Development of a protective vaccine against Helicobacter pylori

Helicobacter pylori, which colonizes the stomach and causes the most common chronic infection in man, is associated with peptic ulceration, gastric carcinoma and gastric lymphoma. Studies in animals demonstrated that mucosal immunization could induce immune response against H. pylori and prevent H. pylori infection only if powerful mucosal adjuvants such as cholera toxin (CT) or heat-labile toxin of E. coli (LT) were used along with an H. pylori protein antigen. Adjuvants such as CT or LT cannot be used for humans because of their toxicity. Finding non-toxic alternative adjuvants/immunomodulators or immunization strategies that eliminates the use of adjuvants is critical for the development of efficacious human Helicobacter vaccines. We investigated whether several new adjuvants such as Muramyl Tripeptide Phosphatidylethonolamine (MTP-PE), QS21 (a Quil A derivative), Monophosphoryl lipid A (MPL) or heat shock proteins (HSP) of Mycobacterium tuberculosis could be feasible to develop a safe and effective mucosal vaccine against H. pylori using a murine model. C57/BL6 mice were immunized with liposomes incorporating each adjuvant along with urease, a major antigenic protein of H. pylori, to test their mucosal effectiveness. Since DNA vaccination eliminates both the use of adjuvants and antigens we also investigated whether immunization with plasmid DNA encoding urease could induce protective immunity to H. pylori infection in the same murine model. We found that oral vaccination with liposomal MTP-PE (6.7 m g) and urease, (100 m g) induced antigen-specific systemic and mucosal immune response and protected mice against H. pylori challenge when compared to control groups. Parenteral and mucosal immunizations with as little as 20 m g naked or formulated DNA encoding urease induced systemic and mucosal immune response against urease and partially protected mice against H. pylori infection. DNA vaccination provided long-lasting immunity and serum anti-urease IgG antibodies were elevated for up to 12 months. No toxicity was detected after immunizations with either liposomal MTP-PE and urease or plasmid DNA and both were well tolerated. We conclude that immunization liposomes containing MTP-PE and urease is a promising strategy deserving further investigation and may be considered for humans. DNA vaccination could be used to prime immune response prior to oral protein vaccination and may reduce the dose of protein and adjuvant needed to achieve protective immunity. ^

The characterization of PKC-isozyme specific EGFR-dependent Erk1/2 activation in androgen-independent prostate cancer cell lines

One growth factor receptor commonly altered during prostate tumor progression is the epidermal growth factor receptor (EGFR). EGFR signaling regulates Erk1/2 phosphorylation through multiple mechanisms. We hypothesized that PKC isozymes play a role in EGFR-dependent signaling, and that through PKC isozyme selective inhibition, EGFR-dependent Erk1/2 activation can be attenuated in AICaP cells. ^ To test the hypothesis, PKC activation was induced by 12-O-tetradecanoyi-phorbol-13-acetate (TPA) in PC-3 cells. As a result, Erk1/2 was activated similarly to what was observed upon EGF stimulation. EGF-induced Erk1/2 activation in PC-3 cells was PKC-dependent, as demonstrated through use of a selective PKC inhibitor, GF109203X. This provides evidence for PKC regulatory control over Erk1/2 signaling downstream of EGFR. Next, we demonstrated that when PKC was inhibited by GF109203X, EGF-stimulated Erk1/2 activation was inhibited in PC-3, but not DU145 cells. TPA-stimulated Erk1/2 activation was EGFR-dependent in both DU145 and PC-3 cells, demonstrated through abrogation of Erk1/2 activation by a selective EGFR inhibitor AG1478. These data support PKC control at or upstream of EGFR in AICaP cells. We observed that interfering with ligand/EGFR binding abrogated Erk1/2 signaling in TPA-stimulated cells, revealing a role for PKC upstream of EGFR. ^ Next, we determined which PKC isozymes might be responsible for Erk1/2 regulation. We first determined that human AICaP cell lines express the same PKC isozymes as those observed in clinical prostate cancer specimens (α, ϵ, &zgr;, ι and PKD). Isozyme-selective methods were employed to characterize discrete PKC isozyme function in EGFR-dependent Erk1/2 activation. Pharmacologic inhibitors implicated PKCα in TPA-induced EGFR-dependent Erk1/2 activation in both PC-3 and DU145 cells. Further, the cPKC-specific inhibitor, Gö6976 decreased viablilty of DU145 cells, providing evidence that PKCα is necessary for growth and survival. Finally, resveratrol, a phytochemical with strong cancer therapeutic potential inhibited Erk1/2 activation, and this correlated with selective inhibition of PKCα. These results demonstrate that PKC regulates pathways critical to progression of CaP cells, including those mediated by EGFR. Thus, PKC isozyme-selective targeting is an attractive therapeutic strategy, and understanding the role of specific PKC isozymes in CaP cell growth and survival may aid in development of effective, non-toxic PKC-targeted therapies. ^

An evaluation of the Cypress Creek watershed for inorganic chemical load as it flows into Lake Houston

Background. The Cypress Creek is one of the main tributaries of Lake Houston, which provides drinking water to 21.4 million customers. Furthermore, the watershed is being utilized for contact and non-contact recreation, such as canoeing, swimming, hiking trail, and picnics. Water along the creek is impacted by numerous wastewater outfalls from both point and non-point sources. As the creek flows into Lake Houston, it carries both organic and inorganic contaminants that may affect the drinking water quality of this important water source reservoir. Objective. This study was carried out to evaluate the inorganic chemical load of the water in Cypress Creek along its entire length, from the headwaters in Waller County and up to the drainage into Lake Houston. The purpose was to determine whether there are hazardous concentrations of metals in the water and what would be the likely sources. Method. Samples were collected at 29 sites along the creek and analyzed for 29 metals, 17 of which were on the Environmental Protection Agency priority pollution list. Public access sites primarily at bridges were used for sample collection. Samples were transported on ice to the University Of Texas School Of Public Health laboratory, spiked with 2 ml HNO3 kept overnight in the refrigerator, and the following day transported to the EPA laboratory for analysis. Analysis was done by EPA Method 200.7-ICP, Method 200.8ICP/MS and Method 245.1-CVAAS. Results. Metals were present above the detection limits at 65% of sites. Concentrations of aluminum, iron, sodium, potassium, magnesium, and calcium, were particularly high at all sites. Aluminum, sodium, and iron concentrations greatly exceeded the EPA secondary drinking water standards at all sites. ^ Conclusion. The recreational water along Cypress Creek is impacted by wastewater from both permitted and non-permitted outfalls, which deposit inorganic substances into the water. Although a number of inorganic contaminants were present in the water, toxic metals regulated by the EPA were mostly below the recommended limits. However, high concentrations of aluminum, sodium, and iron in the Cypress Creek bring forward the issue of unauthorized discharges of salt water from mining, as well as industrial and domestic wastewater.^

APPLICATION OF THE BRAINSTEM EVOKED RESPONSE (BER) TO NEUROTOXICITY EVALUATION OF THE AUDITORY SYSTEM: ASSESSMENT OF INORGANIC LEAD-INDUCED OTOTOXICITY

An experimental procedure was developed using the Brainstem Evoked Response (BER) electrophysiological technique to assess the effect of neurotoxic substances on the auditory system. The procedure utilizes Sprague-Dawley albino rats who have had dural electrodes implanted in their skulls, allowing neuroelectric evoked potentials to be recorded from their brainstems. Latency and amplitude parameters derived from the evoked potentials help assess the neuroanatomical integrity of the auditory pathway in the brainstem. Moreover, since frequency-specific auditory stimuli are used to evoke the neural responses, additional audiometric information is obtainable. An investigation on non-exposed control animals shows the BER threshold curve obtained by tests at various frequencies very closely approximates that obtained by behavioral audibility tests. Thus, the BER appears to be a valid measure of both functional and neuroanatomical integrity of the afferent auditory neural pathway.^ To determine the usefulness of the BER technique in neurobehavioral toxicology research, a known neurotoxic agent, Pb, was studied. Female Sprague-Dawley rats were dosed for 45 days with low levels of Pb acetate in their drinking water, after which BER recordings were obtained. The Pb dosages were determined from the findings of an earlier pilot study. One group of 6 rats received normal tap water, one group of 7 rats received a solution of 0.1% Pb, and another group of 7 rats received a solution of 0.2% Pb. After 45 days, the three groups exhibited blood Pb levels of 4.5 (+OR-) 0.43 (mu)g/100 ml, 37.8 (+OR-) 4.8 (mu)g/100 ml and 47.3 (+OR-) 2.7 (mu)g/100 ml, respectively.^ The results of the BER recording indicated evoked response waveform latency abnormalities in both the Pb-treated groups when midrange frequency (8 kHz to 32 kHz) stimuli were used. For the most part, waveform amplitudes did not vary significantly from control values. BER recordings obtained after a 30-day recovery period indicated the effects seen in the 0.1% Pb group had disappeared. However, those anomalies exhibited by the 0.2% Pb group either remained or increased in number. This outcome indicates a longer lasting or possibly irreversible effect on the auditory system from the higher dose of Pb. The auditory pathway effect appears to be in the periphery, at the level of the cochlea or the auditory (VIII) nerve. The results of this research indicate the BER technique is a valuable and sensitive indicator of low-level toxic effects on the auditory system.^