p56lck (Lck)-dependent manner; Lck+ Jurkat T cells undergo apoptosis in response to rosmarinic acid (RosA) treatment, whereas Lck− Jurkat subclone J.CaM1.6 cells do not. J.CaM1.6 cells with various Lck mutants indicated that Lck SH2 domain, but not Lck kinase activity, was required for RosA-induced apoptosis. RosA induced apoptosis in the absence of a TCR stimulus, and this was not prevented by interruption of the Fas/Fas ligand interaction. Instead, RosA-mediated apoptosis involved a mitochondrial pathway as indicated by cytochrome c release and the complete blockage of apoptosis by an inhibitor of mitochondrial membrane depolarization. Both caspase-3 and -8 were indispensable in RosA-induced apoptosis and work downstream of mitochondria and caspase-9 in the order of caspase-9/caspase-3/caspase-8. In freshly isolated human PBMC, RosA specifically induced apoptosis of Lck+ subsets such as T and NK cells, but not Lck-deficient cells, including B cells and monocytes. Moreover, RosA’s ability to kill T and NK cells was restricted to actively proliferating cells, but not to resting cells. In conclusion, Lck-dependent apoptotic activity may make RosA an attractive therapeutic tool for the treatment of diseases in which T cell apoptosis is beneficial.

f Mycobacterium tuberculosis is the major causative agent of tuberculosis (TB). The gamma interferon (IFN-␥) release assay (IGRA) has been widely used to diagnose TB by testing cell-mediated immune responses but has no capacity for distinguishing between active TB and latent TB infection (LTBI). This study aims to identify a parameter that will help to discriminate active TB and LTBI. Whole-blood samples from 33 active TB patients, 20 individuals with LTBI, and 26 non-TB controls were applied to the commercial IFN-␥ release assay, QuantiFERON-TB Gold In-Tube, and plasma samples were analyzed for interleukin-2 (IL-2), IL-6, IL-8, IL-10, IL-13, tumor necrosis factor-alpha (TNF-␣), IFN-␥, monokine induced by IFN-␥ (MIG), interferon gamma inducible protein 10 (IP-10), interferon-inducible T cell alpha chemoattractant (I-TAC), and monocyte chemoattractant protein 1 (MCP-1) by using a commercial cytometric bead array. The Mycobacterium tuberculosis antigen-specific production of most of the assayed cytokines and chemokines was higher in the active TB than in the LTBI group. The mitogen-induced responses were lower in the active TB than in the LTBI group. When the ratio of TB-specific to mitogen-induced responses was calculated, IL-2, IL-6, IL-10, IL-13, TNF-␣, IFN-␥, MIG, and IP-10 were more useful in discriminating active TB from LTBI. In particular, most patients showed higher IP-10 production to Mycobacterium tuberculosis antigens than to mitogen at the individual level, and the ratio for IP-10 was the strongest indicator of active infection versus LTBI with 93.9% sensitivity and 90% specificity. In conclusion, the ratio of the TB-specific to the mitogen-induced IP-10 responses showed the most promising accuracy for discriminating active TB versus LTBI and should be further studied to determine whether it can serve as a biomarker that might help clinicians administer appropriate treatments. M ycobacterium tuberculosis, the major causative agent for tuberculosis (TB), is among the most successful human pathogens, infecting approximately 8.6 million people and leading to 1.3 million deaths each year (1). It is estimated that 2 billion people live with latent TB infection (LTBI) and are therefore a potential source of active TB (2, 3). Identifying LTBI is necessary in order to reduce the risk of development of the disease, while diagnosis of active TB can enable rapid treatment and disease control. To this end, diagnostic biomarkers that can accurately indicate disease status are needed (4, 5).There is presently no diagnostic gold standard for LTBI. Until recently, the tuberculin skin test (TST) involving the intracutaneous injection of purified protein derivative (PPD) into the forearm was the only available method for diagnosing LTBI. However, PPD cross-reacts with nontuberculous mycobacteria as well as with Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine and has poor sensitivity in immunocompromised patients (6). The interferon gamma (IFN-␥) release assay (IGRA) has been widely used in clinical practice a...

Serum IL-2, IL-9, IL-13, IL-17, TNF-α, sCD40L and VEGF-A levels may be adjunctive biomarkers for differential diagnosis of active TB, LTBI, and NTM disease. Assessment of serum sCD40L and M. tb antigen-specific IFN-γ, TNF-α, and IL-2 levels could help predict successful anti-TB treatment in conjunction with M. tb clearance.

Lck is a T cell-restricted Src family protein tyrosine kinase that plays pivotal roles in TCRmediated signaling. We aimed to identify novel agents that could disrupt the molecular interaction of the Src homology 2-domain of Lck (Lck SH2) with its binding partners, with the expectation that this would impair TCR signaling and generate immunosuppression. Largescale screening of plant extracts indicated that rosmarinic acid (RosA) in extracts of Prunella vulgaris consistently inhibits the interaction between Lck SH2 and a peptide containing its consensus binding sequence (pYEEI). The inhibitory effect of RosA was specific for SH2 domains of Src family protein tyrosine kinase. RosA inhibited TCR-induced-Ca 2+ mobilization and IL-2 promoter activation but not phorbol 12-myristate 13-acetate/ionomycininduced IL-2 promoter activation, indicating its point of inhibition at the membrane proximal site of TCR signaling. Furthermore, RosA inhibited TCR-induced splenocyte proliferation as well as one-way MLR at an IC 50 of 25-50 ? M and inhibited cytokine expression such as IL-2 and IFN-+ . Here, we first report RosA as an inhibitor of TCR-signaling and subsequent T cell proliferation.

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BackgroundBCG immunogenicity in infants differs between populations and these differences have been attributed to various factors. In this study, the influence of geographical location, season of birth, timing of vaccination, micronutrient status (zinc) and inflammatory status (C-reactive protein, CRP) were assessed.MethodsImmunogenicity was assessed by cytokine signature in culture supernatants from diluted whole blood samples stimulated with M. tuberculosis PPD, using a multiplex bead assay. Results were correlated with the plasma zinc and CRP concentrations at the time of sampling, and with interview and household data. BCG vaccinated infants were recruited in Malawi, The Gambia and the UK.ResultsIn Malawi, infants vaccinated within the first week after birth showed lower production of most cytokines measured than those vaccinated later. The number of cytokines showing significant differences between Malawian and Gambian infants decreased after adjusting for season of birth. In Malawi, a proportion of infants had zinc deficiency and elevated plasma CRP (>10 mg/L), but neither zinc deficiency nor high CRP was associated with production of any of the cytokines measured.ConclusionsThe cytokine/chemokine signatures observed in response to M. tuberculosis PPD in infants at 3 months post BCG vaccination were affected by geographical location, season of birth, and timing of vaccination but not associated with the concentration of plasma zinc or inflammatory status. These factors should be considered in future trials of new TB vaccines.

The aim of this study was to evaluate the protective efficacy of MTBK_ 24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis-infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses (P Ͻ 0.05) and recalled gamma interferon (IFN-␥), interleukin-2 (IL-2), IL-6, and IL-17 responses (P Ͻ 0.05 or P Ͻ 0.01) to MTBK_24820. After challenge with the Beijing/K strain, an approximately 0.5 to 1.0 log 10 reduction in CFU in lungs and fewer lung inflammation lesions were observed in MTBK_24820-immunized mice compared to those for control mice. Moreover, MTBK_24820 immunization elicited significantly higher numbers of CD4 ϩ T cells producing protective cytokines, such as IFN-␥ and IL-17, in lungs and spleens (P Ͻ 0.01) and CD4 ϩ multifunctional T cells producing IFN-␥, tumor necrosis factor alpha (TNF-␣), and/or IL-17 (P Ͻ 0.01) than in control mice, suggesting protection comparable to that of BCG against the hypervirulent Beijing/K strain. The dominant immunogenic T-cell epitopes that induced IFN-␥ production were at the N terminus (amino acids 85 to 102 and 217 to 234). Its vaccine potential, along with protective immune responses in vivo, may be informative for vaccine development, particularly in regions where the M. tuberculosis Beijing/K-strain is frequently isolated from TB patients.

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