In Vivo Retargeting of Adenovirus Type 5 to αvβ6 Integrin Results in Reduced Hepatotoxicity and Improved Tumor Uptake following Systemic Delivery

A key impediment to successful cancer therapy with adenoviral vectors is the inefficient transduction of malignant tissue in vivo. Compounding this problem is the lack of cancer-specific targets, coupled with a shortage of corresponding high-efficiency ligands, permitting selective retargeting. The epithelial cell-specific integrin αvβ6 represents an attractive target for directed therapy since it is generally not expressed on normal epithelium but is upregulated in numerous carcinomas, where it plays a role in tumor progression. We previously have characterized a high-affinity, αvβ6-selective peptide (A20FMDV2) derived from VP1 of foot-and-mouth disease virus. We generated recombinant adenovirus type 5 (Ad5) fiber knob, incorporating A20FMDV2 in the HI loop, for which we validated the selectivity of binding and functional inhibition of αvβ6. The corresponding αvβ6-retargeted virus Ad5-EGFP_A20 exhibited up to 50-fold increases in coxsackievirus- and-adenovirus-receptor-independent transduction and up to 480-fold-increased cytotoxicity on a panel of αvβ6-positive human carcinoma lines compared with Ad5-EGFP_WT. Using an αvβ6-positive (DX3-β6) xenograft model, we observed a ∼2-fold enhancement in tumor uptake over Ad5-EGFP_WT following systemic delivery. Furthermore, ∼5-fold-fewer Ad5-EGFP_A20 genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. Warfarin pretreatment, to deplete coagulation factors, did not improve tumor uptake significantly with either virus but did significantly reduce liver sequestration and hepatic toxicity. The ability of Ad5-EGFP_A20 to improve delivery to αvβ6, combined with its reduced hepatic tropism and toxicity, highlights its potential as a prototype virus for future clinical investigation.The aim of cancer gene therapy is to achieve targeted delivery of therapeutic transgenes to malignant tissue, with negligible effects on surrounding healthy tissue. Efforts in the development of adenoviruses as therapeutic agents have been persistent. However, several challenges still remain. Inefficient transduction of diseased tissue and the innate hepatotropism and toxicity of adenovirus type 5 (Ad5) in vivo following intravenous delivery represent major issues to be addressed. Additionally, the use of adenoviral vectors for cancer therapy is thought to be incompatible with the broad distribution of the primary adenovirus receptor, the coxsackievirus and adenovirus receptor (CAR), in normal tissues (6). Furthermore, it recently has emerged that human, but not murine, erythrocytes express CAR on their surface, which promotes the sequestration of Ad5 in the circulation and may represent another restriction to efficient tumor delivery in vivo (8).The predominant adenoviral serotype currently used in gene therapy applications is human Ad5. Ad5 binds to cells through a docking process in which the distal knob domain of the fiber structural protein binds to CAR (6, 23). This is followed by the exposure of an arginine-glycine-aspartate (RGD) motif in the penton base which promotes viral internalization, mediated primarily by αvβ3 and αvβ5 integrins (49). Binding to CAR represents the initial event in cell attachment in vitro, and therefore CAR expression levels long have been thought to be critical in determining the transduction efficiency of Ad5 in vivo. Several studies have reported low expression of CAR in primary carcinoma lines and tumor explants (3, 21, 30, 32, 37), highlighting the necessity for CAR-independent targeting strategies. However, the nonspecific sequestration of Ad5 in the liver remains the major obstacle to achieving high-efficiency tumor targeting following systemic delivery.A preeminent role for coagulation factors (i.e., FVII, FIX, FX, protein C, and C4BP) in directing liver uptake following systemic delivery has been demonstrated in recent years (36, 41, 48), and hepatocyte transduction now has been shown to be mediated predominantly by a direct Ad5 hexon-FX interaction (22, 48). This discovery has prompted the experimental use of anticoagulants, such as warfarin, in an attempt to avoid liver sequestration, with the aim of increasing the bioavailability of the virus for the tumor. However, it recently has emerged that coagulation factors may also be required for efficient tumor delivery in vivo and that the depletion of blood factors may in fact preclude successful tumor uptake (16). Accordingly, Ad vector constructs which combine liver detargeting with high-efficiency, CAR-independent gene delivery to cancer-specific receptors are highly desirable.The epithelial cell-specific integrin αvβ6 generally is undetectable in normal adult tissue but is upregulated significantly in numerous carcinomas, where high expression often correlates with poor prognosis (1, 4, 13). We have shown previously that over 90% of oral squamous cell carcinomas express αvβ6 strongly (35, 45) and that high αvβ6 expression promotes tumor progression (35, 43, 46). Binding to αvβ6 is via the RGD motif in its ligands, which include the latency-associated peptides (LAP) of transforming growth factor β1 (TGF-β1) and TGF-β3 (33), in addition to the VP1 structural protein of foot-and-mouth disease virus (FMDV), for which αvβ6 is a native receptor (20). We have shown that specificity for αvβ6 is dependent on the inclusion of a DLXXL motif in an extended carboxy α-helical loop, with the RGD motif situated at the apex of a hairpin loop domain (10). Functional analysis of known αvβ6 ligands identified a candidate peptide, A20FMDV2, which had high affinity and selective binding to αvβ6 (10). Recent studies have further supported this finding, demonstrating that this peptide forms a highly stable, EDTA-resistant complex with αvβ6 integrin, in a manner analogous to the highly infectious FMDV (11).Over the past decade, significant attempts to increase the delivery of Ad5 to target tissues have been made by modifying structural tropism determinants. Retargeting strategies have included the insertion of RGD motifs (12), polylysine (pK7) motifs (50) and TAT peptide from human immunodeficiency virus type 1 (26) in attempts to improve delivery to malignant tissue or to the endothelial networks that supply the tumors. The resolution of the crystal structure of the Ad5 knob domain by X-ray crystallography identified the HI loop region as being suitable for peptide incorporation (51), and to date this site has been shown to tolerate the insertion of ligands of up to 83 amino acids with negligible effects on structural integrity (5). In order to redirect the native tropism of Ad5 to αvβ6, we genetically incorporated A20FMDV2 into the HI loop region of the fiber knob domain. We hypothesized that Ad5-EGFP_A20 would permit significant improvements in selective delivery to αvβ6, both in vitro and in vivo.Here, we describe the successful subversion of Ad5 infection to αvβ6 expressed on human carcinoma cell lines in vitro and demonstrate that the enhanced transduction observed with Ad5-EGFP_A20 is CAR independent. We have confirmed that the improved infectivity is due to the insertion of the A20FMDV2 peptide and that cell entry is mediated predominantly through αvβ6 integrin. Additionally, using an αvβ6-positive (DX3-β6) xenograft model, we observed ∼2-fold enhancement in tumor uptake over Ad5-EGFP_WT in vivo following systemic delivery. Furthermore, ∼5-fold-fewer Ad5-EGFP_A20 genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. These data show that redirecting Ad5 to αvβ6 can increase tumor delivery while simultaneously limiting hepatotoxicity; this may therefore represent a means of overcoming some of the limitations of current Ad5 therapy.     

Mosquito immune responses and compatibility between Plasmodium parasites and anopheline mosquitoes

Background  

Functional screens based on dsRNA-mediated gene silencing identified several Anopheles gambiae genes that limit Plasmodium berghei infection. However, some of the genes identified in these screens have no effect on the human malaria parasite Plasmodium falciparum; raising the question of whether different mosquito effector genes mediate anti-parasitic responses to different Plasmodium species.     

The flagellar protein FliL is essential for swimming in Rhodobacter sphaeroides

In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides. Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion.     

HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

Background

Direct cellular contact with stimulated T cells is a potent mechanism that induces cytokine production in human monocytes in the absence of an infectious agent. This mechanism is likely to be relevant to T cell-mediated inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Microparticles (MP) generated by stimulated T cells (MP_T) display similar monocyte activating ability to whole T cells, isolated T cell membranes, or solubilized T cell membranes. We previously demonstrated that high-density lipoproteins (HDL) inhibited T cell contact- and MP_T-induced production of IL-1β but not of its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra).

Methodology/Principal Findings

Labeled MP_T were used to assess their interaction with monocytes and T lymphocytes by flow cytometry. Similarly, interactions of labeled HDL with monocytes and MP_T were assessed by flow cytometry. In parallel, the MP_T-induction of IL-1β and sIL-1Ra production in human monocytes and the effect of HDL were assessed in cell cultures. The results show that MP_T, but not MP generated by activated endothelial cells, bond monocytes to trigger cytokine production. MP_T did not bind T cells. The inhibition of IL-1β production by HDL correlated with the inhibition of MP_T binding to monocytes. HDL interacted with MP_T rather than with monocytes suggesting that they bound the activating factor(s) of T cell surface. Furthermore, prototypical pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, CCL3 and CCL4 displayed a pattern of production induced by MP_T and inhibition by HDL similar to IL-1β, whereas the production of CCL2, like that of sIL-1Ra, was not inhibited by HDL.

Conclusions/Significance

HDL inhibit both MP_T binding to monocytes and the MP_T-induced production of some but not all cytokines, shedding new light on the mechanism by which HDL display their anti-inflammatory functions.     

Mechanism of N-methylation by the tRNA m1G37 methyltransferase Trm5

Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N(1) position of G37 directly 3' to the anticodon. While the biological role of m(1)G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one order of magnitude from pH 6.0 to reach a plateau at pH 7.0. This suggests a mechanism involving proton transfer from G37 as the key element in catalysis. Consideration of the kinetic data in light of the Trm5-tRNA-AdoMet ternary cocrystal structure, determined in a precatalytic conformation, suggests that proton transfer is associated with an induced fit rearrangement of the complex that precedes formation of the reactive configuration in the active site. Key roles for the conserved R145 side chain in stabilizing a proposed oxyanion at G37-O(6), and for E185 as a general base to accept the proton from G37-N(1), are suggested based on the mutational analysis.     

Combined effects of long-living chemical species during microbial inactivation using atmospheric plasma-treated water

Electrical discharges in humid air at atmospheric pressure (nonthermal quenched plasma) generate long-lived chemical species in water that are efficient for microbial decontamination. The major role of nitrites was evidenced together with a synergistic effect of nitrates and H(2)O(2) and matching acidification. Other possible active compounds are considered, e.g., peroxynitrous acid.     

Dynamics of clathrin and adaptor proteins during endocytosis

The endocytic adaptor complex AP-2 colocalizes with the majority of clathrin-positive spots at the cell surface. However, we previously observed that AP-2 is excluded from internalizing clathrin-coated vesicles (CCVs). The present studies quantitatively demonstrate that AP-2 disengages from sites of endocytosis seconds before internalization of the nascent CCV. In contrast, epsin, an alternate adaptor for clathrin at the plasma membrane, disappeared, along with clathrin. This suggests that epsin remains an integral part of the CCV throughout endocytosis. Clathrin spots at the cell surface represent a heterogeneous population: a majority (70%) of the spots disappeared with a time course of 4 min, whereas a minority (22%) remained static for 30 min. The static clathrin spots undergo constant subunit exchange, suggesting that although they are static structures, these spots comprise functional clathrin molecules, rather than dead-end aggregates. These results support a model where AP-2 serves a cargo-sorting function before endocytosis, whereas alternate adaptors, such as epsin, actually link cargo to the clathrin coat surrounding nascent endocytic vesicles. These data also support a role for static clathrin, providing a nucleation site for endocytosis. adaptor complex; epsin; total internal reflection fluorescence microscopy     

A 2.5-Mb contig constructed from Angus, Longhorn and horned Hereford DNA spanning the polled interval on bovine chromosome 1

The polled locus has been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. As an intermediate step in our efforts to identify the polled locus and the underlying causative mutation for the polled phenotype, we have constructed a BAC-based physical map of the interval containing the polled locus. Clones containing genes and markers in the critical interval were isolated from the TAMBT (constructed from Angus and Longhorn genomic DNA) and CHORI-240 (constructed from horned Hereford genomic DNA) BAC libraries and ordered based on fingerprinting and the presence or absence of 80 STS markers. A single contig spanning 2.5 Mb was assembled. Comparison of the physical order of STSs to the corresponding region of human chromosome 21 revealed the same order of genes within the polled critical interval. This contig of overlapping BAC clones from horned and polled breeds is a useful resource for SNP discovery and characterization of positional candidate genes.