The mode by which Helicobacter pylori, the causative agent of most gastric ulcers, is transmitted remains undetermined. Epidemiological evidence suggests these organisms are waterborne; however, H. pylori has rarely been grown from potential water sources. This may be due to the ability of this organism to rapidly enter the viable but nonculturable (VBNC) state. Our investigation examines the entrance of H. pylori into this state in laboratory cultures and a natural freshwater environment as well as the relationship between morphology and culturability. To this end, membrane diffusion chambers were utilized to expose the cells to the natural fluctuations of a freshwater stream. In both the laboratory and environment, samples were assayed for culturability using plate counts and stained using a LIVE/DEAD BacLight assay for viability and morphological determinations. Additionally, water samples were collected, six environmental parameters were measured, and resuscitation conditions were examined. H. pylori was observed to lose culturability in the laboratory and stream, although viability was maintained. While the results of our study agree with those of previous studies which suggested that there is a transition in morphology from rods to cocci as culturability is lost, the morphological distribution of cells did not change as culturability was lost in the environment. The majority of cells in the VBNC state in the laboratory are cocci; however, all morphological forms were present in the environment. The results of these studies suggest that H. pylori persists in laboratory cultures and the environment in the VBNC state and that cells in this state represent a public health hazard.Infection by the gram-negative microaerophilic rod Helicobacter pylori is associated with the development of chronic human gastritis, peptic ulcers, and gastric adenocarcinoma (2,5,26). It has been estimated that more that half of the world's population is infected with this organism (18). Despite such a high incidence of infection, the bacterium's reservoir and mode of transmission remain undetermined. Molecular methods have detected the presence of H. pylori DNA in river water, well water, and wastewater as well as in surface and shallow groundwater, suggesting that this organism is waterborne and might be transmitted by the fecal-oral route (12,14,22). However, only a single study has been published which claims that isolation of H. pylori directly from environmental sources had been performed and that the isolation occurred only following immunomagnetic separation of the cells from raw sewage (20).It has been postulated that the inability to culture H. pylori from the environment is due to its entrance into the viable but nonculturable (VBNC) state. Cells that have entered this state are no longer culturable on routine bacteriological media, although they remain viable (25). Entrance of the bacterium into the VBNC state is induced by a variety of adverse conditions, such as temperature downshift or nutrient depletion (25). The entr...

T helper17 (Th17) cells are known to play a critical role in adaptive immune responses to several important extracellular pathogens. Additionally, Th17 cells are implicated in the pathogenesis of several autoimmune and inflammatory disorders as well as in cancer. Therefore, it is essential to understand the mechanisms that regulate Th17 differentiation. Notch signaling is known to be important at several stages of T cell development and differentiation. Here we report that Notch1 is activated in both mouse and human in-vitro polarized Th17 cells and blockade of Notch signaling significantly down-regulates the production of Th17 associated cytokines suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies as well as chromatin immunoprecipitation, that IL-17 and RORγt are direct transcriptional targets of Notch signaling in Th17 cells. Finally, in-vivo inhibition of Notch signaling reduced IL-17 production and Th17 mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Thus, this study highlights the importance of Notch signaling, in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.

Vibrio vulnificus has proven difficult to culture from water or shellfish during winter months, which is attributed to the viable but nonculturable (VBNC) state. Because reactive oxygen species were found to be involved in the low temperature-induced entrance of V. vulnificus into this state, we generated an oxyR mutant which lacks catalase activity. This strain is nonculturable on solid media even at ambient temperature, due to the presence of H(2)O(2) in such media. Low temperature incubation of the parent resulted in loss of catalase activity, making the cells H(2)O(2) sensitive, and paralleling the loss of culturability (entry into the VBNC state). Thus, cells of V. vulnificus in the VBNC state are likely exhibiting this response to low in situ temperature and only when the artificial condition of laboratory culture is attempted are the cells nonculturable due to cold-induced loss of catalase activity. To our knowledge, this is the first study providing direct evidence for the metabolic basis of nonculturability and the viable but nonculturable state.

The Lyme disease spirochete Borrelia burgdorferi is the only known human pathogen that directly activates invariant NKT (iNKT) cells. The number and activation kinetics of iNKT cells vary greatly among different strains of mice. We now report the role of the iNKT cell response in the pathogenesis of Lyme disease using C57BL/6 mice, a strain with optimal iNKT cell activation that is resistant to the development of spirochetal-induced inflammation. During experimental infection of B6 mice with B. burgdorferi, iNKT cells localize to the inflamed heart where they are activated by CD1d-expressing macrophages. Activation of iNKT cells in vivo results in the production of IFN-γ, which we demonstrate ameliorates the severity of murine Lyme carditis by at least two mechanisms. First, IFN-γ enhances the recognition of B. burgdorferi by macrophages, leading to increased phagocytosis of the spirochete. Second, IFN-γ activation of macrophages increases the surface expression of CD1d, thereby facilitating further iNKT activation. Collectively, our data demonstrate that in the resistant background, B6, iNKT cells modulate the severity of murine Lyme carditis through the action of IFN-γ, which appears to self-renew through a positive feedback loop during infection.

Members of the testis-specific serine/threonine kinases (Tssk) family may have a role in sperm differentiation in the testis and/or fertilization. To gain insight into the functional relevance of these kinases, their expression was examined both at the mRNA and protein levels. Quantitative PCR analysis confirmed that all five Tssk mRNAs are almost exclusively expressed postmeiotically in the testis. Recombinant mouse and human Tssks were cloned and used for validation of an array of commercial and custom-made antibodies against Tssks. Immunolocalization in mouse testis, and in mouse and human sperm, showed that Tssk1, Tssk2, Tssk4 and Tssk6, but not Tssk3, were present in mouse sperm and in germ cells from mouse testis. TSSK1, TSSK2 and TSSK6 were also detected in human sperm, while TSSK3 was absent. In both mouse and human sperm, Tssk1 was partially soluble, while Tssk2, Tssk4 and Tssk6 were insoluble in non-ionic detergents. In vitro recombinant TSSK2 activity assays showed maximum enzymatic activity at 5 mM Mg(2+) and a Km for ATP of ∼10 µM. These, observations together with findings that the Tssk1/Tssk2 double knock-out as well as the Tssk6 null mice are sterile without presenting other detectable defects, suggest that these kinases could be used as targets for male contraception.

The interaction of Borrelia burgdorferi, the causative agent of Lyme borreliosis, with phagocytic cells induces the activation of NF-B and the expression of proinflammatory cytokines including tumor necrosis factor alpha (TNF-␣). B. burgdorferi-induced TNF-␣ production is also dependent on the activation of p38 mitogenactivated protein (MAP) kinase. The specific contribution of these signaling pathways to the response of phagocytic cells to the spirochete and the molecular mechanisms underlying this response remain unresolved. We now show that p38 MAP kinase activity regulates the transcriptional activation of NF-B in response to spirochetal lysate stimulation of phagocytic cells. The regulation occurs at the nuclear level and is independent of the translocation of the transcription factor to the nucleus or its capacity to bind to specific DNA target sequences. In RAW264.

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Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.In the United States, Vibrio vulnificus is the leading cause of death associated with consumption of seafood (12, 15). Consumption of raw Gulf Coast oysters from April to November is responsible for nearly all of the cases. Although V. vulnificus is abundant in oysters during that time of year, cases are rare even in the high-risk group (i.e., those with preexisting liver disease or who are immunocompromised) (7). A major obstacle in developing effective control strategies is the inability to identify in the oysters V. vulnificus strains that are capable of causing human illness.Two major research needs identified at a 1994 V. vulnificus workshop sponsored by the Food and Drug Administration (FDA) were to develop methods to distinguish virulent V. vulnificus strains from avirulent strains and to determine the infectious dose (22). Since human volunteer studies with V. vulnificus are not ethical, a consensus approach was proposed to determine the infectious dose by relating disease frequency with exposure. It was also suggested that a collection of strains from oysters and human septicemia cases associated with oyster consumption should be characterized in various assays in an attempt to determine traits that may be linked to virulence. The Centers for Disease Control and Prevention (CDC), FDA, and various state departments of health collected approximately 75 well-characterized clinical strains from human septicemia cases with known sources of oysters consumed, patient histories, etc. A recent study of the abundance of V. parahaemolyticus and V. vulnificus in retail oysters by the Interstate Shellfish Sanitation Conference and the FDA generated a large collection of V. vulnificus cultures that is seasonally and geographically diverse and well defined (3). Molecular characterization and virulence assays of representative V. vulnificus isolates from these tw...