We investigated the cellular basis for secretion of inflammatory cytokines in mice following intravenous administration of adenoviral vectors (Ad). Serum inflammatory cytokines including interleukin-6 (IL-6), IL-12, and tumor necrosis factor-alpha (TNF-alpha) were detected as early as 6 h following intravenous injection of Ad-expressing Escherichia coli beta-galactosidase (Ad-lacZ). Ad-lacZ readily accumulated in the splenic marginal zone 1 h after intravenous infusion, where both dendritic cells (DCs) and macrophages were transduced and activated within 6 h. Flow cytometric analyses showed that the expression of Ia and CD86 antigens was markedly enhanced on splenic DCs indicating their activation in vivo by Ad-lacZ. Upon ex vivo culture, these early-activated splenic DCs spontaneously produced high levels of IL-6 and IL-12. By contrast, activated splenic macrophages spontaneously secreted only IL-6. Elimination of tissue macrophages and splenic DCs in vivo considerably reduced the early release of IL-12, IL-6, and TNF-alpha and significantly blocked the specific cellular immune response to Ad and the transgene product in vivo. Our findings indicate that preferential activation of DCs and macrophages may account for Ad-triggered acute inflammatory response in vivo in mice. Moreover, DCs and macrophages may play different roles in this process in terms of their abilities to produce distinct patterns of inflammatory cytokines.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(⌬PE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY mar , in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY mar in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY mar was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(⌬PE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(⌬PE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(⌬PE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.

Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy. While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality. In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system. Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved. We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors. By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications.

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T cell defect is a common feature in lepromatous or borderline lepromatous leprosy (LL/BL) patients in contrast to tuberculoid or borderline tuberculoid type (TT/BT) patients. Tuberculoid leprosy is characterized by strong Th1-type cell response with localized lesions whereas lepromatous leprosy is hallmarked by its selective Mycobacterium leprae specific T cell anergy leading to disseminated and progressive disease. FoxP3+ Regulatory T cells (Treg) which are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases also dampen proinflammatory T cells that include T helper 17 (Th17) cells. This study is aimed at evaluating the role of Treg cells in influencing other effector T cells and its relationship with the cytokine polarized state in leprosy patients. Peripheral blood mononuclear cells from of BT/TT (n = 15) and BL/LL (n = 15) patients were stimulated with M. leprae antigen (WCL) in presence of golgi transport inhibitor monensin for FACS based intracellular cytokine estimation. The frequency of Treg cells showed >5-fold increase in BL/LL in comparison to BT/TT and healthy contacts. These cells produced suppressive cytokine, IL-10 in BL/LL as opposed to BT/TT (p = 0.0200) indicating their suppressive function. The frequency of Th17 cells (CD4, CD45RO, IL-17) was, however, higher in BT/TT. Significant negative correlation (r = -0.68, P = 0.03) was also found between IL-10 of Treg cells and IL-17+ T cells in BL/LL. Blocking IL-10/TGF-β restored the IL-17+ T cells in BL/LL patients. Simultaneously, presence of Th17 related cytokines (TGF-β, IL-6, IL-17 and IL-23) decreased the number of FoxP3+ Treg cells concomitantly increasing IL-17 producing CD4+ cells in lepromatous leprosy. Higher frequency of Programmed Death-1/PD-1+ Treg cells and its ligand, PDL-1 in antigen presenting cells (APCs) was found in BL/LL patients. Inhibition of this pathway led to rescue of IFN-γ and IL-17 producing T cells. Results indicate that Treg cells are largely responsible for the kind of immunosuppression observed in BL/LL patients. This study also proves that Treg cells are profoundly affected by the cytokine milieu and this property may be utilized for benefit of the host.

Aminoglycosides, amikacin (AK) and kanamycin (KM) are second line anti-tuberculosis drugs used to treat tuberculosis (TB) and resistance to them affects the treatment. Membrane and membrane associated proteins have an anticipated role in biological processes and pathogenesis and are potential targets for the development of new diagnostics/vaccine/therapeutics. In this study we compared membrane and membrane associated proteins of AK and KM resistant and susceptible Mycobacterium tuberculosis isolates by 2DE coupled with MALDI-TOF/TOF-MS and bioinformatic tools. Twelve proteins were found to have increased intensities (PDQuest Advanced Software) in resistant isolates and were identified as ATP synthase subunit alpha (Rv1308), Trigger factor (Rv2462c), Dihydrolipoyl dehydrogenase (Rv0462), Elongation factor Tu (Rv0685), Transcriptional regulator MoxR1(Rv1479), Universal stress protein (Rv2005c), 35kDa hypothetical protein (Rv2744c), Proteasome subunit alpha (Rv2109c), Putative short-chain type dehydrogenase/reductase (Rv0148), Bacterioferritin (Rv1876), Ferritin (Rv3841) and Alpha-crystallin/HspX (Rv2031c). Among these Rv2005c, Rv2744c and Rv0148 are proteins with unknown functions. Docking showed that both drugs bind to the conserved domain (Usp, PspA and SDR domain) of these hypothetical proteins and GPS-PUP predicted potential pupylation sites within them. Increased intensities of these proteins and proteasome subunit alpha might not only be neutralized/modulated the drug molecules but also involved in protein turnover to overcome the AK and KM resistance. Besides that Rv1876, Rv3841 and Rv0685 were found to be associated with iron regulation signifying the role of iron in resistance. Further research is needed to explore how these potential protein targets contribute to resistance of AK and KM.

Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/2-NF-B signaling axis. Furthermore, PE_PGRS11 markedly diminished H 2 O 2 -induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress.