Immunization of rabbits against Pasteurella multocida using a commercial swine vaccine

Elaboration of heat-labile toxin (PMT) is an important virulence factor in some isolates of Pasteurella multocida from rabbits. Previously, we reported that immunization with inactivated PMT (IPMT) stimulated protective immunity to challenges from PMT. To test the hypothesis that immunization with a commercial swine vaccine containing IPMT stimulates similar protective immunity, groups of five rabbits were inoculated twice intramuscularly (i.m.), 10 days apart, with 0.5 ml of sterile saline or a commercial swine P. multocida bacterin-toxoid (BT). In addition, a group was inoculated intranasally with 5 microg of IPMT. Serum and nasal lavage samples were taken on days 0, 7, 14 and 21 after initial immunization and assayed by ELISA for anti-PMT antibody. Serum IgG and nasal lavage IgA were detectable by day 14 in BT and IPMT-immunized rabbits, but not in the saline controls. Groups of similarly inoculated rabbits were then challenged intranasally with 28 microg of PMT 21 days after initial immunization, and necropsied 7 days later, along with control challenged and non-challenged rabbits. Histological lesion severity was graded on a numerical scale. Non-immunized and saline, challenged controls developed more severe pneumonia, pleuritis, nasal turbinate atrophy and testicular atrophy than IPMT and BT-immunized rabbits. The results confirm the hypothesis that immunization with a commercial swine P. multocida BT confers protective immunity in rabbits against challenges from PMT.     

Eimeria stiedai merozoite 49-kDa soluble antigen induces protection against infection

A soluble antigen isolated from Eimeria stiedai merozoites with a molecular mass of 49 kDa was detected in the bile of infected rabbits. Rabbits immunized with the antigen shed a lower number of oocysts than did nonimmunized rabbits postchallenge (p.c.). The immunized rabbits showed a marked and transient increase of alanine-aminotransferase (ALT) activity on day 8 p.c. The blood indocyanine green (ICG) clearance and r-glutamyltransferase (GGT) activity showed no change throughout the experiment However, nonimmunized rabbits showed a gradual increase of ALT and GGT in the plasma and a delay of ICG p.c. Many merozoites were observed in the biliary ducts of the nonimmunized rabbits on day 8 p.c. using standard histology. In contrast, in the immunized rabbits, many inflammatory cells were observed around the biliary ducts, but there were few parasites in the tissue. These results suggest that the 49-kDa soluble protein antigen detected in the bile of the infected rabbits was a merozoite-specific antigen, and the immune reaction to the antigen may induce protective effects against the infection.     

Effect of the delta-sleep peptide on parasympathetic regulation of the cardiac rhythm

Effect of delta-sleep peptide (60 nM/kg) on the parasympathetic regulation of cardiac activity has been studied in the experiments on rabbits. It has been established that intravenous administration of this peptide to voluntary-behaving animals results in heart rate reduction by an average of 16%, that can be eliminated by atropine. Delta-sleep peptide has been demonstrated to intensify negative chronotropic effect in the case of directly irritated wandering nerve. The data obtained explain a protective effect of delta-sleep peptide on the heart under emotional stress.     

Effect of the recombinant vaccinia viruses that express HTLV-I envelope gene on HTLV-I infection.

The human T-lymphotropic virus type I (HTLV-I) is etiologically linked to adult T-cell leukemia (ATL). To develop a vaccine against ATL, we constructed recombinant vaccinia viruses containing the envelope gene of HTLV-I in the vaccinia virus hemagglutinin (HA) gene, a new site where foreign genes can be inserted. A single inoculation of the recombinant virus induced antibodies to the env proteins of HTLV-I in rabbits and had a protective effect against HTLV-I infection.     

Immunoliposomes and PEGylated Immunoliposomes: Possible Use for Targeted Delivery of Imaging Agents

Liposomes can be made target-specific by immobilizing antibodies on their surface against characteristic components of target organ or tissue. Possible schemes of antibody immobilization on liposomes are briefly considered. The use of immunoliposomes for the targeted delivery of diagnostic and therapeutic agents within the cardiovascular system is discussed. Immunoliposomes are shown to be suitable carriers for targeting blood vessel injuries, lung endothelium, and myocardial infarction. The role of polyethylene glycol in the preparation of long-circulation liposomes is investigated, and a hypothesis explaining the mechanism of polymer protective action in terms of physicochemical properties of diluted polymeric solutions is suggested. Polyethylene glycol-coated liposomes are investigated as possible carriers of imaging agents for gamma and MR visualization of different areas of interest in the body, including lymph nodes. The possibility of simultaneous immobilization of protective polymer and antibody on the liposome surface is proved, and the long-circulating targeted immunoliposomes are used for the targeted delivery of radiolabel to necrotic areas in rabbits with experimental myocardial infarction.     

Expression of Major Antigen Domains of Gene of E2 CSFV and Analysis of its Immunological Activity

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.     

炭疽活疫苗家兔免疫力与血清抗芽胞IgG关系的研究

炭疽疫苗是预防炭疽流行和炭疽生物恐怖的重要手段。已有动物实验表明,炭疽活疫苗的保护力优于以保护性抗原为主要成份的无细胞疫苗,但两类现行疫苗都有待重新评价和改进。炭疽疫苗的效力必须用适当的实验室方法进行检测与分析才能了解其性质和细节。试验中力图探寻炭疽活疫苗家兔免疫力与血清抗芽胞抗体水平的关系。用“皮上划痕人用炭疽活疫苗”免疫家兔,以特定制备的炭疽芽胞抗原用ELISA法检测血清抗炭疽芽胞IgG抗体水平,并用强毒炭疽杆菌攻击进行效力试验。免疫家兔血清几何平均抗芽胞IgG滴度在免疫后一个月内持续升高,14d达到206,28d时达到776,这时其抵抗20MLD毒菌攻击的保护率为80％,符合中国生物制品规程要求的保护力。一个月后抗体水平开始下降,42d时滴度降至223。实验所揭示的炭疽减毒活疫苗诱导的家兔抗芽胞IgG抗体与抗炭疽保护力之间的关系,既为评价现行疫苗提供了资料,也为研制新型疫苗建立了参考性指标。     

Isolation from a pregnant woman of a serum protein responsible for inhibiting an in vitro cytotoxicity reaction

A non specific immunosupressive factor able to block an in vitro cytotoxicity reaction is demonstrated in the serum of pregnant women. A solution containing this blocking factor is obtained by gel filtration and precipitation of plasma by polyethylene glycol 4 000. Then after immunisation of rabbits the immune serum can be used for affinity chromatography. An alpha 2 glycoprotein has been separated which inhibits the in vitro cytotoxicity reaction and whose molecular weight determined by gel filtration and polyacrylamide gel is about 200 000 daltons.     

Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles

The recognition that rotaviruses are the major cause of life-threatening diarrheal disease and significant morbidity in young children has focused efforts on disease prevention and control of these viruses. Although the correlates of protection in children remain unclear, some studies indicate that serotype-specific antibody is important. Based on this premise, current live attenuated reassortant rotavirus vaccines include the four predominant serotypes of virus. We are evaluating subunit rotavirus vaccines, 2/6/7-VLPs and 2/4/6/7-VLPs, that contain only a single VP7 of serotype G1 or G3. In mice immunized parenterally twice, G3 virus-like particles (VLPs) induced a homotypic, whereas G1 VLPs induced a homotypic and heterotypic (G3) serum neutralizing immune response. Administration of three doses of G1 or G3 VLPs induced serum antibodies that neutralized five of seven different serotype test viruses. The inclusion of VP4 in the VLPs was not essential for the induction of heterotypic neutralizing antibody in mice. To confirm these results in another species, rabbits were immunized parenterally with two doses of 2/4/6/7-VLPs containing a G3 or G1 VP7, sequentially with G3 VLPs followed by G1 (G3/G1) VLPs, or with live or psoralen-inactivated SA11. High-titer homotypic serum neutralizing antibody was induced in all rabbits, and low-level heterotypic neutralizing antibody was induced in a subset of rabbits. The rabbits immunized with the G1 or G3/G1 VLPs in QS-21 were challenged orally with live G3 ALA rotavirus. Protection levels were similar in rabbits immunized with homotypic G3 2/4/6/7-VLPs, heterotypic G1 2/4/6/7-VLPs, or G3/G1 2/4/6/7-VLPs. Therefore, G1 2/4/6/7-VLPs can induce protective immunity against a live heterotypic rotavirus challenge in an adjuvant with potential use in humans. Following challenge, broad serum heterotypic neutralizing antibody responses were detected in rabbits parenterally immunized with G1, G3/G1, or G3 VLPs but not with SA11. Immunization with VLPs may provide sufficient priming of the immune system to induce protective anamnestic heterotypic neutralizing antibody responses upon subsequent rotavirus infection. Therefore, a limited number of serotypes of VLPs may be sufficient to provide a broadly protective subunit vaccine.     

Mechanisms of telomerase binding to telomeres

There are essentially two alternative mechanisms for the binding of telomerase to telomeres, assuming that a protective component is initially bound to the telomerase binding region on the telomeres. Either the protective (or blocking) agent first dissociates and telomerase binds thereafter, or telomerase binds first and the protective agent then dissociates from the ternary complex. In the limit, this second possibility permits the ternary complex to become a transition complex (creating another possible mechanism). Numerical simulation of both rapid mixing and chemical relaxation is used to study these alternatives. We aim to determine how the mechanisms may be distinguished experimentally and identify an appropriate experimental design. We show that rapid mixing experiments are better than chemical relaxation experiments, since the latter are more affected by the statistics of single molecule kinetics. However, hidden fast steps can only be revealed by chemical relaxation. The detection of mechanistic changes hinges on linking fluorescence reporters to the reaction components, either directly (chemically) or indirectly (via an indicator reaction). Fluorescence is excited by two-photon absorption in a small reaction volume. Various detection strategies and design issues are examined, including limitations imposed by diffusion. Constant rather than stopped flow is shown to be preferable.     

Gene expression analysis in interstitial lung edema induced by saline infusion

To investigate the molecular events taking place during the development of hydraulic interstitial edema, we analyzed by microarray and conventional molecular techniques the variation of gene expression in lung from rabbits treated with slow-rate saline infusions. This analysis indicates that even a condition characterized by a small increase in extravascular water can have a significant influence on the inflammatory milieu. In this regard, cytokines, in particular TNFalpha, can be considered early mediators capable of inducing secondary effects on the injured tissue. Moreover, two MT1 genes were strongly up-regulated, data consistent with their role as protective molecules.     

Pathways of silicomolybdate photoreduction and associated photophosphorylation in tobacco chloroplasts.

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (greater than 5 mg/ml) is associated with Photosystem I, since it supports phosphorylation with a P/e2 value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e2 value routinely observed is about 0.2. Bovine serum albumin (1-2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even if the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.     

Expression of major antigen domains of E2 gene of CSFV and analysis of its immunological activity

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection. Foundation items: The National “973” (2005CB523201); Key Technology R&D Programme (2006BAD06A03).     

Accumulation of platelets at sites of antigen-antibody-mediated injury: a possible role for IgE antibody and mast cells.

Circulating 51Cr-labeled platelets accumulate at skin sites in which a reversed passive Arthus reaction has been induced. The accumulation is biphasic in time and is accompanied by an increased vascular permeability. Increased permeability itself, however, will not produce localization of platelets. A similar platelet accumulation was observed upon injection of compound 48/80 or anti-IgE antibody into the skin and this was not altered in rabbits depleted of complement or neutrophils. Activation of skin mast cells and release of a platelet-activating factor (PAF) is suggested as a mechanism for the effect produced by anti-IgE and compound 48/80. The first phase of platelet accumulation in the Arthus reaction was also unaffected in rabbits depleted of neutrophils or complement, which may suggest a role for IgE antibody and mast cells. The second phase of accumulation was diminished in complement-depleted animals and abrogated in rabbits without neutrophils, suggesting a complement and neutrophil-mediated process but which still might be mediated through mast cell activation by neutrophil cationic protein.     

Management of Hepatitis E Virus (HEV) Zoonotic Transmission: Protection of Rabbits against HEV Challenge following Immunization with HEV 239 Vaccine

Hepatitis E virus (HEV) constitutes a significant health burden worldwide, with an estimated approximately 33% of the world’s population exposed to the pathogen. The recent licensed HEV 239 vaccine in China showed excellent protective efficacy against HEV of genotypes 1 and 4 in the general population and pregnant women. Because hepatitis E is a zoonosis, it is also necessary to ascertain whether this vaccine can serve to manage animal sources of human HEV infection. To test the efficacy of the HEV 239 vaccine in protecting animal reservoirs of HEV against HEV infection, twelve specific-pathogen-free (SPF) rabbits were divided randomly into two groups of 6 animals and inoculated intramuscularly with HEV 239 and placebo (PBS). All animals were challenged intravenously with swine HEV of genotype 4 or rabbit HEV seven weeks after the initial immunization. The course of infection was monitored for 10 weeks by serum ALT levels, duration of viremia and fecal virus excretion and HEV antibody responses. All rabbits immunized with HEV 239 developed high titers of anti-HEV and no signs of HEV infection were observed throughout the experiment, while rabbits inoculated with PBS developed viral hepatitis following challenge, with liver enzyme elevations, viremia, and fecal virus shedding. Our data indicated that the HEV 239 vaccine is highly immunogenic for rabbits and that it can completely protect rabbits against homologous and heterologous HEV infections. These findings could facilitate the prevention of food-borne sporadic HEV infection in both developing and industrialized countries.     

参附注射液对心肌缺血/再灌注损伤血流动力学和心肌酶的影响

目的：探讨参附注射液对家兔心肌缺血/再灌注（I/R）损伤的保护作用及其机制。方法：家兔30只,随机分为3组（n=10）：对照组、心肌缺血/再灌注损伤（MI/RI）组和参附注射液组,统一标准喂养。行药物预处理10 min后,手术结扎家兔冠状动脉左心室支,建立急性心肌缺血再灌注模型,观察急性心肌缺血和再灌注状态下血流动力学及心肌组织中酶的变化。结果：与对照组比较：MI/RI组心脏舒缩功能减退,丙二醛（MDA）含量增高,超氧化物歧化酶（SOD）、谷光甘肽过氧化物酶（GSH-PX）和细胞能源Na＋-K＋-ATP酶及Ca2＋-ATP酶活性降低,乳酸脱氢酶（LDH）、肌酸激酶（CK）大量释放。而与MI/RI组比较：参附注射液组能不同程度的恢复左心室收缩压（LVSP）、心室内压最大变化速率（±dp/dtmax）（P〈0.01）,降低左室舒张末期压（LVEDP）（P〈0.01）,抑制MDA、LDH、CK升高,增强SOD、GSH-PX、Na＋,K＋-ATP及Ca2＋-ATP活力。结论：参附注射液对心肌缺血/再灌注损伤具有明显的保护作用。     

A protective factor of ribosomal shigellosis vaccine

Shigella ribosomal vaccine was shown previously to possess protective properties in the keratoconjunctival test on guinea pigs and to be capable of preventing experimental infection in 90% of challenged monkeys. The presence of the O-specific component (OSC) constituting about 0.5% of the ribosomal preparation by serological activity suggested its importance for the protective effect. This was studied in experiments with two O-specific immunosorbents prepared by coupling anti-O rabbit antibodies with Staphylococcus aureus cells or with CNBr-Sepharose. Ribosomes treated with immunosorbents proved to be lacking the serologically active OSC and lost their ability to induce O-antibody response in rabbits and mice. After the removal of this component ribosomal preparations were incapable of ensuring protection from Shigella kerato-conjunctival infection. The isolated OSC was also inactive in this test. The data obtained in this investigation confirm the hypothesis stating that the protective activity of Shigella ribosomal vaccine is based on the combined action of ribosomes and O-specific factor whose nature and properties require further study.     

Analysis of lymphocyte adhesion and the phenomenon of immunologic adhesion inhibition

Investigations were performed in rabbits to elucidate the cell physiological parameters of the inhibitor reaction of adhesion. For this purpose, an improved method of measuring the cell adhesion was used. The well-known fact that culture cells will reveal a much stronger reaction could be confirmed. This may be due to the high increase of T-lymphocytes because of findings concerning cell differentiation with acid alpha-naphthylacetate esterase (ANAE), whereas no corresponding results could be found for the particular role of macrophages. It could be proved that the inhibitor reaction already began 20 minutes after the antigen contact. On the basis of these facts beta-lymphotoxin might be assumed to play a part in the inhibitor reaction. A direct relationship between oxygen consumption and the inhibitor reaction of adhesion could not be proved. The respiratory depression frequently observed might be due to the cytotoxic effect of excreted lymphotoxins. As the inhibitor reaction of adhesion could be identified even after 1-1 1/2 years, it can be regarded as a longterm immunophenomenon. The findings with metabolic blockers underline that the primary adhesion of lymphocytes is supported by the oxidative metabolism and not by glycolysis which is generally characteristic of granulocytes.