Experiments were performed using the standardized murine model of Helicobacter pylori infection to determine the immunogenicity of H. pylori outer membrane vesicles in immune protection. These vesicles, which are naturally shed from the surface of the bacterium, induce a protective response when administered intragastrically to mice in the presence of cholera holotoxin, despite the absence of the urease enzyme and associated Hsp54 chaperonin. Immunoblotting identified a specific serum immunoglobulin G (IgG) response to an 18-kDa outer membrane protein in a significant number of immunized animals. This commonly expressed, immunodominant protein was subsequently identified as lipoprotein 20 (Lpp20). Hybridoma backpacks secreting an IgG1 subclass monoclonal antibody to Lpp20 were generated in H. pylori-infected mice and were found to significantly reduce bacterial numbers, providing evidence that this surface-exposed antigen is a true vaccine candidate and not merely an antigenic marker for successful, protective immunization.Helicobacter pylori, a bacterium which is estimated to infect more than half the world's population, is associated with peptic ulcer disease (4) and the development of gastric cancer (32). Immunization against this bacterium represents a cost-effective strategy to reduce global gastric cancer rates (5) and would also have a major impact on H. pylori-related peptic ulcer disease. H. pylori vaccine candidates identified to date include the urease enzyme (20,40,51,55) and the urease enzyme chaperonin heat shock protein A (21). Mice immunized with purified VacA cytotoxin are also protected from challenge with a Tox ϩ strain of H. pylori (48). A common factor among these three vaccine candidates is their reported association with the outer membrane of H. pylori (1,16,17,27,36,52,57). The potential of catalase as an H. pylori vaccine candidate has also been identified (58). This enzyme, which is found in both the cytosol and the periplasmic space of H. pylori (28), is also thought to be surface exposed (57). More recently, the screening of recombinant H. pylori antigens (30) has identified another five potential H. pylori vaccine candidates. These include Lpp20, a conserved H. pylori lipoprotein that is membrane associated but not surface exposed (38).In our search for candidate H. pylori vaccine antigens, we have focused on the outer membrane of the bacterium. Like many other gram-negative bacteria (reviewed in reference 25), H. pylori and Helicobacter felis shed part of their outer membrane as vesicles when grown under certain conditions (34). These outer membrane vesicles (OMV) are thought to be formed when the outer membrane of the bacterium expands faster than the underlying peptidoglycan layer, resulting in portions of the membrane blebbing off the surface of growing cells (44). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis reveals that the protein and lipopolysaccharide content of these OMV closely resembles that of a Sarkosyl-insoluble outer membrane preparati...

We have used whole-exome sequencing in ten individuals from four unrelated pedigrees to identify biallelic missense mutations in the nuclear-encoded mitochondrial inorganic pyrophosphatase (PPA2) that are associated with mitochondrial disease. These individuals show a range of severity, indicating that PPA2 mutations may cause a spectrum of mitochondrial disease phenotypes. Severe symptoms include seizures, lactic acidosis, cardiac arrhythmia, and death within days of birth. In the index family, presentation was milder and manifested as cardiac fibrosis and an exquisite sensitivity to alcohol, leading to sudden arrhythmic cardiac death in the second decade of life. Comparison of normal and mutant PPA2-containing mitochondria from fibroblasts showed that the activity of inorganic pyrophosphatase was significantly reduced in affected individuals. Recombinant PPA2 enzymes modeling hypomorphic missense mutations had decreased activity that correlated with disease severity. These findings confirm the pathogenicity of PPA2 mutations and suggest that PPA2 is a cardiomyopathy-associated protein, which has a greater physiological importance in mitochondrial function than previously recognized.

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We found evidence of Helicobacter infection in a significant number of patients presenting for colonoscopy but no specific association between the presence of these bacteria and colon disease. Our finding of disparity between molecular and serological techniques to detect Helicobacter species suggests that future studies should not rely on serology alone to detect these bacteria in the human colon.