Toll-like receptors (TLRs) play a crucial role in host defense against microbial infection. The microbial ligands recognized by TLRs are not unique to pathogens, however, and are produced by both pathogenic and commensal microorganisms. It is thought that an inflammatory response to commensal bacteria is avoided due to sequestration of microflora by surface epithelia. Here, we show that commensal bacteria are recognized by TLRs under normal steady-state conditions, and this interaction plays a crucial role in the maintenance of intestinal epithelial homeostasis. Furthermore, we find that activation of TLRs by commensal microflora is critical for the protection against gut injury and associated mortality. These findings reveal a novel function of TLRs-control of intestinal epithelial homeostasis and protection from injury-and provide a new perspective on the evolution of host-microbial interactions.

A significant decrease in the frequency of posttransfusion FNHTR, but not ATR, for RBCs and PCs followed introduction of 100-percent UPL. The data support the hypothesis that the practice of UPL of RBCs and PCs decreases the frequency of FNHTR and thus improves patient care over the practice of selective leukoreduction.

Oncolytic viruses have been tested against many carcinomas of ectodermal and endodermal origin; however, sarcomas, arising from mesoderm, have received relatively little attention. Using 13 human sarcomas representing seven tumor types, we assessed the efficiency of infection, cytolysis, and replication of green fluorescent protein (GFP)-expressing vesicular stomatitis virus (VSV) and its oncolytically enhanced mutant VSV-rp30a. Both viruses efficiently infected and killed 12 of 13 sarcomas. VSV-rp30a showed a faster rate of infection and replication. In vitro and in vivo, VSV was selective for sarcomas compared with normal mesoderm. A single intravenous injection of VSV-rp30a selectively infected all subcutaneous human sarcomas tested in mice and arrested the growth of tumors that otherwise grew 11-fold. In contrast to other sarcomas, synovial sarcoma SW982 demonstrated remarkable resistance, even to high titers of virus (multiplicity of infection [MOI] of 100). We found no dysfunction in VSV binding or internalization. SW982 also resisted infection by human cytomegalovirus and Sindbis virus, suggesting a virus resistance mechanism based on an altered antiviral state. Quantitative reverse transcriptase (qRT)-PCR analysis revealed a heightened basal expression of interferon-stimulated genes (ISGs). Pretreatment, but not cotreatment, with interferon attenuators valproate, Jak1 inhibitor, or vaccinia virus B18R protein rendered SW982 highly susceptible, and this correlated with downregulation of ISG expression. Jak1 inhibitor pretreatment also enhanced susceptibility in moderately VSV-resistant liposarcoma and bladder carcinoma. Overall, we find that the potential efficacy of VSV as an oncolytic agent extends to nonhematologic mesodermal tumors and that unusually strong resistance to VSV oncolysis can be overcome with interferon attenuators.

Members of the genus IMPORTANCEUnderstanding the interactions between oncolytic viruses and the innate immune system will facilitate employing these viruses as therapeutic agents in cancer patients. The cancer-selective nature of some oncolytic viruses is based on the impaired innate immunity of many cancer cells. The parvoviruses H-1, LuIII, and MVM target cancer cells; however, their relationship with the innate immune system is relatively uncharacterized. Surprisingly, we found that these parvoviruses do not evoke an interferon response in normal human fibroblasts, glia, or melanocytes. Furthermore, unlike most other types of virus, we found that parvovirus infectivity is unaffected by interferon treatment of human normal or tumor cells. Finally, parvoviral replication was unimpaired by interferon in four human tumor types, including those with residual interferon functionality. We conclude that deficits in the interferon antiviral response of cancer cells do not contribute to parvoviral oncoselectivity in human cells. The interferon-resistant phenotype of parvoviruses may give them an advantage over interferon-sensitive oncolytic viruses in tumors showing residual interferon functionality.

Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons.

Members of the rodent subgroup of the genus Parvovirus exhibit lytic replication and spread in many human tumor cells and are therefore attractive candidates for oncolytic virotherapy. However, the significant variation in tumor tropism observed for these viruses remains largely unexplained. We report here that LuIII kills BJ-ELR ‘stepwise-transformed’ human fibroblasts efficiently, while MVM does not. Using viral chimeras, we mapped this property to the LuIII capsid gene, VP2, which is necessary and sufficient to confer the killer phenotype on MVM. LuIII VP2 facilitates a post-entry, pre-DNA-amplification step early in the life cycle, suggesting the existence of an intracellular moiety whose efficient interaction with the incoming capsid shell is critical to infection. Thus targeting of human cancers of different tissue-type origins will require use of parvoviruses with capsids that effectively make this critical interaction.

Minute Virus of Mice (MVM) shares inherent oncotropic properties with other members of the genus Parvovirus. Two elements responsible, at least in part, for this oncoselectivity have been mapped to an Ets1 binding site adjacent to the P4 TATA box of the initiating promoter, P4, and to a more distal cyclic AMP responsive element (CRE), located within the telomeric hairpin stem. Here the CRE overlaps one half-site for the binding of parvoviral initiation factor (PIF), which is essential for viral DNA replication. We used a degenerate oligonucleotide selection approach to show that CRE binding protein (CREB) selects the sequence ACGTCAC within this context, rather than its more generally accepted palindromic TGACGTCA recognition site. We have developed strategies for manipulating these sequences directly within the left-end palindrome of the MVM infectious clone and used them to clone mutants whose CRE either matches the symmetric consensus sequence or is scrambled, or in which the PIF binding site is incrementally weakened with respect to the CRE. The panel of mutants were tested for fitness relative to wildtype in normal murine fibroblasts A9 or transformed human fibroblasts 324 K, through multiple rounds of growth in co-infected cultures, using a differential real-time quantitative PCR assay. We confirmed that inactivating the CRE substantially abrogates oncoselectivity, but found that improving its fit to the palindromic consensus is somewhat debilitating in either cell type. We also confirmed that reducing the PIF half-site spacing by one basepair enhances oncoselectivity, but found that a further basepair deletion significantly reduces this effect.

Vesicular stomatitis virus (VSV) shows promise as vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-β-D-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-β protected mouse brain cells from VSV, as did a combination of ribavirin and chloroquine. Intracranial AAV-mIFN-β protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-β moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-β; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain.