An Oligopeptide Transporter of Mycobacterium tuberculosis Regulates Cytokine Release and Apoptosis of Infected Macrophages

Background: The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppA(MTB). Methodology/Principal Findings: OppA(MTB) was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppA(MTB) confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1 beta, IL-6 and TNF-alpha was also compromised after inactivation of oppD. Conclusions: Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis

Stability of Lentiviral Vector-Mediated Transgene Expression in the Brain in the Presence of Systemic anti-Vector Immune Responses. Hum Gene Ther

Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose-response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 10(2), and maximum expression at 10(6), transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune responses against lentiviral vectors was effective in causing a decrease in transgene expression only if the immune response was directed against the transgene. A systemic immune response against vector components alone did not cause brain inflammation, possibly because vector-derived epitopes were not being presented in the CNS

Cyclophosphamide enhances glioma virotherapy by inhibiting innate immune responses

Clinical trials are testing oncolytic viruses (OVs) as therapies for cancer. We have shown that animals that have brain tumors and are treated with a herpes simplex virus (HSV)-derived OV live significantly longer when cyclophosphamide (CPA) is preadministered. Here, we explore the mechanisms behind this finding. In a syngeneic rat glioma model, intratumoral HSV administration is associated with rapid increase of natural killer cells, microglia/macrophages (CD68(+) and CD163(+)), and IFN-γ. Pretreatment with CPA enhances HSV replication and oncolysis and reduces an HSV-mediated increase in CD68(+) and CD163(+) cells and intratumoral IFN-γ. Molecular imaging shows CPA pretreatment to inhibit HSV-induced infiltration of tumor-associated phagocytic cells. Our results reveal molecular and cellular mechanisms that inhibit intratumoral spread of HSV and suggest a therapeutic path for improving the efficacy of virotherapy as a treatment for cancer

A serum substitute for fed-batch culturing of hybridoma cells

We developed a substitute for serum to produce fed-batch cultures of hybridoma cells in serum-free medium and confirmed that the cells could be successfully cultivated this way. Our substitute consisted of 12 components. The specific production rates of lactate and ammonia, which are harmful byproducts from the cells, were significantly reduced compared with a conventional serum-containing batch culture. This reduction led to a higher cell concentration and a longer production lifetime. As a result, the final concentration of monoclonal antibody was 400 mg/L, or five times greater than that in the conventional serum-containing batch culture. The developed substitute is expected to enable fed-batch cultivation in a serum-free condition

Exploring Post-translational Arginine Modification Using Chemically Synthesized Methylglyoxal Hydroimidazolones

The methylglyoxal-derived hydroimidazolones (MG-Hs, Figure 1A) comprise the most prevalent class of non-enzymatic, post-translational modifications of protein arginine residues found in nature. These adducts form spontaneously in the human body, and are also present at high levels in the human diet. Despite numerous lines of evidence suggesting that MG-H–arginine adducts play critical roles in both healthy and disease physiology in humans, detailed studies of these molecules have been hindered by a lack of general synthetic strategies for their preparation in chemically homogeneous form, and on scales sufficient to enable detailed biochemical and cellular investigations. To address this limitation, we have developed efficient, multi-gram-scale syntheses of all MG-H–amino acid monomers in 2–3 steps starting from inexpensive, readily available starting materials. Thus, MG-H derivatives were readily incorporated into oligopeptides site-specifically using standard solid-phase peptide synthesis (SPPS). Access to synthetic MG-H-peptide adducts has enabled detailed biochemical investigations, which have revealed a series of novel and unexpected findings. First, one of the three MG-H isomers – MG-H3 – was found to possess potent, pH-dependent antioxidant properties in biochemical and cellular assays intended to replicate redox processes that occur in vivo. Computational and mechanistic studies suggest that MG-H3-containing constructs are capable of participating in mechanistically distinct H-atom-transfer and single-electron-transfer oxidation processes. Notably, the product of MG-H3 oxidation was unexpectedly observed to disassemble into the fully unmodified arginine residue and pyruvate in aqueous solution. We believe these observations to reflect meaningfully on the role(s) of MG-H–protein adducts in human physiology, and expect the synthetic reagents reported herein to enable investigations into non-enzymatic protein regulation at an unprecedented level of detail