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Tracking the localization and dynamics of the intracellular bioactive lipid phosphatidic acid (PA) is important for understanding diverse biological phenomena. Although several PA sensors have been developed, better ones are still needed for comprehensive PA detection in cells. We recently found that α-synuclein (α-Syn) selectively and strongly bound to PA in vitro. Here, we revealed that the N-terminal region of α-Syn (α-Syn-N) specifically bound to PA, with a dissociation constant of 6.6 μm. α-Syn-N colocalized with PA-producing enzymes, diacylglycerol kinase (DGK) β at the plasma membrane (PM), myristoylated DGKζ at the Golgi apparatus, phorbol ester-stimulated DGKγ at the PM, and phospholipase D2 at the PM and Golgi but not with the phosphatidylinositol-4,5-bisphosphate-producing enzyme in COS-7 cells. However, α-Syn-N failed to colocalize with them in the presence of their inhibitors and/or their inactive mutants. These results indicate that α-Syn-N specifically binds to cellular PA and can be applied as an excellent PA sensor.

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8+ T cell response to Bacillus Calmette-Guerin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8+ T cells with an rBCG-based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG-Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). We identified two MHC-I (H2-Kd )-restricted epitopes that induce cross-reactive responses to Mtb and other related mycobacteria in both BALB/c (H2d ) and CB6F1 (H2b/d ) mice. The H2-Kd -restricted peptide epitopes elicited polyfunctional CD8+ T cell responses that were also highly cross-reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2-Kd -restricted epitopes elicit distinct CD8+ T cell populations, a result explained by the X-ray structure of the two peptide/H2-Kd complexes. These results suggest that rBCG-Mkan85B vector-based immunization and DNA-Mkan85B boost may enhance CD8+ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.

α-Synuclein (α-syn), which causally links to Parkinson's disease, binds to vesicles containing phosphatidic acid (PA). However, the effects of the fatty acyl chains of PA on its ability to bind to α-syn protein remain unclear. Intriguingly, we reveal that among several PA species, 18:1/18:1-PA is the most strongly bound PA to the α-syn protein. Moreover, 18:1/18:1-PA more strongly enhances secondary structural changes from the random coil form to the α-helical form than 16:0/18:1-PA. Furthermore, 18:1/18:1-PA more markedly accelerates generation of multimeric and proteinase K-resistant α-syn protein compared to 16:0/18:1-PA. These results indicate that among phospholipids examined so far, 18:1/18:1-PA demonstrates the strongest binding to α-syn, as well as the most effective enhancement of its secondary structural changes and aggregation formation.

Although effective liquid chromatography (LC)/mass spectrometry (MS) methods enabling the separation of phospholipid molecular species have been developed, there are still problems with an intracellular signaling molecule, phosphatidic acid (PA). In this study, we optimized LC/MS conditions to improve the quantitative detection of PA molecular species from a cellular lipid mixture. Using the newly developed LC/MS method, we showed that stimulation of CTLL-2 murine T-lymphocytes by interleukin-2 (IL-2) induced a significant increase of 36:1-, 36:2-, 40:5- and 40:6-diacyl-PA. A diacylglycerol kinase (DGK) inhibitor, R59949, attenuated the increase of 36:1-, 40:5-, 40:6-diacyl-PA, suggesting that DGK IL-2-dependently and selectively generated these diacyl-PA species., Highlights ▸ We optimized LC/MS conditions to improve quantitative detection of PA species. ▸ IL-2 induced increase of 36:1-, 36:2-, 40:5- and 40:6-diacyl-PA. ▸ IL-2 stimulation caused DGK-dependent increases of limited molecular species of PA. ▸ The LC/MS method developed in this study will be advantageous for lipidomics studies.

Summary To investigate the pathological and immunological profiles of rat tuberculosis, Lewisfemale rats were infected aerially with Mycobacterium tuberculosis. Histopathology,immunological profiles of mononuclear cells from M. tuberculosis-infected rat lungtissue, and the expression patterns of cytokine and iNOS mRNAs were examined overtime. M. tuberculosis induced granulomatous lesions in the lungs, spleen, lymph nodesand liver, but these lesions lacked central necrosis. Multinucleate giant cells wereobserved in late-phase tuberculosis. CD4 + and CD8 + T cells increased with time andreached a peak 5 weeks after infection, decreasing gradually thereafter. ED1 antigen,suggestive of alveolar macrophages, was expressed at a high level in early phasetuberculosis and remained at the same level even in the late phase. OX62 antigenincreased gradually and reached a peak 5 weeks after infection. Interferon-g, tumournecrosis factor-a and iNOS mRNAs were expressed strongly over time, but theirexpression decreased 12 weeks after infection. Because rat tuberculosis is very similarto murine tuberculosis and it is easy to obtain mononuclear cells from M. tuberculosis-infected rat lung tissue, the rat tuberculosis model appears to be suitable forimmunological studies in vivo.KeywordsFACS analysis, M. tuberculosis, rat tuberculosis, RT-PCRMice, rats, guinea pigs and rabbits have been used as animalmodels of human tuberculosis. Mice show lower tuberculinsensitivity and less caseous necrosis than human beings. Guineapigs, a species with good delayed-type hypersensitivity, arethe most susceptible of the four animals and usually develop ablood-disseminated disease similar to that observed in infantsand immunosuppressed individuals. The rabbit is the commonlaboratory animal in which the disease closely resembles thatfound in humans (Dannenberg 1999). Pulmonary granulomascan be induced with Mycobacterium tuberculosis, M. aviumand M. bovis in these animals (Francis 1958). Guinea pigs andrabbits are not suitable for immunological studies becausecongenic strains are few and sufficient number of monoclonalantibodies against guinea pig and rabbit antigens is not avail-able for immunological analysis. Mice are frequently used forsuch purposes because there are a lot of congenic strainsavailable and murine immunology is well established likehuman immunology. However, to investigate the roles ofcells in M. tuberculosis-infected pulmonary tissue in moredetail, the murine model is not suitable because it is difficultto obtain mononuclear cells for immunological analysis frommurine lung tissue and it is also difficult to perform tuberculin

To investigate the role of TLR in the development of murine tuberculosis in vivo, TLR2 and TLR6 knockout (KO) mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. Both TLR2 and TLR6 KO mice survived until sacrifice at 12 weeks after infection. Infected TLR2 KO mice developed granulomatous pulmonary lesions with neutrophil infiltration, which were slightly larger in size than those in wild-type mice. Pulmonary levels of the mRNAs for inducible nitric oxide synthase (iNOS), TNF-alpha, TGF-beta, IL-1beta, and IL-2 were significantly lower, but levels of the mRNAs for IL-4 and IL-6 were higher, than in wild-type (WT) mice. No significant difference was recognized in cytokine mRNA expression between TLR2 KO and WT mice at 12 weeks after infection. DNA binding by NF-kappaB was low in TLR2 KO mice. On the other hand, TLR6 KO mice were not different from WT mice in terms of pulmonary histopathology, mRNA expression and CFU assay. Therefore, TLR2 does not play an essential role in the pathogenesis of murine tuberculosis, although it is important for defense against mycobacterial infection.

In order to understand the role of IRF-1 in the development of murine tuberculosis in vivo, IRF-1 knockout mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. These knockout mice developed multifocal necrotic lesions in the lung, liver and spleen tissues and died of disseminated tuberculosis within 43 days of infection. Compared with the levels in wild-type mice, the pulmonary inducible NO synthase (iNOS) mRNA expression level was significantly lower, but IL-18 and IL-6 mRNA levels were higher. There was no statistically significant difference in the expression of IFN-gamma and TNF-alpha mRNA between the IRF-1 knockout and wild-type mice. IRF-1 is indirectly responsible for iNOS mRNA expression and plays an important role in the pathogenesis of murine tuberculosis.