The action of a novel fluoroquinolone antibiotic agent antofloxacin hydrochloride on human-ether-à-go-go-related gene potassium channel.

The administration of certain fluoroquinolone antibacterials has recently been linked to QT interval prolongation, raising the clinical concerns over the cardiotoxicity of these agents. In this study, the effects of a novel fluoroquinolone, antofloxacin hydrochloride (AX) on human-ether-à-go-go-related gene (HERG) encoding potassium channels and the biophysical mechanisms of drug action were performed with whole-cell patch-clamp technique in transiently transfected HEK293 cells. The administration of AX caused voltage- and time-dependent inhibition of HERG K+ current (I(HERG/MiRP1)) in a concentration-dependent manner but did not markedly modify the properties of channel kinetics, including activation, inactivation, deactivation and recovery from inactivation as well. In comparison with sparfloxacin (SPX), levofloxacin lactate (LVFX), the potency of AX to inhibit HERG tail currents was the least one, with an IC(50) value of 460.37 microM. By contrast, SPX was the most potent compound, displaying an IC(50) value of 2.69 microM whereas LVFX showed modest potency, with an IC(50) value of 43.86 microM, respectively. Taken together, our data suggest that AX only causes a minor reduction of I(HERG/MiRP1) at the estimated free plasma level.

[Chinfloxacin hydrochloride inhibits HERG potassium channel at open state].

This study is designed to investigate the effects of chinfloxacin hydrochloride (CFX) on the kinetics of HERG K+ channel. Whole cell patch clamp technique was used to record HERG K+ currents from HEK293 cells transiently transfected with cgi-HERG-GFP plasmids and channel kinetics were assessed in the absence and presence of CFX and moxifloxacin hydrochloride (MOX). Results demonstrated that the open state of HERG K+ channel was inhibited by CFX in a concentration- and time-dependent manner, with an IC50 of 162.1 +/- 14.2 micromol x L(-1), two folds higher than its positive control MOX. But there were no significant effects on channel kinetics. In addition, the inhibitory effect of CFX on HERG was enhanced when cells were subjected to altered extracellular K+ concentration.

Promotion of immunity of mice to Pasteurella multocida and hog cholera vaccine by pig interleukin-6 gene and CpG motifs.

A novel oligodeoxynuleotides containing 11 CpG motifs was synthesized and inserted into the VR1020 plasmid containing pig interleukin-6 (IL-6) gene (VPIL6) to construct recombinant plasmid, VPIL6C. The chitosan nanoparticles (CNP) were prepared by ionic cross linkage to entrap the VPIL6C (VPIL6C-CNP), VPIL6 (VPIL6-CNP) and CpG (CpG-CNP). 42-Day old female mice were divided into four groups and intramuscularly injected respectively with 6 pmol VPIL6C-CNP, VPIL6-CNP, CpG-CNP and VR1020-CNP along with the bivalent vaccines against the Pasteurellosis and hog cholera. The blood was weekly collected from mice after vaccination to detect the changes of immunoglobulins, specific antibodies, IL-2, IL-4, IL-6 and immune cells. 28 days after vaccination, the mice were orally challenged with virulent Pasteurella multocida. The results showed that in comparison with those of the control VR1020 group, the content of immunoglobulins, specific antibodies and interleukins significantly increased in the sera from the treated two groups (P<0.05). Meanwhile, the number of lymphocytes and monocytes also remarkably elevated in the treated groups (P<0.05). The immune responses of VPIL6C mice were notably stronger than those of VPIL6 and CpG group. The challenge results proved that the overall immunity was further promoted in the treated mice which resisted the challenge infection; while the control mice manifested evident symptoms and lesions, and died of infection. These suggested that VPIL6C-CNP could better promote the immunity and resistance of mice against Pasteurellosis than VPIL6-CNP and CpG-CNP, and facilitate the development of effective adjuvant to enhance the immunity of animal against infection.

Enhancement of immunity and resistance in mice by pig IL-6 gene and CpG motifs encapsulated in chitosan nanoparticle.

This study was conducted to explore the synergetic effect of a novel plasmid containing a porcine IL-6 gene and CpG motifs on immunity of mice in order to develop an effective adjuvant to boost resistance against infection. The synthetic oligodeoxynucleotide containing 11 CpG motifs was inserted into the reconstructed VR1020 plasmid containing the pig IL-6 gene (VRPIL6), designated VRIL6C, and then encapsulated in chitosan nanoparticles (CNP) prepared by ionic cross linkage, designated VRIL6C-CNP. The 3-week old mice were injected, respectively, with VRIL6C-CNP, VRIL6-CNP, CpG-CNP and VR1020-CNP to detect the changes of immunity. At 28 days post inoculation, the mice were challenged with virulent hemolytic serotype 2 Streptococcus to test their resistance against infection. The results showed that there was a significant increase in immunoglobulins and interleukins in mice receiving VRIL6C-CNP compared with the control groups, as well as an increase in the lymphocytes and monocytes in the inoculated mice, so that the immunity was remarkably improved in the VRIL6C-CNP group. The challenge provoked stronger immunity and protection against infection in the VRIL6C-CNP group than in the control mice that manifested severe symptoms and lesions. This suggests that VRIL6C-CNP could remarkably enhance the nonspecific immunity of mice, and facilitate the development of an effective immunopotentiator to promote the resistance of the animals against infection.

Shuffling of pig interleukin-2 gene and its enhancing of immunity in mice to Pasteurella multocida vaccine.

In order to explore the safe and effective adjuvant for promotion of immunity of animals against infection, the experiment was carried out to shuffle Tibet pig IL-2 cDNA with other IL-2 cDNA from human, yak and mouse, and the effect of shuffled IL-2 (IL-2S) gene in vivo was investigated on immunity of mice to Pasteurella multocida. The IL-2S protein was found to remarkably promote the proliferation of pig lymphoblasts than the native pig IL-2 protein. Then the IL-2S gene was cloned into VR1020 eukaryotic plasmid (VRIL2S) and enwrapped with chitosan nanoparticles (CNP-VRIL2S). Twenty-one day old female Kunming mice were muscularly inoculated respectively with the CNP-VRIL2, CNP-VRIL2S and CNP-VR1020 along with Pasteurella multocida vaccine, and orally challenged with virulent Pasteurella multocida on 28 days post-vaccination. The blood was weekly collected to detect the change of IgG, IgA, IgM, specific antibody, IL-2, IL-4 and IL-6 by ELISA. The immunoglobulins, specific antibody and interleukins significantly increased in CNP-VRIL2S group compared with the control mice after vaccination and challenge, and 9 of 10 immunized mice survived challenge, while the all control mice manifested severe symptoms and lesions, and finally died of infection. These indicated that VRIL2S entrapped with CNP is a novel safe and effective adjuvant to boost the specific immunity and resistance of animal against infectious pathogen, which could facilitate the development of highly promising powerful adjuvant.

Enhancement of immunity to an Escherichia coli vaccine in mice orally inoculated with a fusion gene encoding porcine interleukin 4 and 6.

Experiments were conducted to investigate the effect of a fusion gene of porcine IL-4 and IL-6 (PIL4/IL6) packaged with chitosan nanoparticles (CNPs) in terms of the development of a novel effective adjuvant. The IL4/PIL6 fusion gene was constructed and inserted into a eukaryotic expression vector. The plasmid was bound to CNP and then utilized to orally inoculate 21-day-old female Kunming mice that simultaneously received intramuscular injection of inactivated Escherichia coli vaccine. At 35 days post-vaccination, the mice were challenged by oral feeding with virulent O139: K88 strain EPEC E. coli bacteria. Compared with those of control mice, the content of immunoglobulins and specific antibodies to E. coli increased significantly in the sera of mice immunized with VPIL4/IL6-CNP (P<0.05). Furthermore, the levels of IL-2, IL-4 and IL-6 increased remarkably in the sera of immunized mice (P<0.05). After challenge, these immunological markers were elevated to different degrees in the mice immunized with the fusion gene construct (IL4/VPIL6-CNP) or individual plasmids (VPIL4+VPIL6-CNP). The immunized mice all survived the challenge and did not show any symptoms or lesion, whereas the VR1020-CNP control mice manifested obvious clinical symptoms and hemorrhagic lesions in the digestive tracts. These results demonstrated that VPIL4/IL6 entrapped with CNP is a novel promising adjuvant to promote specific immunity and resistance of animals against infectious pathogen.