Transfer of immunity by means of spleen cells from mice immunized with outer membrane proteins of Shigella flexneri

Intraperitoneal immunization of mice with a single dose (5 micrograms) of outer membrane proteins (OMP) of Shigella flexneri was found to evoke in the spleen the appearance of cells by means of which immunity to lethal dose of Shigella could be transferred into other mice. Active cells capable of transferring immunity appeared in the spleen of the animals as early as on day 3, reached the strongest protective activity on day 4 and disappeared on day 8 after immunization. Active cells from animals immunized with two doses of OMP maintained in the spleens for 19 days. The experiments revealed that immunity to Shigella could be transferred only with lymphocytes; macrophages were found to be inactive.     

Humoral response in mice immunized with outer membrane proteins of Shigella flexneri

Intraperitoneal immunization of mice with outer membrane proteins (OMP) of Sh. flexneri induced in the animals a synthesis of specific antibodies. Their level determined by ELISA test was found to be relatively low in the sera of animals immunized with a single dose (10 micrograms) of OMP; it was markedly higher in mice immunized with two doses of OMP, and very high after three fold immunization. The specific antibodies maintained in the animals for 8-16 weeks after immunization. Anti-OMP sera given to normal mice by intraperitoneal route protected them not only against challenge with homologous Shigella but also against Proteus and Escherichia.     

Humoral response in mice immunized with outer membrane proteins of Hafnia alvei. Protective activity of anti-OMP-antibodies.

Intraperitoneal immunization of mice with outer membrane proteins (OMP) of Hafnia alvei induced in the animals a synthesis of specific antibodies. The antibody levels, determined by ELISA test, were found to be relatively low in the sera of mice immunized with a single dose (5 micrograms) of OMP and after a second immunization. However, they were higher in mice immunized with three doses of OMP. The antibodies were present in circulation for a relatively short time after immunization. Serum containing anti-OMP antibodies given intraperitoneally to normal mice protected them only against challenge with a homologous Hafnia strain.     

Some biological properties of outer membrane proteins of Shigella

Outer membrane proteins (OMP) derived from two antigenically different representatives of Shigella (Sh. flexneri 3a and Sh. sonnei phase I) were tested for the toxicity, pyrogenicity, ability to induce Shwartzman reaction as well as for their influence on the leukocyte system. LD50 dose determined on mice was 28 mg/kg for OMP of Sh. flexneri and 23 mg/kg for OMP of Sh. sonnei. Both preparations injected intravenously to rabbits caused moderate increase of body temperature, expressed by the value 1.8 degrees C. Intravenous administration of protein preparations to rabbits, induced at first leukopenia and then transient leukocytosis. When injected subcutaneously in the dose of 500 micrograms and after 24 h intravenously in the dose 100 micrograms, they produced slight hemorrhagic changes at the site of administration.     

Studies on specificity of protection induced by immunization with outer membrane proteins of Shigella

Immunization of C3H/HeJ mice with outer membrane proteins (OMP) and with peptidoglycan associated proteins (PGP) isolated from Shigella flexneri 3a and from Shigella sonnei phase I protected the animals against lethal dose of homologous and heterologous bacteria and against various serotypes of Shigella flexneri. Neither of the protein preparations protected the animals against challenge with Escherichia, Klebsiella, Citrobacter, Salmonella, Serratia, Proteus and Pseudomonas. OMP preparations however, isolated from these species protected the animals not only against challenge with homologous bacteria but also against Shigella flexneri 3a.     

Studies on nonspecific protection induced with OMP of Shigella flexneri and other genera of Enterobacteriaceae

Outer membrane proteins (OMP) isolated from Shigella flexneri, Escherichia coli, Proteus vulgaris and Salmonella typhimurium were tested for their protective activity. Each OMP preparate given to mice intraperitoneally in a single injection (5 micrograms/per mouse) protected the animals not only in homologous but also in varying intensity in heterologous systems. Evidence was obtained that this nonspecific protection is cell mediated.     

Protection against keratoconjunctivitis shigellosa induced by immunization with outer membrane proteins of Shigella spp.

Active immunization of guinea pigs and rabbits with outer membrane proteins (OMP) isolated from Shigella flexneri 3a and Shigella sonnei phase I protected the animals against keratoconjunctivitis shigellosa induced with the homologous or heterologous strain. Protection was also achieved in rabbits after passive immunization with anti-OMP immune serum. Active immunization with lipopolysaccharide of S. flexneri 3a did not protect rabbits against keratoconjunctivitis shigellosa.     

Effect of outer membrane proteins of Shigella on delayed hypersensitivity in mice to sheep red blood cells

Small doses of outer membrane proteins (OMP) of Shigella flexneri injected intraperitoneally into mice 1 to 3 days before or 3 days after sensitization of animals with sheep erythrocytes were found to increase delayed hypersensitivity as measured by the footpad reaction. In contrast, administration of higher doses of OMP resulted in suppression of hypersensitivity response. Cell transfer experiments showed that the spleen cells from sensitized and OMP treated mice transferred stimulating and suppressing activity to normal recipients. Suppression of hypersensitivity was also observed when recipients were injected with OMP 24 h before they were infused with spleen cells obtained from donor mice sensitized with sheep erythrocytes.     

Effect of outer membrane proteins from Shigella on humoral immunity induced in mice by SRBC.

Shigella flexneri outer membrane proteins (OMP) which had been earlier found to exert immunomodulatory effect on cell mediated immune response were also found to act as immunomodulator of the humoral immune response. Effects of OMP were investigated in the experiments in vitro and in vivo, where the level of humoral immune response, measured as the number of plaque-forming cells (PFC) to SRBC in the spleen was evaluated. We demonstrate that small doses of OMP (1-5 micrograms) stimulate, whereas higher doses (10-50 micrograms) suppress the humoral immunity.     

痢疾多糖-蛋白结合疫苗小鼠母子抗体传递试验

目的　以小鼠为试验动物模型 ,用不同的痢疾多糖 蛋白结合疫苗免疫母鼠 ,观察IgG抗体的母婴传递情况。方法　用福氏 2a痢疾多糖 重组铜绿假单胞菌外毒素A(rEPA)结合疫苗 (F2a O SP rEPA)和宋内痢疾多糖 重组铜绿假单胞菌外毒素A结合疫苗 (S O SP rEPA)分别皮下免疫母鼠 ,待其怀孕、分娩 ,用ELISA分别检测母体、子体小鼠的IgG抗体滴度。结果　子体小鼠出生第 2周的IgG抗体水平较高 ,随着小鼠周龄的增加 ,抗体水平逐渐下降。结论　痢疾多糖 蛋白结合疫苗免疫母体后 ,诱导的特异性抗体可经母体传递给子体 ,抗体滴度随时间的推移而逐渐降低。     

Fraction of spleen cells responsible for suppression of cellular immune response to SRBC in mice treated with outer membrane proteins of Shigella.

Effect of splenocytes isolated from mice immunized with suppressive dose of OMP from Shigella on delayed hypersensitivity, induced in mice with sheep red blood cells was investigated. Only the population of T lymphocytes was found to suppress the delayed hypersensitivity, as measured by the footpad reaction. The results suggest that OMP of Shigella are able to induce in the spleens of animals active T cells which are responsible for the suppression of cellular response induced by SRBC.     

Intranasal immunization with multivalent group A streptococcal vaccines protects mice against intranasal challenge infections

We have previously shown that a hexavalent group A streptococcal M protein-based vaccine evoked bactericidal antibodies after intramuscular injection. In the present study, we show that the hexavalent vaccine formulated with several different mucosal adjuvants and delivered intranasally induced serum and salivary antibodies that protected mice from intranasal challenge infections with virulent group A streptococci. The hexavalent vaccine was formulated with liposomes with or without monophosphorylated lipid A (MPL), cholera toxin B subunit with or without holotoxin, or proteosomes from Neisseria meningitidis outer membrane proteins complexed with lipopolysaccharide from Shigella flexneri. Intranasal immunization with the hexavalent vaccine mixed with these adjuvants resulted in significant levels of antibodies in serum 2 weeks after the final dose. Mean serum antibody titers were equivalent in all groups of mice except those that were immunized with hexavalent protein plus liposomes without MPL, which were significantly lower. Salivary antibodies were also detected in mice that received the vaccine formulated with the four strongest adjuvants. T-cell proliferative assays and cytokine assays using lymphocytes from cervical lymph nodes and spleens from mice immunized with the hexavalent vaccine formulated with proteosomes indicated the presence of hexavalent protein-specific T cells and a Th1-weighted mixed Th1-Th2 cytokine profile. Intranasal immunization with adjuvanted formulations of the hexavalent vaccine resulted in significant levels of protection (80 to 100%) following intranasal challenge infections with type 24 group A streptococci. Our results indicate that intranasal delivery of adjuvanted multivalent M protein vaccines induces protective antibody responses and may provide an alternative to parenteral vaccine formulations.     

Immunogenic and protective properties of outer membrane proteins of Hafnia alvei

Outer membrane proteins (OMP) extracted from antigenically distinct or related strains of Hafnia alvei containing defined composition of major proteins proved to be immunogenic. Intraperitoneal immunization of mice with a single dose of such preparations protected the animals against homologous and heterologous Hafnia strains. The OMP preparations were also found to induce protection with varying intensity against Escherichia, Proteus, Shigella and Salmonella.     

Killing of Listeria monocytogenes by inflammatory neutrophils and mononuclear phagocytes from immune and nonimmune mice

Acquired resistance to the facultative intracellular bacterium Listeria monocytogenes is thought to require immunologically activated macrophages. Using peritoneal exudate cells from nonimmunized mice in a suspension bactericidal assay, however, we found that peritoneal neutrophils obtained early during the inflammatory process (4 hr after elicitation) and macrophages obtained later during inflammation (maximal listericidal activity at 48 hr after elicitation) were able to kill Listeria in vitro. The kinetics of expression of bactericidal activity by inflammatory neutrophils and macrophages against both L monocytogenes and E coli were similar. Although intraperitoneal immunization or intravenous hyperimmunization markedly enhanced resistance of mice to Listeria in vivo, immunization did not increase the ability of inflammatory peritoneal phagocytes to kill Listeria in vitro. However, in response to intraperitoneal injection of proteose-peptone or dead Listeria, immunized mice mobilized more neutrophils and monocytes into the inflamed peritoneum. These data suggest that, rather than systemic activation of mononuclear phagocyte bactericidal activity, increased mobilization of neutrophils and mononuclear phagocytes into sites of infection may be of prime importance in resistance to listeriosis.     

Mechanism of the bactericidal action of myeloperoxidase: increased permeability of the Escherichia coli cell envelope.

A sensitive and highly reproducible assay was utilized to study in vitro interactions of Listeria monocytogenes with resident and activated macrophages. The technique is not compromised by extracellular events and can readily differentiate between the efficiency of ingestion and the postphagocytic fate of bacteria. Heat-labile factors in human or homologous serum markedly enhanced the phagocytosis of Listeria without noticeably affecting the intracellular fate of the microorganisms. The behavior of Listeria within macrophages cultivated from C57BL/6 and BALB/c mouse strains corresponded to previous reports of in vivo growth patterns in inbred mice. Thioglycolate- or caseinate-elicited macrophages, although highly phagocytic, were unable to prevent the proliferation of Listeria. A bactericidal macrophage population was derived from from C57BL/6 mice which had been immunized intraperitoneally with a sublethal dose of L. monocytogenes and subsequently boosted with heat-killed homologous organisms. Elicitation of immune animals produced an increase in the percentage of peroxidase-positive macrophages, but this activity could not be correlated with restriction of intracellular bacterial growth.     

Cigarette smoke and phagocyte function: effect of chronic exposure in vivo and acute exposure in vitro.

Phagocytic function was studied in mice chronically exposed to cigarette smoke, and the effects of in vitro exposure to cigarette smoke on macrophage activity were also assessed. Cultures of radiolabeled Pseudomonas aeruginosa were employed to investigate phagocyte activity in vivo and in vitro. Mice were exposed on weekdays to fresh cigarette smoke for periods up to 37 weeks and the bactericidal and clearance activity of their lungs was measured. Both pulmonary clearance and bactericidal activity was impaired. The clearance of intravenously injected bacteria from the blood of smoke-exposed mice occurred at the same rate as in control mice, but the accumulation of radiolabel by the liver was decreased. In addition, the rate of elimination of radiolabel from the liver was less than the controls. Macrophages exposed to cigarette smoke in vitro initially had a depressed phagocytic rate, but if phagocytosis over a prolonged period was measured it was eventually enhanced over the rate of control macrophages. The vapor phase of cigarette smoke could also transiently inhibit and then enhance the phagocytic activity.     

Effect of Dihydrostreptomycin on Phagocytosis of Mouse-Peritoneal Macrophages In Vitro

The effect of the antibiotic dihydrostreptomycin on the phagocytic and bactericidal ability of peritoneal macrophages obtained from mice has been investigated. In subliminal concentrations which did not influence the bacterial growth, the drug caused macrophages to ingest and kill bacteria (Escherichia coli) at a higher rate than did macrophages without antibiotic. The differences for phagocytosis and intracellular killing of E. coli with and without a subliminal amount of dihydrostreptomycin were statistically significant. Macrophages pretreated with the antibiotic did not demonstrate any enhancement of phagocytosis.     

Shigella flexneri is trapped in polymorphonuclear leukocyte vacuoles and efficiently killed.

We examined the bactericidal activity of polymorphonuclear leukocytes (PMN) against an invasive wild-type strain of Shigella flexneri (M90T) and a plasmid-cured noninvasive derivative (BS176). Both Shigella strains, as well as a rough strain of Escherichia coli, were killed with similar efficiencies by intact inflammatory PMN in room air and under N2 (i.e., killing was O2 independent). Bacterial killing by PMN extracts was substantially inhibited by antibodies to the bactericidal/permeability-increasing protein (BPI). Whereas wild-type Shigella escapes from the phagosome to the cytoplasm in epithelial cells and macrophages, wild-type Shigella was trapped in the phagolysosome of PMN as visualized by electron microscopy. The efficient killing of Shigella by PMN suggests that these inflammatory cells may not only contribute initially to the severe tissue damage characteristic of shigellosis but also ultimately participate in clearance and resolution of infection.     

Characterization of monoclonal antibodies with specificity for the core oligosaccharide of Shigella lipopolysaccharide

Monoclonal antibodies (MAbs) were prepared against different strains of Shigella, following immunization of BALB/c mice with a heat-killed preparation of Shigella. Antibody-producing hybridomas were screened in an indirect enzyme-linked immunoadsorbent assay (ELISA) and epitope specificity determined using chemically defined lipopolysaccharide, lipid, and KDO fragments. Five MAbs were characterized and the following specificities identified: 2C32E6 and 4D64B9 (reactive to S. flexneri and S. boydii), 5E45D8 (reactive with S. flexneri), 4B33D10 and 1B52F10 (all species of Shigella). The properties of 1B52F10 revealed its potential importance in immunological detection of Shigella from unknown samples, as it was able to bind to all strains of Shigella.     

热休克蛋白gp96-肽复合物诱导的抗肿瘤免疫

目的: 用不同种类肿瘤提取的热休克蛋白 (HSP)gp96 肽复合物纯化后免疫动物,观察其诱导抗肿瘤免疫的效应,并初步探讨其机制。方法: 从肿瘤细胞中制备HSPgp96 肽复合物, 观察其免疫诱导作用。用不同剂量及不同来源的gp96 肽复合物免疫 6组小鼠后, 用H22癌细胞攻击, 观察各组小鼠的肿瘤发生率、瘤重及瘤组织的形态学变化并与对照组相比较。观察gp96 肽复合物对小鼠腹腔巨噬细胞分泌一氧化氮 (NO)和杀瘤活性的影响。结果: 获得的纯化HSPgp96 肽复合物的Mr为 96 000。gp96 肽复合物的免疫效果与其剂量有关, 以 18μg/只的效果最明显。交叉免疫实验证明, 免疫小鼠能抵抗同种肿瘤细胞的攻击, 而对异种肿瘤细胞的则无免疫保护作用。gp96 肽复合物可刺激小鼠腹腔巨噬细胞分泌NO同时杀瘤活性增高。结论: 从肿瘤细胞中分离纯化的HSPgp96 肽复合物免疫小鼠后, 可获得特异性的抗肿瘤作用; 其对肿瘤细胞的杀伤作用与gp96 肽复合物能增强小鼠腹腔巨噬细胞分泌NO增多有关。