Objective Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular T helper (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently identified that interleukin 21 (IL-21) promoted Tfh differentiation in autoimmune BXD2 mice that develop spontaneous GCs. The objective of this study was to determine the modulatory effects of IL-21 on Tfr and the Tfr/Tfh balance in BXD2 mice. Methods The percentage and phenotype of Tfr were determined in BXD2 and BXD2-Il21−/− mice. The effects of IL-21 on Tfr and the ratio of Tfr/Tfh were evaluated. Sorted Tfr cells from BXD2-Il21−/− mice were co-cultured with Tfh and B cells, or transferred into BXD2 mice to determine their function. Results GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-Il21−/− mice than in WT-BXD2. Adenovirus-IL-21 administration to BXD2-Il21−/− mice decreased Tfr and the ratio of Tfr/Tfh but increased GC B cells in the spleen. rmIL-21 suppressed Foxp3 and significantly reduced Tgfb1, Il2 and Gitr but enhanced Il21, Il6, Pd1, Cxcr5 and Icos in Tfr cells. IL-21 also counteracted Tfr-mediated inhibition of antibody secretion in the Tfh-B cell co-culture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased autoantibody-producing B cells. Conclusion High levels of IL-21 selectively enhanced Tfh differentiation but inhibited Tfr commitment and their suppressive function on Tfh and B cells, suggesting that IL-21 skews the balance from Tfr to Tfh to promote autoreactive GC reactions in BXD2 mice.

A major obstacle to the success of gene therapy strategies with ME180 tumors. Treatment of tumors with Ad.TK RC that directly target cancer cells is the poor vector distriwithout GCV resulted in a similar antitumor effect, conbution within solid tumors. To address this problem, we firming that the replicating vector has an oncolytic effect. developed an E1b 55 kDa attenuated, replicationWhen GCV was initiated 3 days after Ad.TK RC injection, competent adenovirus (Ad.TK RC ) which expresses the hersurvival of mice with each tumor type was greatly propes simplex-1 thymidine kinase (HSVtk) gene to sensitize longed, with 60% of animals with ME180 tumors surviving tumors to ganciclovir (GCV). Efficacy of this combined for over 160 days. These results confirm that both the strategy was tested in nude mice with subcutaneous oncolysis caused by a replicating virus and suicide/prodrug human A375 melanoma and ME180 cervical carcinomas.gene therapy with HSVtk/GCV have potent antitumor Intratumoral injection of a replication-defective adenoviral effects. When combined, these two approaches are compvector expressing HSVtk (Ad.TK) followed by GCV treatlementary resulting in a significantly improved treatment ment resulted in doubling of the survival time of mice bearoutcome. ing A375 tumors and 20% long-term survival of mice

In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.

Background-Heat-shock protein 70 (HSP 70) plays a role in myocardial protection. No studies are available, however, to show that direct gene transfer of HSP 70 reduces myocardial infarction in vivo. Methods and Results-Rabbit hearts were injected with vehicle or Ad.HSP70 at 3 sites (1.5ϫ10 9 pfu, 50 L/site) in the left ventricle (LV). Four days later, hearts were removed, and expression of inducible (HSP 70) and constitutive (HSC 70) proteins was measured in the LV and right ventricle (RV). Subsets of 5 to 7 animals in the vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups were subjected to 30 minutes of ischemia and 3 hours of reperfusion. Infarct size was measured by tetrazolium staining. Increased expression of HSP 70 was observed in LV injected with Ad.HSP70 compared with vehicle-treated hearts. HSP 70 was undetectable in RV, the noninjected region of the heart. The expression of HSC 70 remained unchanged in hearts treated with vehicle or Ad.HSP70. Infarct size (% risk area) decreased to 24.5Ϯ2.8 in Ad.HSP70-injected hearts compared with 41.9Ϯ2.8 and 42.7Ϯ2.5 in the vehicle-and Ad.LacZ-treated hearts (PϽ0.01). The infarct size was not different between the vehicle-and Ad.LacZ-treated hearts (PϾ0.05). The risk areas (% of LV) were not different among the 3 groups, ie, 50.1Ϯ5.2, 47.7Ϯ3.5, and 53.3Ϯ2.9 in vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups (PϾ0.05). Conclusions-Direct gene delivery of HSP 70 in vivo reduces the severity of ischemic injury in the heart. (Circulation.2001;103:877-881.)

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients.

Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time-consuming, and unexpected or unknown viruses may escape detection. Therefore, a novel random PCR approach was developed that allows sequence-independent amplification of viral nucleic acids from virus isolation-positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3 end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1-infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particleassociated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it clearly demonstrates the applicability of the PAN-PCR approach. This method should be particularly useful for characterizing virus isolates that have tested negative for all expected viruses and for identifying unknown viruses.Recent identification of human metapneumovirus (13), the severe acute respiratory syndrome (SARS) coronavirus (4), and HCoNL63 coronavirus (5, 14) suggests that other viruses pathogenic to humans have either not yet been discovered or might newly emerge. This notion is further supported by the observation that in ca. 30 to 45% of patients with clinical signs of lower respiratory tract infections an etiological agent cannot be identified by laboratory diagnosis even if sensitive PCR methods are used (9, 10). In the recent past, one successful approach to identify novel viruses has been virus isolation. After exclusion of known viruses, the virus isolates were subsequently characterized by molecular biological approaches, leading to the identification of genomic fragments of the virus. In addition to the use of degenerate primers from h...

We studied the susceptibility of primary ovarian cancer cells to oncolytic adenoviruses. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, we discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to coxsackie-adenovirus receptor or CD46. Specifically, we found that these adenovirus receptors were trapped in tight junctions and not accessible for virus binding. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-to-mesenchymal transition (EMT). We showed that specific, thus far little-explored adenovirus serotypes (Ad3, Ad7, Ad11, and Ad14) that use receptor(s) other than coxsackie-adenovirus receptor and CD46 were able to trigger EMT in epithelial ovarian cancer cells and cause efficient oncolysis. Our studies on ovarian cancer cultures and xenografts also revealed several interesting cancer cell biology features. Tumors in situ as well as tumor xenografts in mice mostly contained epithelial cells and cells that were in a hybrid stage where they expressed both epithelial and mesenchymal markers (epithelial/mesenchymal cells). These epithelial/mesenchymal cells are the only xenograft-derived cells that can be cultured and with passaging undergo EMT and differentiate into mesenchymal cells. Our study provides a venue for improved virotherapy of cancer as well as new insights into cancer cell biology. [Cancer Res 2009;69(12):5115-25]

Exosomes have been proposed as candidates for therapeutic immunization. The present study demonstrates that incorporation of the G protein of vesicular stomatitis virus (VSV-G) into exosome-like vesicles (ELVs) enhances their uptake and induces the maturation of dendritic cells. Targeting of VSV-G and ovalbumin as a model antigen to the same ELVs increased the cross-presentation of ovalbumin via an endosomal acidification mechanism. Immunization of mice with VSV-G and ovalbumin containing ELVs led to an increased IgG2a antibody response, expansion of antigen-specific CD8 T cells, strong in vivo CTL responses, and protection from challenge with ovalbumin expressing tumor cells. Thus, incorporation of VSV-G and targeting of antigens to ELVs are attractive strategies to improve exosomal vaccines.