Inhibition of P. falciparum growth in human erythrocytes by monoclonal antibodies

Malaria is increasing in incidence and prevalence in most tropical areas and is a major problem for both individuals and communities. Current malaria research is aimed at developing vaccines and, for this, it may be useful to define Plasmodium antigen(s) related to the development of a protective immune response in the host. Monoclonal antibodies have recently been shown to interfere with rodent malaria infection (Plasmodium berghei) at the sporozoite or merozoite stage. We have now raised monoclonal antibodies against single antigenic determinant(s) of Plasmodium falciparum and report that some of them inhibit the growth of erythrocytic forms of P. falciparum in vitro.     

Development of virus non-producer lymphosarcomas in pet cats exposed to FeLv

Naturally occurring oncoviruses of several species are transmitted contagiously and cause lymphosarcoma (LSA) or leukaemia in their hosts. All naturally occurring oncoviruses replicate in vivo in the tumours they induce or, as with bovine leukaemia virus, can be isolated from tumour cells grown in short-term cell culture. However, we have shown that feline leukemia virus (FeLV) is not present in a significant minority of pet cats that develop LSA. Unlike experimentally induced virus-negative leukaemias and sarcomas of other species, LSA cells from FeLV-negative LSA cats lack any FeLV proteins, including p15 or p12, and complete functional copies of FeLV provirus and thus do not produce FeLV when grown in cell culture. Thus, except for FeLV, the naturally occurring animal leukaemogenic oncoviruses seem to induce only virus-producing lymphoid tumours. Our earlier findings prompted a study to determine the frequency of occurrence of FeLV non-producer (NP) LSA in pet cats and whether NP LSAs develop in cats exposed to FeLV. We report here epidemiological data which indicate that development of NP LSAs in pet cats is associated with exposure to FeLV and suggest that FeLV may be the aetiological agent for FeLV NP feline LSAs. Thus, feline NP LSAs may be suitable for studying the potential viral aetiology and mechanism of leukaemogenesis of human lymphoid tumours in which no oncoviruses have, as yet, been proved to cause the disease.     

Intrathymic inoculation of donor liver specific antigen alleviates rejection of liver allotransplantation

Use and effects of liver specific antigen in orthotopic liver transplantations were researched in this study. Group I: syngeneic control (Wistar-to-Wistar); Group II: acute rejection (SD-to-Wistar); Group III: Thymic inoculation of SD rat LSA day 7 before transplantation. The observation of common situation and survival time, rejection grades, NF-KappaB activity of splenocytes and IL-2mRNA expression of grafted liver were used to analyze acute rejection severity and immune state of animals in different groups. The common situation of group I was very well after transplantation and no signs of rejection were found. Recipients of group II lost body weight progressively. All dead within day 9 to day 13 posttransplantation; median survival time was 10.7+/-0.51 days. It was an optimal acute rejection control. As for group III, 5 out of 6 recipients survived for a long time and common situation was remarkably better than that of group II. Its rejection grades were significantly lower than that of group II(P < 0.05). NF-KappaB activity was only detected in group I at day 5 and day 7 after transplantation, whereas high activity of NF-KappaB was detected at all time points in group II and the low NF-KappaB activity detected in group III was significantly lower than that of group II(P < 0.05). No IL-2mRNA expression was detected at any time point in group I, whereas high level expression was detected at all time points in group II and the low level expression only detected at day 3 in group III was significantly lower than that of group II(P < 0.05). Conclusion: LSA is an important transplantation antigen which is involved directly in the immunorejection of liver transplantation. We report here for the first time that intrathymic inoculation of LSA can alleviate the rejection of liver allotransplantation; and that grafts can survive for a long time thereby, thus leading to a novel way to achieve liver transplantation immunotolerance.     

Inhibition of antigen-induced T-cell clone proliferation by antigen-specific antibodies

Attempts to inhibit the recognition of soluble antigens by T lymphocytes using antibodies specific for the antigen in question have been uniformally unsuccessful, in contrast to the observed specific inhibition of antibody generation by B cells. One exception is the unique situation whereby anti-hapten antisera inhibit the T-cell proliferative responses observed when hapten-specific T lymphocytes or clones are cultured with hapten-derivatized cells or proteins. The inability to inhibit T-cell functions by antigen-specific antibodies has been interpreted in several ways: (1) T cells possess a different repertoire from B cells; (2) the antibodies tested recognize epitopes present on the native antigen, whereas T cells recognize non-native (processed) structures; (3) the antigenic determinant(s) recognized by T cells on the surface of antigen presenting cells are either not accessible to antibodies, or are present in low amounts. The development of antigen-specific T-cell clones and monoclonal antibodies both specific for the same antigenic determinants now allows this question to be investigated definitively. Here, we report for the first time the specific inhibition of antigen-induced T-cell clone proliferation by a monoclonal antibody directed against the relevant soluble protein antigen.     

Major surface antigen gene of a human malaria parasite cloned and expressed in bacteria

The late blood stages of the human malaria parasite, Plasmodium falciparum, carry a major surface antigen, p190, of molecular weight (Mr) 190,000. This antigenically variable protein is actively processed, first as the parasite matures and again when it is released into the blood stream and invades a new erythrocyte to initiate a cycle of growth. It elicits a strong immune response in man; all tested adult sera from endemic areas have antibodies against this protein. Our evidence indicates that purified p190 can alter the course of parasitaemia in monkeys with falciparum malaria. We have also succeeded in cloning part of the gene for p190 and expressing it in Escherichia coli. To this end we have developed a new technique, antibody select, which greatly simplifies final identification of expressing clones.     

Evidence for immunological cross-reaction between sporozoites and blood stages of a human malaria parasite

Malaria parasites (Plasmodium spp.) show a complex pattern of development in the mammalian host and many studies support the view that the surface of the sporozoite, injected by the mosquito, has no antigens in common with the erythrocytic stage of development. For example, immunization with the erythrocytic parasites generates antisera with negligible titre by indirect immunofluorescence to the sporozoite surface. Although monoclonal antibodies prepared against erythrocytic stages were reported to show cross-reaction to the sporozoite stage, this appeared to be due to cytoplasmic antigens exposed by the method of sporozoite preparation, and in Plasmodium knowlesi, a cDNA clone coding for the circumsporozoite antigen, the major protein of the sporozoite surface, showed no hydridization to mRNA isolated from the erythrocytic stages. Here, however, we present evidence for an antigenic determinant shared by the sporozoite surface and the erythrocytic stages of the human malaria parasite, P. falciparum. Moreover, our studies show that the antigen(s) elicit a strong immune response in man.     

Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi

The malarial sporozoite, the infective stage found in the salivary gland of the insect vector, bears highly immunogenic surface antigen(s). Repeated exposure to irradiated sporozoites induces protection against malaria in several host species, including man. Further, monoclonal antibodies that confer passive immunity react with the immunogenic surface determinants of different sporozoite species. One approach to prevent malaria, therefore, would be to produce a vaccine that induces high titres of circulating antibodies against the sporozoite surface determinant(s). However, production of such a vaccine has not been possible since sporozoites cannot be cultivated in vitro and, therefore, only limited amounts of surface antigen may be obtained. To overcome this problem, we have prepared mRNA from Plasmodium knowlesi-infected mosquitoes to construct a cDNA library. From this library we have isolated a clone that expresses the sporozoite surface antigen as a beta-lactamase fusion protein in the plasmid pBR322. This is the first potentially protective malarial antigen to be cloned by recombinant DNA technology.     

江西省龙南县滑坡易发性评价

区域滑坡易发性评价是国内外地质灾害研究的重点和热点。目前,国内外学者已提出了支持向量机(support vector machine,SVM)、BP神经网络和随机森林等多种模型并成功用于滑坡易发性评价。但在利用这些机器学习模型评价滑坡易发性时,存在着参数选取困难、建模效率低、模型训练时间长和对评价指标解释能力弱等问题。为简化建模过程、提高预测精度及增强模型的可解释性,提出了基于频率比分析和偏最小二乘回归法(partial least squares regression,PLSR)的滑坡易发性评价模型。PLSR模型很好地发挥了主成分分析和回归分析的优势,考虑了评价指标间的内在联系,具有建模过程简洁、可解释性强的优点。将结合频率比法的PLSR模型应用于江西省龙南县滑坡易发性评价,并与BP神经网络、SVM模型的易发性评价结果进行对比。研究表明:PLSR模型的预测精度优于BP神经网络,且与SVM模型预测精度接近;另外,在综合考虑建模效率、预测精度和模型解释能力的情况下,PLSR模型具有更高的实用性。     

Immunization of Aotus monkeys with recombinant proteins of an erythrocyte surface antigen of Plasmodium falciparum

Recent studies have identified and characterized a ring-infected erythrocyte surface antigen (RESA) of the human malaria parasite Plasmodium falciparum with a relative molecular mass (Mr) of approximately 155,000 (refs 1-7). RESA is localized in the micronemes of merozoites and also the membrane of red cells infected with ring-stage parasites. It is thought to be released through the apical pore from the rhoptry at the time of merozoite invasion. Because antibodies directed against this antigen strongly inhibit parasite growth in vitro, RESA may be useful in developing a vaccine against this parasite Here we describe an immunization trial using Aotus monkeys and Escherichia coli-derived fused polypeptides corresponding to various regions of the RESA molecule. Some monkeys in all test groups, but not in the control group, were protected against overwhelming infection. Strikingly, protection correlated with antibody responses to either of two different repetitive sequences in RESA.     

Structural basis for Duffy recognition by the malaria parasite Duffy-binding-like domain

Molecular processes that govern pathogenic features of erythrocyte invasion and cytoadherence in malaria are reliant on Plasmodium-specific Duffy-binding-like domains (DBLs). These cysteine-rich modules recognize diverse host cell-surface receptors during pathogenesis. DBLs of parasite erythrocyte-binding proteins mediate invasion, and those from the antigenically variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) have been implicated in cytoadherence. The simian and human malarial parasites, P. knowlesi and P. vivax, invade human erythrocytes exclusively through the host DARC receptor (Duffy antigen receptor for chemokines). Here we present the crystal structure of the P. knowlesi DBL domain (Pkalpha-DBL), which binds to DARC during invasion of human erythrocytes. Pkalpha-DBL retains the overall fold observed in DBLs from P. falciparum erythrocyte-binding antigen (EBA)-175 (ref. 4). Mapping the residues that have previously been implicated in binding highlights a fairly flat but exposed site for DARC recognition in subdomain 2 of Pkalpha-DBL; this is in sharp contrast to receptor recognition by EBA-175 (ref. 4). In Pkalpha-DBL, the residues that contact DARC and the clusters of residues under immune pressure map to opposite surfaces of the DBL, and suggest a possible mechanism for immune evasion by P. vivax. Our comparative structural analysis of Pkalpha-DBL and P. falciparum EBA-175 provides a framework for the understanding of malaria parasite DBLs, and may affect the development of new prophylactic and therapeutic strategies.     

Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum

Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor-ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor-ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Ok(a-) erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor-ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.     

Simultaneous ultrastructural demonstration of multiple peptides in endocrine cells by a novel immunocytochemical method.

Current immunocytochemical techniques detect all antibodies that react with tissue. Unfortunately, some of these antibodies may react with antigens other than those intended. Such problems are minimised by using sets of antibodies detecting different regions of the desired antigen. However, as immunocytochemical methods can now detect very low antibody concentrations, the purity of antisera is critical. Furthermore, although antisers may be purified by affinity chromatography, difficulties in recovering high-avidity antibodies cause most affinity-purified antisera to be enriched in low-avidity antibodies, which may be dislodged during staining. We have therefore developed, and describe here, a new ultrastructural post-embedding staining technique, based on the divalency of IgG molecules and using antigen-coated colloidal gold granules. Previously, colloidal gold-labelled antibodies have been used for post-embedding staining. Unlike our gold-labelled antigen detection (GLAD) technique, however, these methods do not differentiate between specific and nonspecific antibodies. The GLAD method detects only specific antibodies and does not select against high-avidity antibodies, and in this it resembles the radioimmunocytochemical method. However, the GLAD method differs from the latter in that it is useful for ultrastructural studies, does not require autoradiography and allows simultaneous detection of multiple antigens. Moreover, specific activity compared with background may be quantitated.     

Antibody-based protection against HIV infection by vectored immunoprophylaxis

Despite tremendous efforts, development of an effective vaccine against human immunodeficiency virus (HIV) has proved an elusive goal. Recently, however, numerous antibodies have been identified that are capable of neutralizing most circulating HIV strains. These antibodies all exhibit an unusually high level of somatic mutation, presumably owing to extensive affinity maturation over the course of continuous exposure to an evolving antigen. Although substantial effort has focused on the design of immunogens capable of eliciting antibodies de novo that would target similar epitopes, it remains uncertain whether a conventional vaccine will be able to elicit analogues of the existing broadly neutralizing antibodies. As an alternative to immunization, vector-mediated gene transfer could be used to engineer secretion of the existing broadly neutralizing antibodies into the circulation. Here we describe a practical implementation of this approach, which we call vectored immunoprophylaxis (VIP), which in mice induces lifelong expression of these monoclonal antibodies at high concentrations from a single intramuscular injection. This is achieved using a specialized adeno-associated virus vector optimized for the production of full-length antibody from muscle tissue. We show that humanized mice receiving VIP appear to be fully protected from HIV infection, even when challenged intravenously with very high doses of replication-competent virus. Our results suggest that successful translation of this approach to humans may produce effective prophylaxis against HIV.     

A late-differentiation antigen associated with the helper inducer function of human T cells

T lymphocytes possessing helper function produce soluble factors that greatly augment B-cell proliferation and differentiation into antibody-secreting cells. In humans the subset of T lymphocytes bearing the T4 surface antigen comprises most of the cells that display helper activity and recognize class II antigens of the major histocompatibility complex (MHC), while the subset bearing the T8 antigen comprises T cells recognizing class I MHC antigens and exhibiting cytotoxic or suppressor function. Monoclonal antibodies to T4 or T8 greatly inhibit the cognitive and effector function of cells with the corresponding phenotype. This function/phenotype correlation is not absolute, however, for there are many examples of T8-positive clones that recognize MHC class II antigens and have helper activity, as well as of T4-positive clones with suppressor or cytotoxic function. Recently a family of cell-surface neoantigens, which might be relevant to T-cell function and which are present on activated but not on resting T lymphocytes, has been identified in mouse and humans using monoclonal antibodies. Some of these antibodies block the cytolytic activity of alloreactive T-cell clones, suggesting the possible involvement of such molecules in the activation of cytotoxic T-cell clones or in the lytic process itself. We now describe a similar late-differentiation antigen (LDA1) that is expressed by human T lymphocytes only following activation and is recognized by a monoclonal antibody that inhibits the antibody-inducing helper function of T lymphocytes.     

治疗性乙型肝炎疫苗研究反思

 “治疗性乙肝疫苗”是以乙肝病毒(HBV)的表面抗原(s抗原)为基础的生物制剂,目的是激发抗s抗原免疫反应,终止HBV慢性感染.HBV的e抗原与s抗原无抗原性关联,对e抗原的反应也与病毒及感染细胞的清除无关,因此以II期临床试验者血清有关病毒e抗原的数据结果,尚不能判断“治疗性乙肝疫苗”是否有效.疫苗的应用是抵御病毒入侵,而治疗性乙肝疫苗是在病毒已经进入人体后应用,在患者肝细胞可能广泛受累的情况下,疫苗一旦打破耐受激发抗s抗原免疫反应,除能清除血清中游离的病毒和s抗原颗粒外,将直接攻击被感染的肝细胞.由于无法估计慢性HBV感染者肝细胞感染程度,所以无法推测免疫反应发生后,免疫病理所致的肝损程度以及相应的风险.在应用上隐含如此风险,是“治疗性乙肝疫苗”走不出实验室的重要因素之一.     

Immunization to a syngeneic sarcoma by a monoclonal auto-anti-idiotypic antibody

Idiotypic networks regulate the immune response to a variety of antigens. Antibodies generated against other antibodies, called anti-idiotypic antibodies, can themselves mimic antigen and elicit a specific immune response. They have been shown to induce delayed-type hypersensitivity (DTH) to model antigens in the mouse. As anti-idiotypic antibodies are thought to be involved in the response to tumour-associated antigens we tested whether injection of monoclonal antibodies derived from mice hyperimmunized to a syngeneic, chemically induced sarcoma could mimic antigen and induce DTH to the sarcoma in naive mice. One of the monoclonal antibodies, 4.72, primed BALB/c mice for DTH to the sarcoma but not for DTH to another sarcoma or to sheep erythrocytes. Antibody 4.72 did not induce DTH in mice of immunoglobulin allotype congeneic strains nor did it bind to the sarcoma cells. As antibodies specific for this sarcoma have not been detected, we do not know whether idiotype on immunoglobulin molecules is recognized by antibody 4.72. However, as the response induced by antibody 4.72 was both antigen-specific and allotype-restricted, analogous to those induced by anti-idiotypic antibodies in other systems, we propose that antibody 4.72 is an anti-idiotypic antibody.     

Climate change and the resurgence of malaria in the East African highlands

The public health and economic consequences of Plasmodium falciparum malaria are once again regarded as priorities for global development. There has been much speculation on whether anthropogenic climate change is exacerbating the malaria problem, especially in areas of high altitude where P. falciparum transmission is limited by low temperature. The International Panel on Climate Change has concluded that there is likely to be a net extension in the distribution of malaria and an increase in incidence within this range. We investigated long-term meteorological trends in four high-altitude sites in East Africa, where increases in malaria have been reported in the past two decades. Here we show that temperature, rainfall, vapour pressure and the number of months suitable for P. falciparum transmission have not changed significantly during the past century or during the period of reported malaria resurgence. A high degree of temporal and spatial variation in the climate of East Africa suggests further that claimed associations between local malaria resurgences and regional changes in climate are overly simplistic.     

潜语义分析语言模型及其在汉语大词汇连续语音识别中的应用

分析了潜语义分析语言模型在建模和解码过程中的主要问题, 实现了潜语义分析语言模型的建模, 并提出一种在连续语音识别系统一遍解码框架中融合的方法. 实验结果表明, 该方法可有效地提高大词汇汉语连续语音识别系统的性能.      

Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites

A 12 amino-acid synthetic peptide (NANP)3 comprising the immunodominant epitope of Plasmodium falciparum circumsporozoite protein was conjugated to tetanus toxoid (TT), adjuvanted with aluminium hydroxide, and administered intramuscularly in three doses at monthly intervals to 35 healthy males as a malaria vaccine. No significant adverse reactions were noted, with mild soreness at the injection site the only common symptom. Seroconversions against NANP occurred in 53% and 71% of recipients of 100 or 160 micrograms, respectively, measured by enzyme-linked immunosorbent assay (ELISA). Most ELISA-positive sera reacted with sporozoites by indirect immunofluorescence (IFA). Three vaccinees with the highest ELISA and IFA titres and four unimmunized controls were challenged with P. falciparum sporozoites introduced via the bites of infective Anopheles mosquitoes. Blood stage parasites were detected in all controls by 10 days (mean 8.5 days, range 7-10). In contrast, the two vaccinees who became infected did not manifest parasitaemia until day 11 and the third vacinee showed neither parasites nor symptoms during the 29 day observation period. This first synthetic peptide parenteral vaccine against a communicable disease tested in man is safe and stimulates biologically active antibodies. These observations encourage the development of improved vaccine formulations which, by enhancing immunogenicity, may lead to practical vaccines to assist in the control of falciparum malaria.     

Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence

Plasmodium falciparum infected erythrocytes containing mature trophozoites and schizonts sequester along venular endothelium and are not in the peripheral circulation of patients with malaria. Knobs appear on infected erythrocytes and are the points of attachment to endothelium. Sequestration may protect the parasite from splenic destruction and may play a role in the pathogenesis of cerebral malaria. Correlates of sequestration have been developed in vitro using cultured human endothelium and an amelanotic melanoma cell line. Knobless strains (K-) of P. falciparum fail to sequester in vivo and to bind to cells in vitro. We now present evidence that the receptor for cytoadherence is the glycoprotein, thrombospondin. Aotus monkey or human erythrocytes containing knobby (K+) but not Aotus erythrocytes containing knobless strains of P. falciparum bind to immobilized thrombospondin. Neither binds to the adhesive proteins laminin, fibronectin, factor VIII/von Willebrand factor or vitronectin. Both soluble thrombospondin and anti-thrombospondin antibodies inhibit binding of parasitized Aotus erythrocytes to immobilize thrombospondin and to melanoma cells which secrete thrombospondin.