Role of the Abcg2 transporter in plasma levels and tissue accumulation of the anti-inflammatory tolfenamic acid in mice.

The Breast Cancer Resistance Protein (BCRP/ABCG2) is an ATP-binding cassette efflux transporter that is expressed in the apical membrane of cells from relevant tissues involved in drug pharmacokinetics such as liver, intestine, kidney, testis, brain and mammary gland, among others. Tolfenamic acid is an anti-inflammatory drug used as an analgesic and antipyretic in humans and animals. Recently, tolfenamic acid has been repurposed as an antitumoral drug and for use in chronic human diseases such as Alzheimer. The aim of this work was to study whether tolfenamic acid is an in vitro Abcg2 substrate, and to investigate the potential role of Abcg2 in plasma exposure, secretion into milk and tissue accumulation of this drug. Using in vitro transepithelial assays with cells transduced with Abcg2, we showed that tolfenamic acid is an in vitro substrate of Abcg2. The in vivo effect of this transporter was tested using wild-type and Abcg2-/- mice, showing that after oral and intravenous administration of tolfenamic acid, its area under the plasma concentration-time curve in Abcg2-/- mice was between 1.7 and 1.8-fold higher compared to wild-type mice. Abcg2-/- mice also showed higher liver and testis accumulation of tolfenamic acid after intravenous administration. In this study, we demonstrate that tolfenamic acid is transported in vitro by Abcg2 and that its plasma levels as well as its tissue distribution are affected by Abcg2, with potential pharmacological and toxicological consequences.

Development of an indirect ELISA for detecting humoral immunodominant proteins of Mycoplasma hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of porcine enzootic pneumonia, which has been associated with economic losses due to reduced daily weight gain and feed efficiency. Although it has a small genome and no more than 1000 genes, M. hyopneumoniae can be cultured in cell free media. However, some proteins were not expressed or were only expressed in negligible amounts under culture conditions. Nevertheless, some of these proteins can be expressed at a high level and induce a strong and rapid immune response after M. hyopneumoniae infection. The unexpressed or less expressed proteins may play critical roles in pathogenesis and/or immune response. In order to find the differentially expressed proteins of M. hyopneumoniae between culture condition and infected animals, we established an indirect ELISA for the detection of humoral immunodominant proteins which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera by using Mhp366 protein which did not react with sera from bacterin-immunized pigs, but revealed a strong immunoreaction with porcine convalescent sera. RESULTS: The checkerboard titration method was done by using porcine convalescent sera as positive sera and inactivated bacterin-induced hyperimmune sera as negative sera. The bacterial lysates of fusion proteins and free GST protein without dilution were the optimal coating antigens. The optimal blocking buffer was PBS with 10% FBS and 2.5% skimmed milk. In the checkboard ELISAs, when the sera were diluted at 1:500 and the HRP-labeled rabbit anti-pig IgG were diluted at 1:20000, most positive result was obtained for the assay. CONCLUSIONS: This established indirect ELISA can be used as a tool for the detection of humoral immunodominant proteins of M. hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

Vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) at a low temperature leads to a low antibody response against Aeromonas salmonicida.

We present a study on the effect of water temperature on immunization of Atlantic lumpfish. In total, 360 fish were vaccinated with either 50 µl of an oil-based injection vaccine (VAX), with Aeromonas salmonicida and Vibrio salmonicida antigens, or PBS. Fish were vaccinated at three different water temperatures, 5°C, 10°C and 15°C, and sorted into six groups (N = 60). Lumpfish were weighed every 3 weeks after vaccination, sampled at 3, 6, 9 and 18 weeks post-immunization (wpi) and evaluated by modified Speilberg score, ELISA and immunoblotting. Vaccinated fish showed low antibody response against V. salmonicida. Fish vaccinated at 5°C showed significantly lower antibody response against A. salmonicida throughout the study. At higher temperatures, vaccinated fish showed significantly increased antibody responses, at 18 wpi for 10°C and at 6 and 18 wpi for 15°C. Immunoblotting demonstrated specific response against the LPS antigen of A. salmonicida in the 10°C and 15°C VAX groups. Mean body weight increased in all groups throughout the study. Vaccinated fish had low Speilberg scores with no melanization of abdominal tissue. Our results show that vaccinating lumpfish at a lower water temperature may lead to a low antibody response against A. salmonicida.

Proteomic profiling of ovine serum by SELDI-TOF MS: optimisation, reproducibility and feasibility of biomarker discovery using routinely collected samples.

The diagnosis of infectious diseases in animals may be enhanced by study of the serum proteome in which myriad components are influenced by physiological and pathological processes. Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) has the capacity to detect known and unknown immunologically relevant molecules in the serum proteome. Optimum combinations of ProteinChip array surfaces, energy absorbing molecules, sample dilutions and instrument settings were determined for spectral generation from whole ovine sera. The coefficient of variation for within and between chip mass/charge and peak intensity were <0.03% and <23%, respectively. There were minor alterations in spectra associated with storage of chips or machine drift. Clotting times of 30 min to 3h did not greatly alter protein spectra although storage of sera at -20 degrees C led to alterations. However, routinely collected serum samples stored at -20 degrees C were useful for identification of biomarkers associated with vaccination with a bacterial antigen. This information will inform future studies on serum proteome profiling in livestock, but independent assessments are recommended for each species.

Live vaccine strain Francisella tularensis is detectable at the inoculation site but not in blood after vaccination against tularemia.

INTRODUCTION: Live vaccine strain (LVS) Francisella tularensis is a live, attenuated investigational tularemia vaccine that has been used by the US Army for decades to protect laboratory workers. Postvaccination bacterial kinetic characteristics of LVS at the inoculation site and in the blood are unknown and, therefore, were assessed in a prospective study. LVS vaccination of laboratory workers provided the opportunity to compare culture with polymerase chain reaction (PCR) for the detection of F. tularensis in human clinical samples. METHODS: Blood and skin swab samples were prospectively collected from volunteers who received the LVS tularemia vaccine at baseline (negative controls) and at 5 specified time points (days 1, 2, 7 or 8, 14 or 15, and 35 after vaccination). Bacterial culture and PCR of whole blood samples (17 volunteers) and inoculation site swabs (41 volunteers) were performed. RESULTS: The culture and PCR results of all blood samples were negative. Results of real-time PCR from the inoculation site samples were positive for 41 (100%) of 41 volunteers on day 1, for 40 (97.6%) of 41 volunteers on day 2, for 24 (58.5%) of 41 on day 7 or 8, for 6 (16.7%) of 36 on day 14 or 15, and for 0 (0%) of 9 on day 35. Positive results of bacterial cultures of the inoculation site samples occurred significantly less frequently, compared with PCR testing, with 4 (9.8%) of 41 volunteers having positive results on day 1 (P<.001) and 4 (9.8%) of 41 on day 2 (P<.001); all results from subsequent days were negative. CONCLUSIONS: F. tularensis LVS genomic DNA was detected in the majority of samples from the inoculation site up to 1 week after LVS vaccination, with real-time PCR being more sensitive than culture. Our data suggest that bacteremia does not occur after LVS vaccination in normal, healthy human volunteers.

Oral immunization of mice with attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit.

OBJECTIVE: To establish attenuated Salmonella typhimurium producing Helicobacter pylori (H. pylori) urease subunit B (UreB) and determine whether it could be used as an oral vaccine against H. pylori. METHODS: H. pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector pTC01 after sequencing, and then transformed into attenuated Salmonella typhimurium SL3261 to acquire SL3261/pTC01-UreB. The expression of H. pylori UreB in SL3261 was detected by Western blot. Twelve weeks after oral immunization of mice, antibody responses were evaluated using serum and intestinal fluid by ELISA assay. Interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in the supernatant of spleen cells culture were also assessed by ELISA. In vitro stability of pTC01-UreB plasmid in SL3261 was confirmed by growing in Luria Broth (LB) medium to 80 generations. RESULTS: The UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB as evidenced by sequence analysis. Enzyme digestion revealed that the correct pTC01-UreB was obtained. Western blot showed that a 61kDa protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum. After 80 generations of continuous culture, the recombinant plasmid pTC01-UreB was stable in SL3261 and had no obvious toxicity. Multiple oral immunizations with SL3261/pTC01-UreB could significantly induce H. pylori-specific mucosal IgA response as well as serum IgG response. Moreover, there were significant increases of IFN-gamma and IL-10 in the SL3261/pTC01-UreB group. Finally, no obvious side effects for mice and no change in gastric inflammation were observed. CONCLUSION: Attenuated Salmonella typhimurium expressing H. pylori UreB may be used as oral vaccine against H. pylori infection.

A rapid and sensitive chemiluminescence assay for evaluation of functional opsonic activity of Haemophilus influenzae type b-specific antibodies.

Luminol-enhanced chemiluminescence (CL) of heterologous neutrophils was used to assess the capacity of a 1-ng/ml concentration of Haemophilus influenzae type b (Hib)-specific antibodies to induce opsonization of Hib with autologous heat-inactivated sera from children immunized with Hib capsular polysaccharide-polyribosylribitolphosphate (Hib-PRP) conjugate vaccine. Serum samples from 15 of 36 children (42%) vaccinated with Hib-PRP conjugate vaccine had protective levels of Hib-specific antibodies of > or = 1,000 ng/ml. Ten of these 15 (67%) had poor or nonfunctional opsonic activity. Of the 10 children whose sera lacked opsonic activity, 5 (50%) presented with recurrent Hib infection. In contrast, none of the sera of 20 healthy adults lacked opsonic capability. CL intensity was proportional to the concentration of anti-Hib antibodies used for opsonization. Furthermore, the titers of Hib-PRP-specific antibody in children and adults did not correlate with opsonic activity. These results suggest that luminol-enhanced CL as described here with minute concentrations of antibody for opsonization can be used to assess functional capacity of anti-Hib antibodies after vaccination or natural infection in the evaluation of patients with recurrent infections.