Simultaneous activation of complement and coagulation by MBL-associated serine protease 2

The complement system is an important immune mechanism mediating both recognition and elimination of foreign bodies. The lectin pathway is one pathway of three by which the complement system is activated. The characteristic protease of this pathway is Mannan-binding lectin (MBL)-associated serine protease 2 (MASP2), which cleaves complement proteins C2 and C4. We present a novel and alternative role of MASP2 in the innate immune system. We have shown that MASP2 is capable of promoting fibrinogen turnover by cleavage of prothrombin, generating thrombin. By using a truncated active form of MASP2 as well as full-length MASP2 in complex with MBL, we have shown that the thrombin generated is active and can cleave both factor XIII and fibrinogen, forming cross-linked fibrin. To explore the biological significance of these findings we showed that fibrin was covalently bound on a bacterial surface to which MBL/MASP2 complexes were bound. These findings suggest that, as has been proposed for invertebrates, limited clotting may contribute to the innate immune response.     

Rapid evolution of immunoglobulin superfamily C2 domains expressed in immune system cells

To test the hypothesis that proteins expressed in cells of the vertebrate immune system evolve unusually rapidly, 107 orthologous immunoglobulin C2 domains were compared between human and murine rodent. The analysis showed that the rate of nonsynonymous (amino-acid- altering) nucleotide substitution in these domains was correlated with factors associated with protein structure and with breadth of tissue expression, as well as with the rate of synonymous substitution. However, when such factors were controlled for statistically, there remained a strong positive association between expression in the immune system and nonsynonymous rate, with the highest rates being seen in genes expressed in the immune system only. Certain immune system genes are known to be subject to positive selection favoring diversity at the amino acid level; most of these genes encode receptors that interact directly with foreign antigens. The observed acceleration of the rate of nonsynonymous evolution in C2 domains of immune system proteins may be explained by either (1) reduced constraint at the amino acid level on molecules interacting with immune system receptors that are themselves evolving rapidly due to positive diversifying selection or (2) positive selection favoring amino acid changes correlated with changes in the immune system receptors.      

Expression and delivery of tetanus toxin fragment C fused to the N-terminal domain of SipB enhances specific immune responses in mice

Live attenuated bacteria can be used as a carrier for the delivery of foreign antigens to a host's immune system. The N-terminal domain of SipB, a translocon protein of the type III secretion system of Salmonella enterica serovar Typhimurium, is required for secretion and outer membrane localization. In the present study, vaccine plasmids for antigen delivery in which the non-toxic tetanus toxin fragment C (TTFC), which contains a T cell epitope, is fused to the N-terminal 160 amino acids of SipB were developed. It was found that the recombinant proteins are secreted into the culture media and localized to the bacterial surface. TTFC-specific antibody responses are significantly increased in mice orally immunized with attenuated S. Typhimurium BRD509 strains carrying TTFC delivery plasmids. When the TTFC delivery cassettes were introduced into a low copy vector, the plasmid was stably maintained in the BRD509 strain and induced an immune response to the TTFC antigen in mice. These results suggest that expression and delivery of heterologous antigens fused to the N-terminus of SipB enhance the induction of antigen-specific immune responses, and that the N-terminal domain of SipB can be used as a versatile delivery system for foreign antigens.     

Formation of antibodies against the antigen-recognizing receptors of T-lymphocytes in a syngenous system]

As shown, formation of antibodies to the antigen-recognition receptors of T-lymphocytes was possible in a syngeneic system. The antiserum of CBA mice given intravenous injections of CBA lymphocytes, immune to C57BL cells, proved to specifically inhibit in a mixed culture blasttransformation of CBA T-lymphocytes only against the C57BL cells. The same antiserum failed to influence the proliferative activity of CBA T-lymphocytes reacting to the "foreign" antigen (DBA/2 cells). No antibodies against the C57BL cells were revealed in the antireceptor antiserum. It is assumed that the autoantireceptor antibodies had a regulatory effect on the immune response.     

The complement system in teleosts

Complement, an important component of the innate immune system, is comprised of about 35 individual proteins. In mammals, activation of complement results in the generation of activated protein fragments that play a role in microbial killing, phagocytosis, inflammatory reactions, immune complex clearance, and antibody production. Fish appear to possess activation pathways similar to those in mammals, and the fish complement proteins identified thus far show many homologies to their mammalian counterparts. Because information about complement proteins, regulatory proteins, and complement receptors in fish is far from complete, it is unclear whether all the complement functions that have been identified in mammals also occur in fish. However, it has been clearly demonstrated that fish complement can lyse foreign cells and opsonise foreign organisms for destruction by phagocytes. There are also indications that complement fragments participate in inflammatory reactions. Fish possess multiple isoforms of several complement proteins, such as C3 and factor B. It has been hypothesised that the function of this diversity in complement proteins serves to expand their innate immune recognition capacity and response. Understanding the functions of complement in fish and the roles the individual proteins, including the various isoforms, play in host defence, is important not only for understanding the evolution of this system but also for the development of new strategies in fish health management.     

Cytotoxic immune cells with specificity for defined soluble antigens. I. Assay with antigen-coated target cells

An in vitro cytotoxic system is described, in which immune cells specific for a given soluble antigen exert a specific cytotoxic effect on target cells to which this antigen has been covalently linked. The nature of the target cell is important in this system. When antigen-coated P 815-X2 mastocytoma cells and antigen-coated chicken red blood cells were incubated for several hours in culture medium at 37 °C, the presence of membrane-bound antigen could still be demonstrated on the latter, but not on the former target cells. This might be the reason why antigen-specific target cell destruction by specific immune cells was observed only with antigen-coated chicken red blood cells as target cells. The specificity of the cytotoxic effect was controlled in each experiment in a criss-cross way by using two non cross-reacting antigens both as immunogens and for coating the target cells. Specific cytotoxicity was demonstrable with both guinea pig and mouse immune cells and with different kinds of antigens: foreign proteins, hapten-heterologous protein conjugates and hapten-autologous protein conjugates.     

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.     

重组金丝雀痘病毒载体疫苗研制进展

金丝雀痘病毒(canarypox virus,CNPV)引起的雀痘是一种禽类传染性疾病。CNPV感染后可在宿主体内诱导细胞、体液和黏膜免疫应答,经过改造可开发为一种候选疫苗载体,其可表达病毒和寄生虫的多种蛋白,因此,研制重组CNPV载体疫苗进行免疫预防具有重要的经济意义。国外学者已构建了多种类似疫苗,如马流感病毒(equine influenza virus,EIV)、犬瘟热病毒(canine distemper virus,CDV)、麻疹病毒(measles virus,MV)、尼帕病毒(Nipah virus,NiV)、艾滋病毒(human immunodeficiency virus type 1,HIV-1)、非洲马病病毒 4 型(African horse sickness virus type 4,AHSV-4)、蓝舌病毒 17 型(bluetongue virus serotype 17,BTV-17)、丙型肝炎病毒(hepatitis C virus,HCV)、巨细胞病毒(cytomelovirus,CMV)、狂犬病毒(rabies virus)和婴儿利什曼原虫(Lei...     

Innate immunity and its evasion and suppression by hymenopteran endoparasitoids

Recent studies suggest that insects use pattern recognition molecules to distinguish prokaryotic pathogens and fungi from "self" structures. Less understood is how the innate immune system of insects recognizes endoparasitic Hymenoptera and other eukaryotic invaders as foreign. Here we discuss candidate recognition factors and the strategies used by parasitoids to overcome host defense responses. We suggest that host-parasitoid systems are important experimental models for studying how the innate immune system of insects recognizes foreign invaders that are phylogenetically more closely related to their hosts. The strategies used by parasitoids suggest that insects may employ "hidden-self" recognition molecules for attacking foreign objects intruding the open circulatory system. BioEssays 23:344-351, 2001.     

Chemokines provide the sustained inflammatory bridge between innate and acquired immunity

In this review we focus on the role of chemokines in discreet areas of innate immunity and demonstrate that chemokines are key participants to not only the early inflammatory response to a foreign agent, but important to the sustained immune reaction. Our studies support the concept that a concerted and interactive innate and acquired immune reaction is key for an automatic, dynamic, sustained, and regulated response toward clearing foreign stimuli. It is imperative that the in vivo concept of innate and acquired immunity be considered a continuum of a global assault on a foreign agent and not as modes, which are independent of one another.     

DNA vaccination strategies against infectious diseases

DNA immunisation represents a novel approach to vaccine and immunotherapeutic development. Injection of plasmid DNA encoding a foreign gene of interest can result in the subsequent expression of the foreign gene products and the induction of an immune response within a host. This is relevant to prophylactic and therapeutic vaccination strategies when the foreign gene represents a protective epitope from a pathogen. The recent demonstration by a number of laboratories that these immune responses evoke protective immunity against some infectious diseases and cancers provides support for the use of this approach. In this article, we attempt to present an informative and unbiased representation of the field of DNA immunisation. The focus is on studies that impart information on the development of vaccination strategies against a number of human and animal pathogens. Investigations that describe the mechanism(s) of protective immunity induced by DNA immunisation highlight the advantages and disadvantages of this approach to developing vaccines within a given system. A variety of systems in which DNA vaccination has resulted in the induction of protective immunity, as well as the correlates associated with these protective immune responses, will be described. Particular attention will focus on systems involving parasitic diseases. Finally, the potential of DNA immunisation is discussed as it relates to veterinary medicine and its role as a possible vaccine strategy against animal coccidioses.     

Intratumoral DNA electroporation induces anti-tumor immunity and tumor regression

In situ expression of a foreign antigen and an immune-modulating cytokine by intratumoral DNA electroporation was tested as a cancer therapy regimen. Transgene expression in the tumors was sustained for 2–3 weeks after intratumoral electroporation with mammalian expression plasmid containing firefly luciferase cDNA. Electroporation with cDNA encoding tetanus toxin fragment C (TetC) induced tetanus toxin-binding antibody, demonstrating immune recognition of the transgene product. Intratumoral electroporation with TetC and IL-12 cDNA after mice were treated with CD25 mAb to remove regulatory T cells induced IFN-γ producing T-cell response to tumor-associated antigen, heavy inflammatory infiltration, regression of established tumors and immune memory to protect mice from repeated tumor challenge. Intratumoral expression of immune-modulating molecules may be most suitable in the neoadjuvant setting to enhance the therapeutic efficacy and provide long-term protection.     

Comparative immunological analysis of echinoderm cellular and humoral defense factors

Coelomocyte are found in the fluid filling coelomic cavity of echinoderms and depending on species can be a mixture of several morphologically different types. There are among them: granular and agranular amoebocytes, morula cells, vibratile and lymphocyte-like cells. All these cells take part in cellular response to immune challenges through phagocytosis, clotting, encapsulation of foreign particles, cytotoxicity, and the production of antimicrobial agents, such as reactive oxygen and nitric oxide. The data are given on a variety of humoral factors found in the coelomic fluid, including different types of lectines, agglutinins, hemolysins, acute phase proteins and antimicrobial factors. The discussion on cooperation between cellular and humoral arms of defense reactions during inflammation reveals the crucial role of coelomocytes in immune response. It is suggested that the sea urchin complement system (that is homologous to the alternative pathway in vertebrates) is appeared initially in echinoderms as a protein cascade that points to opsonization of foreign cells and particles, augmenting their phagocytosis and subsequent destruction by coelomocytes. So the identification of a simple complement system as a part of the echinoderm immune response shows that these animals as well as all invertebrate deuterostomes share innate immune system homologies with vertebrates. Studying the simpler immune response demonstrated by echinoderms is important for understanding the ancestral deuterostome defense system and reconstructing the evolution of immune system in higher vertebrates.     

Breaking T cell tolerance with foreign and self co-immunogens. A study of autoimmune B and T cell epitopes of cytochrome c.

The initiation of autoimmune B cell and T cell responses by self Ag or by foreign pathogens (molecular mimics) is not well understood. In the present study, cytochrome c (cyt c) was used as a model autoantigen to investigate how self-proteins are involved in the priming of autoimmune T cell responses. Immunization with foreign cyt c has been extensively analyzed in previous studies as a model for both humoral and cellular immune responses. Mice do not, however, make antibody or T cell responses to immunization with self (mouse) cyt c. In addition, T cell tolerance can be broken by autoreactive B cells that are readily elicited by immunization with cross-reactive foreign cyt c. These immune B cells presumably bind self cyt c and process and present the self Ag to stimulate an autoreactive T cell response. Autoreactive T cell clones derived by this mechanism are all specific for determinants within amino acids 1-80 of the cyt c protein presented by I-Ek. No T cell responses were observed to the carboxyl terminal 81-104 fragment that dominates the response to foreign cyt c. All clones derived in this study are stimulated by a polypeptide encompassing amino acids 54-68 and utilized the V beta 8.2 TCR gene. In contrast, T cells stimulated by foreign cyt c did indeed respond to fragment 81-104 and appear to utilize alternate TCR genes. Our data demonstrate that B cells specific for linear determinants distributed along the entire length of the foreign cyt c molecule can provide the stimulus required for breaking T cell tolerance to self cyt c. The applications of this work to understanding the mechanisms of autoimmune disease are discussed.     

The role of CXCR3 and its ligands in renal transplant outcome

Chemokines and their corresponding receptors serve as pro-inflammatory and migratory signals for immune cells. CXCR3 and its corresponding ligands, CXCL9, CXCL10 and CXCL11, participate in the induction of immune responses against several foreign antigens. Numerous cells, including macrophages, NK cells and T lymphocytes, express CXCR3 and thus, expression of the receptor and its ligands can induce activity of these important immune cells against foreign antigens, including allogeneic grafts. Several parameters of the immune system participate in the induction and stimulation of powerful immune responses against allogeneic grafts. A thorough understanding of the parameters that regulate these responses can provide insights into new methods for immunotherapy during organ transplantation. The aim of this review is to address the most recent information regarding the roles played by CXCR3 and its corresponding ligands in the outcome of renal transplantation.     

Constitutive competition by self proteins for antigen presentation can be overcome by receptor-enhanced uptake

The cells recognize a bimolecular ligand composed of a self Ia molecule and a fragment of foreign Ag that has been processed by an APC. The effect of self proteins on the processing and presentation of foreign Ag was examined in order to ascertain the mechanisms for competition between foreign and self Ag. How this competition can be overcome to allow an efficient immune response was also examined. Normal mouse serum proteins (NMS) compete for the processing and presentation of the foreign Ag bovine RNase by APC. This competition could have occurred at any of three levels in the APC: 1) Ag uptake, 2) Ag processing, or 3) the binding of Ag to an Ia molecule. No competition for either the uptake or the processing of RNase by self proteins could be demonstrated. However, self peptides do compete with foreign Ag by binding directly to Ia molecules, as has been shown previously. Thus, the observed inhibition by NMS of Ag presentation occurred because of competition for binding to the Ia molecule. We hypothesized that during the generation of an immune response this competition is overcome by enhanced uptake of foreign Ag. To test this, we compared the ability of NMS to compete for the presentation of RNase when it entered the APC via fluid-phase pinocytosis or through receptor-mediated uptake via the mannose receptor. When the RNase entered the APC through the mannose receptor, the ability of NMS to compete was dramatically reduced. Thus, self proteins constitutively compete for the presentation of foreign Ag at the level of binding to an Ia molecule, and this competition can be overcome by receptor-mediated uptake of the Ag.     

Cellular immune competence of a Drosophila mutant with neoplastic hematopoietic organs

In the Tum^l mutant of Drosophila melanogaster, the larval hematopoietic organs undergo neoplastic changes and release into circulation large numbers of blood cells. The lamellocytes, and to a lesser extent the plasmatocytes from which they are derived, are the cells that encapsulate various endogenous tissues and form melanotic tumors. The mutation is temperature sensitive, with maximum gene expression manifested at 29°C. The ability of Tum^l larvae to encapsulate eggs of the wasp parasite Leptopilina heterotoma is dependent not only on temperature, with host larvae much more immune reactive at 29°C than at lower temperatures (15° or 21°C), but also on the interval of time following infection when temperature shift experiments are performed. When the shift of parasitized larvae from 21° to 29°C is delayed by 18 hr the hosts are not as immune reactive as those shifted immediately after infection. Since Tum^l larvae are potentially highly immune reactive at the time of infection (with sufficient numbers of lamellocytes in circulation to encapsulate parasites), the low degree of immune competence in hosts shifted to 29°C after 18 hr or maintained at lower temperatures suggests that the increased capacity of blood cells to react against foreign surfaces is dependent on the cells acquiring new or altered recognition and adherence properties at 29°C. The 18-hr delay may provide the parasite with an opportunity to interfere with the acquisition of these specific cellular alterations. Differential hemocyte counts from parasitized larvae show abnormally low lamellocyte counts in susceptible hosts, indicating that successfully developing parasites interfere with the differentiation of hemocytes.     

Immunotropic properties of the causative agent of viral hepatitis C

In this review the immunomodulating properties of the causative agent of viral hepatitis C are characterized on the basis of experimental data, obtained by Russian and foreign researchers during recent 3-5 years. The short characterization of the causative agent is presented and a number of adaptation mechanisms making it possible for hepatitis C virus to resist the action of the immune protective system of the host are considered. The role of individual protein products of the virus in the immunopathogenesis of the disease and the mechanisms of their action on the molecular level are discussed in detail on the basis of the results of mouse and in vitro experiments.     

Viral mimicry of the complement system

The complement system is a potent innate immune mechanism consisting of cascades of proteins which are designed to fight against and annul intrusion of all the foreign pathogens. Although viruses are smaller in size and have relatively simple structure, they are not immune to complement attack. Thus, activation of the complement system can lead to neutralization of cell-free viruses, phagocytosis of C3b-coated viral particles, lysis of virus-infected cells, and generation of inflammatory and specific immune responses. However, to combat host responses and succeed as pathogens, viruses not only have developed/adopted mechanisms to control complement, but also have turned these interactions to their own advantage. Important examples include poxviruses, herpesviruses, retroviruses, paramyxoviruses and picornaviruses. In this review, we provide information on the various complement evasion strategies that viruses have developed to thwart the complement attack of the host. A special emphasis is given on the interactions between the viral proteins that are involved in molecular mimicry and the complement system.     

Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination.

Viewing the immune system as a molecular recognition device designed to identify “foreign shapes”, we estimate the probability that an immune system with N_Ab monospecific antibodies in its repertoire can recognize a random foreign antigen. Furthermore, we estimate the improvement in recognition if antibodies are multispecific rather than monospecific. From our probabilistic model we conclude: (1) clonal selection is feasible, i.e. with a finite number of antibodies an animal can recognize an effectively infinite number of antigens; (2) there should not be great differences in the specificities of antibody molecules among different species; (3) the region of a foreign molecule recognized by an antibody must be severely limited in extent; (4) the probability of recognizing a foreign molecule, P, increases with the antibody repertoire size N_Ab; however, below a certain value of N_Ab the immune system would be very ineffectual, while beyond some high value of N_Ab further increases in N_Ab yield diminishing small increases in P; (5) multispecificity is equivalent to a modest increase (probably less than 10) in the antibody repertoire size N_Ab, but this increase can substantially improve the probability of an immune system recognizing a foreign molecule.Besides recognizing foreign molecules, the immune system must distinguish them from self molecules. Using the mathematical theory of reliability we argue that multisite recognition is a more reliable method of distinguishing between molecules than single site recognition. This may have been an important evolutionary consideration in the selection of weak non-covalent interactions as the basis of antigen-antibody bonds.