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Adenovirus (Ad)-based vectors have shown considerable promise for gene therapy. However, Ad requires the coxsackievirus and adenovirus receptor (CAR) to enter cells efficiently and low CAR expression is found in many human cancers, which hinder adenoviral gene therapies. Here, cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-folate liposomes (Df) encapsulating replication-deficient Ad were synthesized, which showed improved transfection efficiency in various CAR-deficient cell lines, including epithelial and hematopoietic cell types. When encapsulating replication-competent oncolytic Ad (TAV255) in DOTAP-folate liposome (TAV255-Df), the adenoviral structural protein, hexon, was readily produced in CAR-deficient cells, and the tumor cell killing ability was 5× higher than that of the non-encapsulated Ad. In CAR-deficient CT26 colon carcinoma murine models, replication-competent TAV255-Df treatment of subcutaneous tumors by intratumoral injection resulted in 67% full tumor remission, prolonged survival, and anti-cancer immunity when mice were rechallenged with cancer cells with no further treatment. The preclinical data shows that DOTAP-folate liposomes could significantly enhance the transfection efficiency of Ad in CAR-deficient cells and, therefore, could be a feasible strategy for applications in cancer treatment.

Infection with human immunodeficiency virus-1 (HIV-1) causes brain injury. Type I interferons (IFNα/β) are critical mediators of any anti-viral immune response and IFNβ has been implicated in the temporary control of lentiviral infection in the brain. Here we show that transgenic mice expressing HIV-1 envelope glycoprotein 120 in their central nervous system (HIVgp120tg) mount a transient IFNβ response and provide evidence that IFNβ confers neuronal protection against HIVgp120 toxicity. In cerebrocortical cell cultures, neuroprotection by IFNβ against gp120 toxicity is dependent on IFNα receptor 1 (IFNAR1) and the β-chemokine CCL4, as IFNAR1 deficiency and neutralizing antibodies against CCL4, respectively, abolish the neuroprotective effects. We find in vivo that IFNβ mRNA is significantly increased in HIVgp120tg brains at 1.5, but not 3 or 6 months of age. However, a four-week intranasal IFNβ treatment of HIVgp120tg mice starting at 3.5 months of age increases expression of CCL4 and concomitantly protects neuronal dendrites and pre-synaptic terminals in cortex and hippocampus from gp120-induced damage. Moreover, in vivo and in vitro data suggests astrocytes are a major source of IFNβ-induced CCL4. Altogether, our results suggest exogenous IFNβ as a neuroprotective factor that has potential to ameliorate in vivo HIVgp120-induced brain injury.

The neuropathogenesis of HIV associated neurocognitive disorders (HAND) involves disruption of mitochondrial homeostasis and increased neuroinflammation. However, it is unknown if alterations in mitochondrial biogenesis in the brain underlie the neuropathogenesis of HAND. In this study, neuropathological and molecular analyses of mitochondrial biogenesis and inflammatory pathways were performed in brain specimens from a well-characterized cohort of HIV+ cases that were on antiretroviral regimens. In vitro investigations using primary human astroglia and neurons were used to probe the underlying mechanisms of mitochondrial alterations. In frontal cortices from HAND brains compared to cognitive normal brains, total levels of transcription factors that regulate mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and transcription factor A, mitochondrial (TFAM) were decreased. Immunohistochemical analyses revealed that TFAM was decreased in neurons and increased in astroglia. These changes were accompanied by decreased total mitochondrial DNA per cell and increased levels of messenger RNA for the proinflammatory cytokine interleukin (IL)-1β. To determine how IL-1β affects astroglial bioenergetic processes and mitochondrial activity, human astroglial cultures were exposed to recombinant IL-1β. IL-1β induced mitochondrial activity within 30 min of treatment, altered mitochondrial related gene expression, altered mitochondrial morphology, enhanced adenoside triphosphate (ATP) utilization and increased the expression of inflammatory cytokines. WIN55,212-2 (WIN), an aminoalkylindole derivative and cannabinoid receptor agonist, blocked IL-1β-induced bioenergetic fluctuations and inflammatory gene expression in astroglia independent of cannabinoid receptor (CB)1 and peroxisome proliferator-activated receptor (PPAR) γ. A PPARα antagonist reversed the anti-inflammatory effects of WIN in human astroglia. These results show that mitochondrial biogenesis is differentially regulated in neurons and astroglia in HAND brains and that targeting astroglial bioenergetic processes may be a strategy to modulate neuroinflammation.

A crucial step in the arenavirus life cycle is the biosynthesis of the viral envelope glycoprotein (GP) responsible for virus attachment and entry. Processing of the GP precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexin-isozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infection and production of infectious virus. Here, we sought to evaluate arenavirus GPC processing by S1P as a target for antiviral therapy using a recently developed peptide-based S1P inhibitor, decanoyl (dec)-RRLL-chloromethylketone (CMK), and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). To control for off-target effects of dec-RRLL-CMK, we employed arenavirus reverse genetics to introduce a furin recognition site into the GPC of LCMV. The rescued mutant virus grew to normal titers, and the processing of its GPC critically depended on cellular furin, but not S1P. Treatment with the S1P inhibitor dec-RRLL-CMK resulted in specific blocking of viral spread and virus production of LCMV. Combination of the protease inhibitor with ribavirin, currently used clinically for treatment of human arenavirus infections, resulted in additive drug effects. In cells deficient in S1P, the furin-dependent LCMV variant established persistent infection, whereas wild-type LCMV underwent extinction without the emergence of S1P-independent escape variants. Together, the potent antiviral activity of an inhibitor of S1P-dependent GPC cleavage, the additive antiviral effect with ribavirin, and the low probability of emergence of S1P-independent viral escape variants make S1P-mediated GPC processing by peptide-derived inhibitors a promising strategy for the development of novel antiarenaviral drugs.

The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped viruses that use clathrin-mediated endocytosis for entry. However, rLCMV-LASVGP entered cells predominantly via a clathrin-, caveolin-, and dynamin-independent endocytotic pathway similar to the one recently described for LCMV. Productive infection of rLCMV-LASVGP was only mildly affected by a dominant negative mutant of Rab5 and was independent of Rab7, suggesting an unusual mechanism of delivery to endosomes. In addition, rLCMV-LASVGP infection was independent of actin but required intact microtubules. Our data indicate that LASV enters cells via a pathway distinct from the one used by human-pathogenic New World arenaviruses.

Summary. The in vivo productive infection by the ovine Visna/maedi lentivirus (VISNA) is restricted to cells of the monocyte/macrophage lineage. The basis for this restriction is not understood. Although the VISNA envelope (Env) glycoprotein is the main target for virus neutralization, studies on the role of this protein in virus infection are limited. A vaccinia virus recombinant (VV-env-MV) containing the entire VISNA env sequence was generated and shown to produce in infected cells a protein of about 165 kDa (referred to as gp150). During VV-envMV infection, expression of env caused extensive cell-to-cell fusion in cell lines of different origins. Pulse-chase and Western blot analyses revealed that gp150 is not cleaved in VV-env-MV infected cells. The glycoprotein gp150 formed oligomers held by disulfide bonding. Cell-to-cell fusion was prevented in the presence of the inhibitor of glycosilation, tunicamycin, but it was markedly enhanced by an inhibitor of proteoglycan synthesis, β-D-xyloside. These findings showed that the receptor for VISNA Env is widely distributed within cells, that fusion-from-within of cells can occur in the apparent absence of proteolytic cleavage of gp150, and that fusion require a glycosylated Env but not the addition of proteoglycan chains at the cell surface. This recombinant virus could have utility as a potential vaccine against VISNA.

Autoantibodies against p53 have been observed in many cancers, often linked with abnormalities in the TP53 gene. Since p53 mutations and deletions at chromosome 17p are known to occur in CLL, we measured anti-p53 autoantibodies by ELISA in plasma samples from patients with normal cytogenetics as well as those with 13q, 11q, and 17p deletions as well as trisomy 12. Anti-p53 autoantibodies were detected in over half of the patients with a 17p deletion but in very few of the others. There was no correlation between the levels of anti-p53 antibodies and the percentage of cells with 17p abnormalities. The levels of the anti-p53 autoantibodies remained stable for most patients with serial samples. Increased levels of antibodies that bound to two peptide fragments of p53 were also seen in patients with 17p deletions. At least on case with high levels of anti-p53 autoantibodies had a heterozygotic mutation known to result in a dominant negative phenotype, suggesting that aberrant expression of p53 may contribute to the development of autoantibodies and suggests that these autoantibodies may reflect biological features relevant to prognosis.

To study the prognosis (recurrence and mortality) of patients with ischemic colitis (IC).This study was conducted in four Spanish hospitals, participants in the Ischemic Colitis in Spain study We analyzed prospectively 135 consecutive patients who met criteria for definitive or probable IC according to Brandt criteria, and follow up these patients during the next five years, retrospectively. Long-term results (recurrence and mortality) were evaluated retrospectively after a median interval of 62 mo (range 54-75 mo).Estimated IC recurrence rates were 2.9%, 5.1%, 8.1% and 9.7% at years 1, 2, 3 and 5 years, respectively. Five-year survival was 69% (93 of 135) and 24% (10 of 42 patients) died for causes related to the IC. Among these 10 patients, 8 died in their first episode at hospital (4 had gangrenous colitis and 4 fulminant colitis) and 2 due to recurrence.The five-year recurrence rate of IC was low. On the other hand, mortality during follow-up was high and was not associated with ischemic colitis.

Purpose Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations. Methods Alemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunofluorescent-based assay. Results Peptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 μg/ml, and the assay for alemtuzumab had a limit of detection of 1 μg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner. Conclusion Phage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and sufficient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies. Electronic supplementary material The online version of this article (doi:10.1007/s00280-009-1240-1) contains supplementary material, which is available to authorized users.