Research Note: The immune enhancement ability of inulin on ptfA gene DNA vaccine of avian Pasteurella multocida.

To evaluate the ability of inulin to enhance the immune response of a ptfA gene DNA vaccine for avian Pasteurella multocida, inulin was added as an adjuvant to the ptfA-DNA vaccine, obtaining an inulin-adjuvant DNA vaccine. The DNA vaccine was administered to chickens; a fimbria protein vaccine and an attenuated live vaccine were used as positive controls. The levels of the serum antibody and concentrations of interferon-γ (IFN-γ), interleukin-2 (IL-2), and interleukin-4 (IL-4) were determined, and a lymphocyte proliferation assay was performed. After being challenged with virulent P. multocida, the protective efficacy was evaluated. The results showed that the serum antibodies induced by the ptfA-DNA vaccine were not enhanced by inulin. The stimulation index values and the concentrations of IL-2 and IFN-γ in chickens vaccinated with inulin-adjuvant DNA vaccine were significantly higher than those in chickens vaccinated with the DNA vaccine, those with the fimbria protein vaccine, and the chickens gavaged with inulin. The concentrations of IL-4 in the inulin-adjuvant DNA vaccine group and the fimbria protein vaccine group were higher than those in the DNA vaccine group and the inulin-gavage group. The protective efficacy rates of the attenuated live vaccine group, the fimbria protein vaccine group, the DNA vaccine group, the inulin-adjuvant DNA vaccine group, and the inulin-gavage group were 90, 70, 55, 65, and 55%, respectively.

Immune efficacy of DNA vaccines based on oprL and oprF genes of Pseudomonas aeruginosa in chickens.

Pseudomonas aeruginosa (P. aeruginosa) is a zoonotic pathogen that can infect a variety of animals, including poultry. However, as there is no commercial vaccine available it is imperative that new and effective vaccines are developed. In this study, 2 monovalent DNA vaccines (pOPRL and pOPRF), one divalent combination DNA vaccine (pOPRL+pOPRF) and one fusion DNA vaccine (pOPRLF) were constructed based on the oprL and oprF genes of P. aeruginosa. These vaccines were administered to chickens, an outer membrane protein vaccine (OMP vaccine) and inactivated vaccine used as positive controls. The serum antibody, interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4) concentrations were determined and lymphocyte proliferation assays were performed. After challenging with virulent P. aeruginosa, protective efficacy was evaluated. Following vaccination, serum antibodies, stimulation index (SI) values, concentrations of IL-2 and IFN-γ in chickens vaccinated with the bivalent combination DNA vaccine and fusion DNA vaccine were found to be significantly higher than in those chickens vaccinated with the 2 monovalent DNA vaccines. Moreover, the immune indexes in the bivalent combination DNA vaccine group were higher than those in the fusion DNA vaccine group. However, the concentrations of IL-4 in the 4 DNA vaccine groups were of no significant difference. The protective efficacy rate provided by pOPRL, pOPRF, pOPRLF, pOPRL+pOPRF, inactivated vaccine and OMP vaccine were 53.3%, 40%, 66.7%, 80%, 93.3%, and 80%, respectively. The results indicate that DNA vaccines constructed with the oprL and oprF genes of P. aeruginosa, particularly the divalent combination DNA vaccine, represent better potential vaccines. This study has laid a foundation for the design and application of future DNA vaccines of P. aeruginosa.

Construction of a ptfA chitosan nanoparticle DNA vaccine against Pasteurella multocida and the immune response in chickens.

The aim of this study was to evaluate the effect of chitosanon the immune response induced by a DNA vaccine based on the ptfA gene of avian Pasteurella multocida. Naked DNA vaccine was packed with chitosanmolecules, resulting in a chitosannanoparticle DNA vaccine. The encapsulation efficiency, shape, size and resistance to DNA degradation were determined. The vaccine was administered to chickens and serum antibody, interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4) concentrations were determined and lymphocyte proliferation assays were performed. After challenge with virulent avian P. multocida, protective efficacy was evaluated. The encapsulation efficiency of the chitosan nanoparticle DNA vaccine was 95.3%. The particle size was approximately 200nm and close to spherical in shape and it could effectively resist degradation by DNases. Following vaccination, serum antibodies, stimulation index (SI) value and concentrations of IFN-γ and IL-2 in chickens vaccinated with the chitosan nanoparticle DNA vaccine were significantly higher than those that were vaccinated with the naked DNA vaccine (P-values are 0.026, 0.045, 0.039 and 0.024, respectively). However, the concentrations of IL-4 in the two DNA vaccines group were no significant difference (P=0.157). The protective efficacy rate provided by naked DNA vaccine, chitosan nanoparticle DNA vaccine and the attenuated live vaccine were 56%, 68% and 88%, respectively. The results indicated that chitosan was able to enhance the immune response to a naked DNA vaccine based on the ptfA gene of P. multocida.

Evaluation of immunogenicity and protective efficacy of recombinant ptfA of avian Pasteurella multocida.

Avian Pasteurella multocida is the causative agent of fowl cholera, a disease much affecting the poultry industry. In order to study the efficacy of the recombinant subunit vaccine constructed with ptfA gene of avian P. multocida, the ptfA gene fragment amplified by PCR from avian P. multocida was cloned into the prokaryotic expression vector pET32a and the recombinant plasmid pET32a-ptfA was obtained. The pET32a-ptfA was expressed in Escherichiacoli BL21(DE3) and the target protein rPtfA was purified. The purified protein was then mixed with Freund's adjuvant and the recombinant subunit vaccine was obtained. Three groups of chickens labeled as rPtfA, attenuated live vaccine and PBS were vaccinated with the recombinant subunit vaccine, attenuated live vaccine and PBS, respectively. Serum antibodies, peripheral blood lymphocyte proliferation (PBLP) and interferon-γ (IFN-γ) level secreted by peripheral blood lymphocyte were tested. The immunized chickens were finally challenged with virulent avian P. multocida and the protection rate was counted. Indirect ELISA showed the levels of antibodies in rPtfA and attenuated vaccine groups were most significantly higher than the other groups (P<0.01), and the former was slightly lower than the latter. Peripheral blood lymphocyte proliferation experiments and IFN-γ experiments indicated that SI value and the levels of IFN-γ induced by ConA in the two vaccine groups were significantly higher than those of the PBS groups (P<0.01), and that the attenuated vaccine group was higher than the rPtfA group. The protection rates of rPtfA and attenuated live vaccines were 45% and 75%, respectively. The results indicated that the PtfA recombinant subunit vaccine was capable of improving the immunity level and inducing a protective effect for the vaccinated chickens, but it was barely satisfactory.

Structure and nano-mechanical characteristics of surface oxide layers on a metallic glass.

Owing to their low elastic moduli, high specific strength and excellent processing characteristics in the undercooled liquid state, metallic glasses are promising materials for applications in micromechanical systems. With miniaturization of metallic mechanical components down to the micrometer scale, the importance of a native oxide layer on a glass surface is increasing. In this work we use TEM and XPS to characterize the structure and properties of the native oxide layer grown on Ni(62)Nb(38) metallic glass and their evolution after annealing in air. The thickness of the oxide layer almost doubled after annealing. In both cases the oxide layer is amorphous and consists predominantly of Nb oxide. We investigate the friction behavior at low loads and in ambient conditions (i.e. at T = 295 K and 60% air humidity) of both as-cast and annealed samples by friction force microscopy. After annealing the friction coefficient is found to have significantly increased. We attribute this effect to the increase of the mechanical stability of the oxide layer upon annealing.

Age effects on ectopic bone formation induced by purified bone morphogenetic protein.

A mixture of heparin-Sepharose-purified bovine bone morphogenetic protein and type I atellocollagen was implanted in the subcutaneous tissues of 4-week, 10-month and 18-month-old rats. The implants were removed at 7, 14 and 21 days after implantation. The effects of rat age on ectopic bone formation were evaluated on the explants using haematoxylin-eosin staining, morphometric analysis, alkaline phosphatase activity and calcium content determination, as well as immunohistochemical staining of type IV collagen present in the basement membrane of blood vessels. On day 14 and 21, bone was observed in 4-week and 10-month-old rats, but the amount of bone formed in the latter was less than in the 4-week-old rats. In 18-month-old rats, bone was first found focally in very limited regions of the explants on day 21 and the amount of bone was much less than in 4-week-old rats. At all periods, alkaline phosphatase activity was higher in younger rats. On day 7, there were more blood vessels in the explants of 4-week-old rats than in those of 10-month or 18-month-old rats. On day 14 and 21, more blood vessels were found in the central regions of the explants in 4-week-old rats than in the same regions in 10-month or 18-month-old rats. The findings in the present study indicate that the rate and quantity of ectopic bone formation are reduced in aged rats, and suggest that the difference in blood vessel distribution is related to this reduction in ectopic bone formation.

Cloning and in vitro expression of TPK3, a Toxoplasma gondii homologue of shaggy/glycogen synthase kinase-3 kinases.

As an initial effort to dissect the signaling pathways responsible for pathogenesis of Toxoplasma gondii infection, we report the cloning and in vitro functional studies of TPK3 (Toxoplasma protein kinase-3), a homologue of shaggy/glycogen synthase kinase-3 (GSK-3) kinases. The shaggy/GSK-3 family of kinases are highly conserved protein kinases that play important roles in cell fate determination, nuclear signaling and hormonal regulation. The TPK3 gene was isolated by RT-PCR with degenerate primers corresponding to highly conserved regions of serine/threonine protein kinases. The complete sequences of genomic and cDNA clones indicated the open reading frame, 1185 bp in size, is interrupted by five introns. The predicted protein sequence of TPK3 shows 54% identity to shaggy/GSK-3 over the catalytic domains. Southern analysis revealed TPK3 is a single copy locus in the Toxoplasma genome. Antisera to other GSK-3 proteins from other species recognized GST-TPK3 and a protein of the predicted size in parasites lysates. In vitro kinase assays with GST-TPK3 indicated that TPK3 autophosphorylates and phosphorylates protein phosphatase inhibitor-2 (I-2), a specific substrate of GSK-3 kinase.

Cloning, localization, and functional expression of a human brain inward rectifier potassium channel (hIRK1).

We have cloned a novel human brain inward rectifier K+ channel (hIRK1), which shares approximately 60% amino acid identity with another human inward rectifier (hIRK2) but 98% identity with the mouse IRK1. The hIRK1 mRNA is expressed in several human tissues: skeletal muscle > placenta > heart > brain > lung > kidney. In human brain, the hIRK1 mRNA is uniformly distributed (except for a higher level in the corpus callosum, which contains white matter and glial cells), whereas the hIRK2 mRNA is expressed in major regions of the basal ganglia and limbic system. Xenopus oocytes injected with hIRK1 cRNA expressed an inwardly rectifying K+ current that was blocked by extracellular Ba2+. The hIRK1 channel carried a significant outward current when membrane potential was more positive than the K+ equilibrium potential (EK) and therefore had an "N-shape" current-voltage relation, resembling that of the native cardiac IRK channel. The resting membrane potential was near EK in oocytes expressing hIRK1, but was approximately -40 mV in H2O-injected or non-injected oocytes. The ability of hIRK1 to set the resting membrane potential depended on the outward current. Single-channel conductance of hIRK1 was 32 pS measured with 150 mM KCl in the patch pipette, significantly higher than 23 pS measured for mouse IRK1 and approximately 10 pS for hIRK2.

[Pharmacological studies on the effects of huanglian decoction on experimental gastric lesions in rats and antiemetic in pigeons].

The results showed that Huanglion decoction has protective effect on ethanol-,HCl- and aspirin-induced gastric hemorrhagic lesions in rats and antemetic effect on CuSO4-induced vomiting in pigeons. A dose of 27g/(kg.d) po applied in mice showed no toxic action. This dose is 400 times that of clinical application.

[Experimental studies on Uncaria sinensis (Oliv.) Havil and Achyranthes bidentata Blume and their compacibility].

Pharmacological studies were conducted on Uncaria sinensis and Achyranthes bidentata both separately and combined. Comparison was made on the hypotensive effect on normal and renal-type hypertensive rats as well anti-spasmodic and sedative effects in mice. The results showed that Uncaria sinensis and Achyranthes bidentata have obvious synergic action in compatibility.

Clear cell carcinoma of salivary glands: immunohistochemical evaluation of clear tumor cells.

A total of 14 cases of clear cell carcinoma of salivary glands were evaluated by immunohistochemical methods using monoclonal antibodies to cytokeratin (K1.1 and K8.12), vimentin, S-100 alpha and beta subunits, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), MAM-3 and MAM-6 antigens and proliferating cell nuclear antigen (PCNA), as well as polyclonal antibodies to lysozyme (Ly), lactoferrin (la) and Alpha-1-antichymotrypsin (alpha 1-Ach). Histopathologically, the carcinoma was characterized by round or polygonal tumor cells with cytoplasm that does not stain with hematoxylin and eosin, nuclei with little pleomorphism and few or no mitotic figures, and growing in solid sheets, small nests or cords with collagenous stroma. Cytokeratin KL1 and K8.12 was present in few tumor cells with almost negligible to strong reaction in all cases, vimentin in 6, GFAP in 5 cases with multiple-expression of cytokeratin K8.12, vimentin and GFAP in 5 cases. S-100 protein immunoreactivity was the most prominent feature with more intense reaction of S-100 beta than S-100 alpha subunit. NSE reactivity was seen in 6 cases. Ly, La, a1-ch, MAM-3 and MAM-6 antigens were localized in clear cells with various reaction intensities. The authors conclude that the clear tumor cells in clear cell carcinoma of salivary glands are not myoepithelial in origin but epithelial or neuroectodermal/neural crest in origin, showing ductal differentiation at the immunohistochemical level.