Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.

Surface properties of Helicobacter pylori urease complex are essential for persistence.

The enzymatic activity of Helicobacter pylori's urease neutralises stomach acidity, thereby promoting infection by this pathogen. Urease protein has also been found to interact with host cells in vitro, although this property's possible functional importance has not been studied in vivo. To test for a role of the urease surface in the host/pathogen interaction, surface exposed loops that display high thermal mobility were targeted for inframe insertion mutagenesis. H. pylori expressing urease with insertions at four of eight sites tested retained urease activity, which in three cases was at least as stable as was wild-type urease at pH 3. Bacteria expressing one of these four mutant ureases, however, failed to colonise mice for even two weeks, and a second had reduced bacterial titres after longer term (3 to 6 months) colonisation. These results indicate that a discrete surface of the urease complex is important for H. pylori persistence during gastric colonisation. We propose that this surface interacts directly with host components important for the host-pathogen interaction, immune modulation or other actions that underlie H. pylori persistence in its special gastric mucosal niche.

rAAV.sFlt-1 gene therapy achieves lasting reversal of retinal neovascularization in the absence of a strong immune response to the viral vector.

PURPOSE: To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. METHODS: trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. RESULTS: After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45(+) leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. CONCLUSIONS: The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye.

Antitumor efficacy of the novel chemotherapeutic agent coramsine is potentiated by cotreatment with CpG-containing oligodeoxynucleotides.

Coramsine is a novel chemotherapeutic agent isolated from Solanum linnaeanum (devil's apple). Topical treatment provides clinical benefit for skin tumors. To evaluate the potential broader applicability of the drug, its in vivo anticancer efficacy in a murine model of malignant mesothelioma and its mode of action were investigated. Systemic administration of coramsine slowed tumor growth and prolonged survival time. Importantly, the antitumor efficacy of coramsine was enhanced when treatment was combined with stimulation of innate immunity using unmethylated CpG-containing oligodeoxynucleotides (ODNs). Combination treatment further slowed tumor growth and provided a survival benefit. Coramsine seems to kill tumor cells by direct cell lysis. Using 2 different assays to detect apoptosis (caspase activation and DNA fragmentation), we found no evidence that coramsine induces any form of programmed cell death. The fact that the efficacy of coramsine is potentiated by CpG ODNs suggests that coramsine-induced cell death is an immunologic null event.

Cutting edge: tumor-specific CTL are constitutively cross-armed in draining lymph nodes and transiently disseminate to mediate tumor regression following systemic CD40 activation.

The cross-arming of effector CTL in response to cross-presented tumor Ags is predicted to fail in the absence of CD40 stimulation. However, questions remain regarding the role of CD40 signaling and additional CD4+ T cell-derived signals in this process. To address this, we have analyzed the cross-arming of tumor-specific CTL effectors in vivo in a mouse model of established tumor and tumor regression following CD40 activation. We found that tumor-specific CTL were constitutively cross-armed in tumor-draining lymph nodes during tumor growth and that systemic CD40 activation did not alter CTL cross-arming in the tumor-draining lymph nodes. Rather, CD40 activation induced peripheral dissemination of tumor-specific CTL effectors that required continual CD40 stimulation to maintain peripheral CTL and tumor regression. These data indicate that CD40 activation enhances the peripheral survival of constitutively cross-armed CTL and that persistent CD4+ T cell signals are required for their long-term activity.