Mechanisms of Fetal Demise in Pregnant Mice Immunized to Murine Alpha-Fetoprotein

ABSTRACT: Nya: NYLAR mice were immunized to murine alpha-fetoprotein (AFP) by active immunization with rat AFP in Freund's adjuvant emulsion before mating or by passive immunization with a high or low dose of whole rabbit antimouse AFP serum or rabbit anti-AFP IgG at 8–20 days of gestation. In the passively immunized group, anti-AFP serum or purified anti-AFP IgG administered at the end of the second week of gestation produced abortion after 24 hours of 41 and 48% of fetuses, respectively. Although abortion did not occur in the low-dose group, the anti-AFP serum produced fetal death in 32%, as did the anti-AFP IgG in 26%, in 72 hours. In the actively immunized group rat AFP produced developmental arrest, but not abortion, in mothers bearing autologous antibodies to mouse AFP. Histopathologic analysis revealed that fetal death resulted from separation of fetal and maternal tissues of the placenta due to subplacental hemorrhages. Immunofluorescent localization of the rabbit IgG implicated both the chorioallontois and yolk sac placenta as target tissues.     

日本血吸虫紫外线致弱尾蚴免疫小鼠攻击感染后的组织细胞反应

目的观察日本血吸虫紫外线致弱尾蚴疫苗（UVC）免疫动物攻击感染后的局部组织免疫病理变化。方法将70只C57BL／6小鼠随机分为疫苗免疫组和感染对照组。疫苗组小鼠接种紫外线致弱日本血吸虫尾蚴后5周,再经腹部皮肤攻击感染正常日本血吸虫尾蚴（800＋50）条;感染对照组经皮肤感染同量尾蚴。于攻击感染后6～120h的不同时间点各剖杀小鼠5只,取攻击部位皮肤及／或肺组织,进行病理学观察。结果UVC疫苗免疫小鼠攻击感染日本血吸虫尾蚴后,皮肤炎症反应较感染对照出现早、反应强烈、持续时间长,EOS百分比高,但肺部出血斑点出现时间（72h）迟于感染对照组（48h）。72～120h,疫苗组小鼠肺部局灶性炎症明显,肉芽肿样结节形成,肺泡壁多正常,而感染对照组小鼠肺组织炎症轻,但肺泡壁水肿明显,且有较多红细胞渗出。结论紫外线致弱尾蚴疫苗免疫增强了小鼠皮肤及肺组织的细胞反应及其杀虫作用。     

The Role of Superoxide Dismutation in Malaria Parasites

Oxidant stress is associated with the generation of reactive oxygen species that are responsible for the damage of a variety of cellular components. The prevention of such biological damage can be achieved by dismutation of superoxide to H2O2 which in turn is removed by catalase and GSH peroxidase. However, redox-active iron released during the development of plasmodia in the erythrocyte can mediate the conversion of H2O2 to hydroxyl radical which is more reactive. The roles of SOD and the nitroxide SOD mimic 4-OH,2,2,6,6,tetramethyl piperidine-N-oxyl (Tempol) were examined in P. falciparum grown in vitro. Both compounds did not prevent the interference with growth inflicted by various inducers of oxidant stress. Moreover, Tempol inhibited parasite growth, in agreement with previous experiments depicting accelerated mortality in SOD overexpressing mouse model of malaria. Probably, effective defense against ROS requires balanced increments in antioxidant enzymes and is not necessarily improved by an increase in the activity of one enzyme.     

Synthesis of Cytokines During Tumour Development in Mice Immunized with the Mycobacterial Antigen Complex A60

The authors have previously reported on the ability of A60, an immunodominant antigenic complex of Mycobacterium bovis BCG, to prevent cancer development in mice challenged with EMT 6 tumour cells. Such effect proved to rely on neoplastic cell lysis by cytolytic T lymphocytes and activated macrophages. The involvement of cytokines in triggering the immune response leading to tumour rejection is analysed in the present work. The synthesis of IL-2, IFN-α and TNF-α was strongly increased in A60-primed mice. Cancer development depressed the blood levels of these three cytokines. In vitro cultures of lymphocytes from lymph nodes and blood of A60-primed mice produced higher levels of these cytokines in the presence of A60, as  compared to cultures lacking A60. Such effect was inhibited by co-incubation of lymphocytes with EMT  6  tumour cells. In vitro cultures of macrophages yielded higher levels of TNF-α in the presence of A60 and co-incubation of these cells with EMT 6 tumour cells also inhibited TNF-α production. The enhanced synthesis of IL-2 and IFN-α, which promote activation of cytolytic T lymphocytes and macrophages, accounts for the increased tumour cell lysis induced in vivo by A60. The A60-promoted synthesis of TNF-α is partly responsible for the latter effect. The inhibitory action of EMT-6 tumour cells on cytokine synthesis is a powerful mechanism of tumour escape from the immune system's control.     

Proteasome Inhibitors Prevent Caspase-1-Mediated Disease in Rodents Challenged with Anthrax Lethal Toxin

NOD-like receptors (NLRs) and caspase-1 are critical components of innate immunity, yet their over-activation has been linked to a long list of microbial and inflammatory diseases, including anthrax. The Bacillus anthracis lethal toxin (LT) has been shown to activate the NLR Nalp1b and caspase-1 and to induce many symptoms of the anthrax disease in susceptible murine strains. In this study we tested whether it is possible to prevent LT-mediated disease by pharmacological inhibition of caspase-1. We found that caspase-1 and proteasome inhibitors blocked LT-mediated caspase-1 activation and cytolysis of LT-sensitive (Fischer and Brown-Norway) rat macrophages. The proteasome inhibitor NPI-0052 also prevented disease progression and death in susceptible Fischer rats and increased survival in BALB/c mice after LT challenge. In addition, NPI-0052 blocked rapid disease progression and death in susceptible Fischer rats and BALB/c mice challenged with LT. In contrast, Lewis rats, which harbor LT-resistant macrophages, showed no signs of caspase-1 activation after LT injection and did not exhibit rapid disease progression. Taken together, our findings indicate that caspase-1 activation is critical for rapid disease progression in rodents challenged with LT. Our studies indicate that pharmacological inhibition of NLR signaling and caspase-1 can be used to treat inflammatory diseases.Innate immunity plays a critical role in controlling microbial infections. Activation of the inflammasome, an integral part of the innate immune response, has been linked to cell death and morbidity triggered by Salmonella, Francisella, Listeria, and Staphylococcus.^1,2,3 The inflammasome has also been linked to macrophage killing triggered by lethal toxin (LT), a major virulence factor released by the Gram-positive bacterium Bacillus anthracis.^4,5,6,7,8 In fact, when injected into small animals, LT alone is sufficient to reproduce the majority of the symptoms of the anthrax disease. Small molecule inhibitors of LT have been shown to enhance survival of rodents challenged with B. anthracis.^9,10The NOD-like receptor (NLR) Nalp1b controls LT susceptibility in murine macrophages.^4,11 Nalp1b is highly polymorphic in mice, and macrophages from strains expressing a dominant allele of Nalp1b are susceptible to rapid necrosis after LT exposure.^4,12,13 Conversely, murine macrophages expressing a recessive Nalp1b allele undergo slow, caspase-1 independent apoptosis in response to LT challenge.^6,13 We have shown that stimulation of the Nalp1b inflammasome by LT results in caspase-1 activation in susceptible murine macrophages.^6,14,15 Moreover, caspase-1 activation in these cells is essential for LT killing.^4,7,13,16 We also demonstrated that LT-mediated caspase-1 activation and subsequent necrosis in susceptible murine macrophages are controlled by proteasome activity.^5,6,7,17 In fact, proteasome inhibitors are the most efficient inhibitors of LT/caspase-1-mediated macrophage killing identified.^5,6,7,17,18As observed in mice, the susceptibility to LT in rats is strain-dependent. Rats are divided into susceptible strains that are rapidly killed after LT challenge, and strains that are resistant to rapid disease progression.¹⁹ In contrast to mice, however, the in vivo LT susceptibility of rat strains closely mimics the in vitro susceptibility of their corresponding macrophages.¹⁹ Backcrossing experiments using susceptible and resistant rat strains suggest that the (in vivo and in vitro) LT susceptibility in rats is controlled by a single dominant gene.¹⁹ After LT administration, rats develop pulmonary edema, and die from a vascular collapse, a hallmark of human anthrax.^20,21,22 Due to greater similarities with human anthrax, the rat model of LT-induced disease may be superior to the murine model.We hypothesized that macrophage killing and cytokine release are critical events in the death of susceptible rats. Due to the correlation in rats between in vivo and in vitro LT susceptibility, we proposed that LT-mediated macrophage killing controls the rapid disease progression seen in susceptible rats. Due to the correlation between the murine and rat systems, we hypothesized that the single dominant gene controlling rat susceptibility is the rat homologue to murine Nalp1b. If true, caspase-1 activation would also control macrophage killing. We found that caspase-1 and proteasome inhibitors prevented caspase-1 activation and macrophage cytolysis mediated by LT. Furthermore, the proteasome inhibitor NPI-0052 prevented disease progression and mortality in LT-treated susceptible rats. Proteasome inhibitors also increased survival in BALB/c mice challenged with LT. Taken together, we demonstrated that caspase-1 activation not only controls macrophage killing, but also disease progression in susceptible rats challenged with LT. Our findings suggest that drugs controlling the inflammasome and caspase-1 are potential therapeutics for inflammatory diseases mediated by microbial pathogens.     

Immunization of Mice with Live-Attenuated Late Liver Stage-Arresting Plasmodium yoelii Parasites Generates Protective Antibody Responses to Preerythrocytic Stages of Malaria

Understanding protective immunity to malaria is essential for the design of an effective vaccine to prevent the large number of infections and deaths caused by this parasitic disease. To date, whole-parasite immunization with attenuated parasites is the most effective method to confer sterile protection against malaria infection in clinical trials. Mouse model studies have highlighted the essential role that CD8⁺ T cells play in protection against preerythrocytic stages of malaria; however, there is mounting evidence that antibodies are also important in these stages. Here, we show that experimental immunization of mice with Plasmodium yoelii fabb/f⁻ (Pyfabb/f⁻), a genetically attenuated rodent malaria parasite that arrests late in the liver stage, induced functional antibodies that inhibited hepatocyte invasion in vitro and reduced liver-stage burden in vivo. These antibodies were sufficient to induce sterile protection from challenge by P. yoelii sporozoites in the absence of T cells in 50% of mice when sporozoites were administered by mosquito bite but not when they were administered by intravenous injection. Moreover, among mice challenged by mosquito bite, a higher proportion of BALB/c mice than C57BL/6 mice developed sterile protection (62.5% and 37.5%, respectively). Analysis of the antibody isotypes induced by immunization with Pyfabb/f⁻ showed that, overall, BALB/c mice developed an IgG1-biased response, whereas C57BL/6 mice developed an IgG2b/c-biased response. Our data demonstrate for the first time that antibodies induced by experimental immunization of mice with a genetically attenuated rodent parasite play a protective role during the preerythrocytic stages of malaria. Furthermore, they highlight the importance of considering both the route of challenge and the genetic background of the mouse strains used when interpreting vaccine efficacy studies in animal models of malaria infection.     

Arming of Macrophages with Lymphocytes from Mice Immunized with Tumor Tissue

C57BL/6 macrophages are rendered specifically cytotoxic (“armed”) by incubation with spleen cells from C57BL/6 mice sensitized in vivo with allogeneic P-815 mastocytoma cells. Data are presented which suggest that the cytotoxic activity of the macrophages is not due to the adherence of sensitized lymphocytes. The results indicate that it is the T-lymphooyte which is primarily involved in arming. Furthermore it is the cytotoxic and not the proliferating T-cell which is responsible for arming.     

痢疾杆菌免疫小鼠不同部位淋巴组织回忆反应观测

为探索痢疾杆菌感染是否产生免疫记忆，本文以福氏痢疾杆菌２ａ经口及腹腔免疫小鼠两次（相隔１４ｄ），间隔三个月后再刺激，应用ＢＡ-ＥＬＩＳＰＯＴ法检测了小鼠Ｐｅｙｅｒ（ＰＰ）淋巴结、肠系膜淋巴结（ＭＬＮ）及脾脏（ＳＰＬ）中特异性ＩｇＡ、ＩｇＧ、ＩｇＭ抗体分泌细胞（ＡＳＣ）的变化；并以ＭＴＴ比色法测定了ＣｏｎＡ诱导的淋巴细胞增殖反应；还观察了小肠粘膜组织切片。实验表明：口服途径能诱导ＰＰ中ＡＳＣ及淋转的回忆反应，小肠粘膜可见淋巴滤泡显著增生；而腹腔途径只在ＳＰＬ中诱导出ＡＳＣ及淋转回忆反应。结果提示：痢疾杆菌感染存在免疫记忆反应。     

Killing of Blood-Stage Murine Malaria Parasites by Hydrogen Peroxide

Both nonlethal Plasmodium yoelii and lethal Plasmodium berghei were killed in vitro by hydrogen peroxide at concentrations as low as 10⁻⁵ M. Higher concentrations were required in the presence of added normal erythrocytes. Injection of hydrogen peroxide in vivo significantly reduced P. yoelii parasitemia but had less effect on P. berghei.     

Lymphocyte Perturbations in Malawian Children with Severe and Uncomplicated Malaria

Lymphocytes are implicated in immunity and pathogenesis of severe malaria. Since lymphocyte subsets vary with age, assessment of their contribution to different etiologies can be difficult. We immunophenotyped peripheral blood from Malawian children presenting with cerebral malaria, severe malarial anemia, and uncomplicated malaria (n = 113) and healthy aparasitemic children (n = 42) in Blantyre, Malawi, and investigated lymphocyte subset counts, activation, and memory status. Children with cerebral malaria were older than those with severe malarial anemia. We found panlymphopenia in children presenting with cerebral malaria (median lymphocyte count, 2,100/μl) and uncomplicated malaria (3,700/μl), which was corrected in convalescence and was absent in severe malarial anemia (5,950/μl). Median percentages of activated CD69⁺ NK (73%) and γδ T (60%) cells were higher in cerebral malaria than in other malaria types. Median ratios of memory to naive CD4⁺ lymphocytes were higher in cerebral malaria than in uncomplicated malaria and low in severe malarial anemia. The polarized lymphocyte subset profiles of different forms of severe malaria are independent of age. In conclusion, among Malawian children cerebral malaria is characterized by lymphocyte activation and increased memory cells, consistent with immune priming. In contrast, there are reduced memory cells and less activation in severe malaria anemia. Further studies are required to understand whether these immunological profiles indicate predisposition of some children to one or another form of severe malaria.     

复方咳喘停治疗蛔虫变应原致喘豚鼠后血液流变性的动态变化

目的：观察蛔虫变应原致喘豚鼠后及平喘中药治疗后血液流变性的动态变化。方法：把豚鼠分为阴性对照、阳性激发和复方咳喘停中药治疗组。用蛔虫变应原诱发哮喘后,观察各组血液流变性的动态变化。结果：阳性激发组的血浆粘度、红细胞聚集指数及高切还原粘度增高,显著高于阴性组（Ｐ＜０．０１）;中药组上述指标显著低于阳性激发组（Ｐ＜０．０１）,与阴性组相比无显著性差异（Ｐ＞０．０５）。各组红细胞压积均无明显变化。结论：蛔虫变应原致喘豚鼠后,血液出现高粘滞状态;咳喘停能有效改善局部微循环、恢复流态。     

Anti-Horcnal and Anti-Nuclear Antibody (ANA) Responses of Mice Immunized with Estradiol

Sera of female mice, immunized with estradiol-albumin conjugates, were tested for restricted heterogeneity of the anti-estradiol antibody response and for the development of antinuclear antibody (ANA). We found sera with the highest estradiol binding capacity gave clear evidence of clonal dominance when analyzed by isoelectric focusing. A sub-set of the immunized animals, approximately one-third, developed positive ANA serologies within 12 weeks of the primary immunization. All positive sera gave homogeneous patterns of staining in an avidin-biotin amplified indirect immunofluorescence assay. Our results point to a spontaneous production of ANA in at least some animals immunized with a ligand which binds to a nuclear receptor.     

Functional Characterization of Anopheles Matrix Metalloprotease 1 Reveals Its Agonistic Role during Sporogonic Development of Malaria Parasites

The ability to invade tissues is a unique characteristic of the malaria stages that develop/differentiate within the mosquitoes (ookinetes and sporozoites). On the other hand, tissue invasion by many pathogens has often been associated with increased matrix metalloprotease (MMP) activity in the invaded tissues. By employing cell biology and reverse genetics, we studied the expression and explored putative functions of one of the three MMPs encoded in the genome of the malaria vector Anopheles gambiae, namely, the Anopheles gambiaeMMP1 (AgMMP1) gene, during the processes of blood digestion, midgut epithelium invasion by Plasmodium ookinetes, and oocyst development. We show that AgMMP1 exists in two alternative isoforms resulting from alternative splicing; one secreted (S-MMP1) and associated with hemocytes, and one membrane type (MT-MMP1) enriched in the cell attachment sites of the midgut epithelium. MT-MMP1 showed a remarkable response to ookinete midgut invasion manifested by increased expression, enhanced zymogen maturation, and subcellular redistribution, all indicative of an implication in the midgut epithelial healing that accompanies ookinete invasion. Importantly, RNA interference (RNAi)-mediated silencing of the AgMMP1 gene revealed a postinvasion protective function of AgMMP1 during oocyst development. The combined results link for the first time an MMP with vector competence and mosquito-Plasmodium interactions.     

Genetic Control of the Production of Anti-Thyroid Hormone Antibodies in Mice Immunized with Human Thyroglobulin

Various strains of mice with different H-2 and Igh-I allotypes were immunized with human thyroglobulin (HTg) and genetic control of the production of anti-thyroid hormone antibodies was examined. Anti-HTg antisera obtained from different strains of mice were examined for the presence of antithyroid hormone antibodies. At least one Ir-gene which controls the production of anti-T4 antibodies by immunization with HTg was identified in the I-A subregion. The presence of Ir-genes outside the H-2 was also suspected. Concerning the production of anti-T3 antibodies, only three strains (B10.A, C3H.SW, BALB/cJ) were high responders and therefore we were unable to identify the Ir-gene for them. These results indicate the presence of Ir-gene which controls the immune response in mice against thyroid hormone and suggest that the anti-thyroid hormone antibodies observed in various thyroidal and non-thyroidal disorders could be anti-HTg antibodies. The significance of genetic control of the production of anti-thyroid hormone antibodies in mice immunized with HTg is discussed.     

Surface Properties of Extracellular Malaria Parasites: Morphological and Cytochemical Study

Morphological and cytochemical surface characteristics of isolated malaria parasites (Plasmodium berghei) and host erythrocytes were compared by electron microscopy by using thin section and carbon replica techniques. Erythrocytes were uniform in shape and had fine, granular surfaces. In contrast, free parasites exhibited a variety of sizes, shapes, and surface textures. Fine surface stippling was a common topographical feature of isolated parasites. Small, infective forms often had patterned surfaces resulting from the protuberance of an underlying thick intermediate layer. Results of cytochemical analysis using a sialophilic colloidal iron stain indicated that the malaria parasite's surface lacked exposed sialic acid groups which would normally give rise to a net negative surface charge common to erythrocytes. Biochemical assay demonstrated that malaria parasites contained about one-half the amount of sialic acid per unit weight as did control red cell extracts. Similarly, external acidic mucopolysaccharide coats of free parasites, as revealed by ruthenium red staining were extremely thin as compared with the thick glycocalyx layer of red cells. Lipid plaques at the surface of parasites and red cells were localized by lipophilic iron colloid staining. Although the gross patchwork distribution of plaques was somewhat similar for the two cell types, the parasites were stained more intensely and had a closer-knit patchwork pattern than those exhibited by the erythrocytes. Such findings indicate that there are slight differences in the arrangement of phospholipids at the surfaces of limiting membranes of host cells and parasites. The significance of the above cytochemical surface properties of the malaria parasite (which are seemingly akin to those of intracellular organelles is discussed in relation to certain host-parasite interactions, such as parasite adhesion to target cells and enhanced clearance of extracellular parasites.