Genomic characterization of the RUNX2 gene of Fugu rubripes

Inducibility of the mouse gene imap38 in the spleen has been recently described to correlate with resistance to Plasmodium chabaudi malaria. Here, we characterize the human ortholog gene himap1. The HIMAP1 34 kDa protein is localizable at the endoplasmic reticulum in transfected cells. It contains a GTP-binding domain, but it does not bind GTP, in contrast to mouse IMAP38. The himap1 gene belongs to a gene family clustered on chromosome 7q32-36 within a region highly syntenic to the mouse imap38 locus on chromosome 6B. The himap genes 1, 2, 3, and 4 display a conserved intron/exon structure. The mRNA of the himap1 gene is predominantly expressed in the spleen, in lymph nodes to a lesser extent, and only at very low levels in diverse cancer cell lines. In accordance, imap-like genes in mice and plants are associated with proliferative and apoptotic events suggesting a role in the control of cell death/survival.     

Plasmodium chabaudi chabaudi: B-1 cell expansion correlates with semiresistance in BALB/cJ mice

The largest obstacle impeding the development of an effective malaria vaccine is the incomplete understanding of how the immune response is regulated during infection. B-1a cells, a poorly understood subcategory of B lymphocytes, produce nonpathologic autoantibodies of low affinity which have been shown to have distinct immunoregulatory capabilities. What the exact activity of B-1a cells are during the course of malaria has yet to be determined. By use of flow cytometry, it was observed that B-1a cells significantly expand by day 3 postinfection in the spleen and peritoneum of Plasmodium chabaudi chabaudi semiresistant BALB/cJ mice, but not until day 8 postinfection in the spleen of P. chabaudi chabaudi fully susceptible BALB/cByJ mice. The activation of B-1a cells was also demonstrated by the measurement of natural autoantibody IgM production from the serum and cultured peritoneal B-1a cells. Infected semiresistant BALB/cJ mice generated higher levels of anti-ssDNA IgM antibodies than infected fully susceptible BALB/cByJ mice. The preliminary data presented here suggest a possible roll of B-1 cells in contributing to the successful survival of murine malarial infection.     

Ten families of variant genes encoded in subtelomeric regions of multiple chromosomes of Plasmodium chabaudi,a malaria species that undergoes antigenic variation in the laboratory mouse

The chromosome ends of human malaria parasites harbour many genes encoding proteins that are exported to the surface of infected red cells, often being involved in host-parasite interactions and immune evasion. Unlike other murine malaria parasites Plasmodium chabaudi undergoes antigenic variation during passage in the laboratory mouse and hence is a model suitable for investigation of switching mechanisms. However, little is known about the subtelomeric regions of P. chabaudi chromosomes and its variable antigens. Here we report 80 kb of sequence from an end of one P. chabaudi chromosome. Hybridization of probes spanning this region to two dimensional pulsed field gels of the genome revealed 10 multicopy gene families located exclusively in subtelomeric regions of multiple P. chabaudi chromosomes, interspersed amongst multicopy intergenic regions. Hence all chromosomes share a common subtelomeric structure, presumably playing a similar role in spatial positioning as the P. falciparum Rep20 sequence. Expression in blood stages, domains characteristic of surface antigens and copy numbers between four and several hundred per genome, indicate a functional role in antigenic variation for some of these families. We identify members of the cir family, as well as novel genes, that although clearly homologous to cir have large low complexity regions in the predicted extracellular domains. Although all families have homologues in other rodent Plasmodium species, four were previously not known to be subtelomeric. Six have homologues in human and simian malarias.     

Genetic control of resistance to murine malaria

Strain variation in the level of resistance to malaria was investigated in inbred mice after infection with Plasmodium chabaudi. Following intraperitoneal infection with the typing dose of parasitized erythrocytes, mice of 11 inbred strains could be separated using survival time as the criterium into resistant and susceptible groups. Genetic analysis of F₁ hybrid and backcross progeny derived from one of the most resistant (B10.A) and from the most susceptible (A/J) strains as parents suggested that host resistance in this strain combination was genetically controlled by a dominant, non-H-2-linked, autosomal gene or closely linked genes. Analysis of the mechanisms of resistance to P chabaudi showed (1) phenotypic expression of the resistance gene was apparent within 6 days of infection as a significant difference between resistant and susceptible mice in the level of parasitemia; (2) the level of host NK cell activity was not related to the level of host resistance to malaria; (3) compared with susceptible A/J mice, resistant B1O.A hosts had an augmented erythropoietic response during the course of malaria as well as during phenylhydrazine-induced anemia and (4) treatment with BCG or P acnes resulted in an equal degree of protection, measured by parasitemia and survival, in both resistant and susceptible mice.     

Testosterone responsiveness of spleen and liver in female lymphotoxin beta receptor-deficient mice resistant to blood-stage malaria

Disrupted signaling through lymphotoxin beta receptor (LTbetaR) results in severe defects of the spleen and even loss of all other secondary lymphoid tissues, making mice susceptible to diverse infectious agents. Surprisingly, however, we find that female LTbetaR-deficient mice are even more resistant to blood stages of Plasmodium chabaudi malaria than wild-type C57BL/6 mice. Higher resistance of LTbetaR-deficient mice correlates with an earlier onset of reticulocytosis, and the period of anemia is shorter. After surviving fulminant parasitemias of about 35%, mice develop long-lasting protective immunity against homologous rechallenge, with both spleen and liver acting as anti-malaria effectors. Testosterone suppresses resistance, i.e. all mice succumb to infections during or shortly after peak parasitemia. At peak parasitemia, testosterone does not essentially affect cellularity and apoptosis in the spleen, but aggravates liver pathology in terms of increased cell swelling, numbers of apoptotic and binucleated cells and reduced serum alkaline phosphatase levels, and conversely, reduces inflammatory lymphocytic infiltrates in the liver. In the spleen, hybridization of cDNA arrays identified only a few testosterone-induced changes in gene expression, in particular upregulation of INFgamma and IFN-regulated genes. By contrast, a much larger number of testosterone-affectable genes was observed in the liver, including genes involved in regulation of the extracellular matrix, in chemokine and cytokine signaling, and in cell cycle control. Collectively, our data suggest that testosterone dysregulates the inflammatory response in spleen and liver during their differentiation to anti-malaria effectors in malaria-resistant female LTbetaR-deficient mice, thus contributing to the testosterone-induced lethal outcome of malaria.     

Differential T cell responses to Plasmodium chabaudi chabaudi in peripheral blood and spleens of C57BL/6 mice during infection

The definition of the immune status of a person is often taken as the responses obtained from lymphocytes isolated from peripheral blood. We therefore analyzed in a mouse model of Plasmodium chabaudi chabaudi the response of T lymphocytes taken from peripheral blood and compared it with the spleen during and after a primary erythrocytic infection. Using limiting dilution conditions, no malaria-specific T cell responses could be measured in the peripheral blood for up to 21 days after infection with P. chabaudi, whereas T cells responding to malaria Ag were readily detected in the spleen. This was true for T cells providing help and for those producing IFN-gamma. After clearance of the parasitemias to subpatent levels (75 days), qualitatively similar T cell responses were found in both compartments of the immune system, i.e., the Th cell response predominated over the inflammatory response. These data suggest that during an active infection with Plasmodium, T cell responses in peripheral blood are not necessarily indicators of the immune status.     

Haptoglobin and malaria

Haptoglobin gene knockout mice and wild-type controls were infected with Plasmodium berghei ANKA or Plasmodium chabaudi. The peak parasitaemia and parasite burden were higher in Hp-/- mice than in Hp+/+ mice. The increase in spleen weight following malaria infection was smaller in Hp-/- mice than in Hp+/+ animals. The occurrence of cerebral malaria in P. berghei ANKA infection was not different in Hp gene knockout mice and their controls.     

Lymphocyte expression in transgenic trout by mouse immunoglobulin promoter/enhancer

Two groups of transgenic rainbow trout (Oncorhynchus mykiss, Walbaum) have been produced and compared. One group harbored the reporter gene of chloramphenicol acetyltransferase (CAT) associated with mouse immunoglobulin (Ig) promoter/enhancer (pUCL-CAT-E). The other group carried the same reporter gene under the control of the cytomegalovirus promoter/enhancer (pCMV-CAT). Slot blot analysis of DNA from blood cells and other tissues from pUCL-CAT-E fish showed variation of copy number between the major tissues but not between red and white blood cells. Southern blot analysis indicated that multiple copies organized in concatemers were incorporated into the genome. The pCMV-CAT fish had a pronounced expression of CAT in both white and red blood cells. In contrast, activity of CAT was found in the white blood cells of all pUCL-CAT-E fish but not in their red blood cells. Expression in white blood cells was found preferentially in sIg⁺ cells, indicating that B cells are the major expressors. High expression was also found in spleen and kidney, but the activity found in thymocytes was equal to the background level. Analysis of some major tissues showed high white blood cell expression associated with low tissue expression, except that liver (known to contain lymphoid tissue in fish) was higher. Thus the regulatory elements of the Ig gene from mouse induce a tissue-specific expression in fish.     

Plasmodium chabaudi: rosetting in a rodent malaria model

Rosetting is a property of many malaria parasite species that has been linked to virulence in the major species infecting humans, Plasmodium falciparum. Here, the basic properties of rosettes in the rodent malaria laboratory model, P. chabaudi, were studied with a view to future studies on the role of rosetting in malaria parasite virulence and transmission. Rosetting occurred in 14 out of the 15 P. chabaudi clones studied, varied consistently between clones, and ranged between 9 and 37% at full parasite maturity. Rosetting frequency markedly declined after the mouse reached peak parasitemia, possibly due to host immunity. Consistent with P. falciparum and P. vivax, rosettes in P. chabaudi were disrupted by treatment with trypsin and EDTA. However, P. chabaudi rosettes were insensitive to sulfated glycoconjugates (heparin, heparan sulfate and fucoidan). The molecular basis of rosetting in P. chabaudi is unknown at present, but the results suggest that the molecules involved may differ from those in human-infecting species.     

Defects in tumor cell immunogenicity: lymphokine signals in in vitro cytolytic responses to tumor alloantigens

The B16 melanoma of C57BL/6 mice illustrates a deficiency in immunostimulation which may be important in some host-tumor relationships. B16 immunizes very poorly, even against its own major histocompatibility complex (MHC) antigens. We have compared the anti-MHC cytolytic response induced in vitro by B16 and by other tumors of both lymphoid and nonlymphoid origin. We have also studied the role of indomethacin and exogenous lymphokines in facilitating these responses and examined the relationship of specific and nonspecific effector cells induced. In contrast to normal lymphoid cells and two lymphoid tumor cells (EL4 and WEHI-265), the three nonlymphoid tumors, B16, Lewis lung tumor (3LL), and MC-2 fibrosarcoma, failed to induce primary cytolytic responses by themselves. MC-2 and B16 represented two different defects in immunogenicity. MC-2, which we have shown previously to induce an in vivo cytolytic response, could also immunize in vitro provided that prostaglandin production was blocked with indomethacin. In contrast B16, which is poorly immunogenic in vivo, immunized in vitro only if a concanavalin A-induced lymphokine supernatant (CS) was added as an exogenous source of "signal 2." High concentrations of the interleukin 2-containing Con A-induced spleen cell culture supernatant-induced non-H-2b-specific lymphokine-activated killer (LAK) cells in the absence of B16 stimulator cells. However, lymphokine concentrations too low to induce LAK cells enabled the otherwise nonimmunogenic B16 cells to induce specific cytolytic activity.     

Rat T lymphocyte-specific antigens and their cross-reactivity with mouse T cells.

Anti-rat T lymphocyte serum (ATLS)2 was prepared by immunizing rabbits with purified T cells from rat mesenteric nodes and absorbed with rat red cells and syngeneic sarcoma cells. The specificity of ATLS for rat T cells was confirmed by the following reasons: a) ATLS was not toxic for bone marrow cells but lysed most of the thymocytes and a number of spleen and lymph node cells, which were inversely correlated to the percentage of cells with B cell characteristics in respective organs; b) anatomical localization of ATLS-reactive cells in lymphoid organs coincided to the thymus-dependent areas, i.e. the paracortex of lymph node and the periarteriolar region of spleen; c) spleen cells treated with ATLS and complement failed to respond to phytohemagglutinin but normally responded to bacterial lipopolysaccharide; d) those cells treated with ATLS and complement could not induce a graft-vs-host reaction in F1 hosts, whereas the same treatment did not affect direct plaque-forming cells. All of these data confirm the specificity of ATLS and indicate that ATLS recognizes rat T lymphocyte-specific antigens (RTLA). Absorption studies showed that RTLA were present in higher concentration on medullary thymocytes and peripheral T cells than on cortical thymocytes, but absent from bone marrow, liver, and brain tissues. When the cross-reactivity of RTLA with mouse T cells was studied by C-dependent cytotoxicity and immunofluorescence, it was found that mouse T cells shared at least one determinant of RTLA with rat T cells, and that distribution pattern of the cross-reacting antigens in mouse lymphoid tissues was essentially the same as that of RTLA in rat lymphoid organs.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.     

CD4+CD25+Foxp3hi细胞在夏氏疟原虫感染DBA/2小鼠作用研究

利用调节性T细胞消除的致死型夏氏疟原虫(Plasmodium chabaudi chabaudi AS,P.c chabaudi AS)感染鼠疟模型,探讨DBA/2小鼠对P.c chabaudi AS感染易感性的原因。DBA/2小鼠对P.c chabaudi AS易感,伴随原虫血症增加CD4+CD25+Foxp3+细胞数量明显增加,且以CD4+CD25+Foxp3hi增加更为明显。原虫血症达峰值时CD4+CD25+Foxp3hi细胞数量亦达到峰值。相比,Treg消除鼠的原虫出现时间和疟血症峰值时间均明显延迟,且在疟血症达峰值前(5～8 d)原虫血症水平明显低于对照组。与之相应,CD4+CD25+Foxp3hi细胞数量明显处于低水平。同时,Treg消除鼠生存期明显延长。由此提示,P.c chabaudi AS感染导致Foxp3表达增加,扩增的CD4+CD25+Foxp3hi细胞有利于疟原虫复制和逃避宿主免疫应答,进而影响疟疾感染的进程和最终结局。     

致死型夏氏疟原虫感染BALB/c小鼠CD4~＋ CD25~＋ Foxp3~＋调节性T细胞在Th2免疫应答极化中的作用研究

为探讨CD4＋ CD25＋ Foxp3＋调节性T细胞（Treg细胞）在疟疾感染过程中对Th2极化的调控作用,利用Treg细胞消除的致死型夏氏疟原虫（Plasmodium chabaudi chabaudi AS,P.c chabaudi AS）感染鼠疟模型进行研究。结果显示,对照组小鼠在感染后8 d原虫血症达到峰值40.5%,随后迅速下降,于感染后18 d小鼠自愈。相比,Treg细胞消除组于感染后10 d,原虫血症水平迅速上升至32%,随后小鼠相继死亡。在感染后8～10 d,Treg细胞消除小鼠脾脏CD4＋ CD25＋ Foxp3＋细胞占CD4＋细胞百分比含量明显低于对照组。同时,血清疟原虫特异性抗体IgG1和IgG2a水平均明显降低。结果提示,P.c chabaudi AS感染中CD4＋ CD25＋ Foxp3＋细胞参与调控Th2型免疫应答的极化,进而干预疟原虫清除。     

Recombinant viral-vectored vaccines expressing Plasmodium chabaudi AS apical membrane antigen 1: mechanisms of vaccine-induced blood-stage protection

Apical membrane Ag 1 (AMA1) is one of the leading candidate Ags for inclusion in a subunit vaccine against blood-stage malaria. However, the efficacy of Ab-inducing recombinant AMA1 protein vaccines in phase IIa/b clinical trials remains disappointing. In this article, we describe the development of recombinant human adenovirus serotype 5 and modified vaccinia virus Ankara vectors encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost regimen in BALB/c mice, are capable of inducing strong transgene-specific humoral and cellular immune responses. We show that this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect was investigated by in vivo depletion of CD4(+) T cells and adoptive-transfer studies into naive and immunodeficient mice. Depletion of CD4(+) T cells led to a loss of vaccine-induced protection. Adoptive-transfer studies confirmed that efficacy is mediated by both CD4(+) T cells and Abs functioning in the context of an intact immune system. Unlike previous studies, these results confirm that Ag-specific CD4(+) T cells, induced by a clinically relevant vaccine-delivery platform, can make a significant contribution to vaccine blood-stage efficacy in the P. chabaudi model. Given that cell-mediated immunity may also contribute to parasite control in human malaria, these data support the clinical development of viral-vectored vaccines that induce both T cell and Abs against Plasmodium falciparum blood-stage malaria Ags like AMA1.     

Linkage group selection: towards identifying genes controlling strain specific protective immunity in malaria

Protective immunity against blood infections of malaria is partly specific to the genotype, or strain, of the parasites. The target antigens of Strain Specific Protective Immunity are expected, therefore, to be antigenically and genetically distinct in different lines of parasite. Here we describe the use of a genetic approach, Linkage Group Selection, to locate the target(s) of Strain Specific Protective Immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi. In a previous such analysis using the progeny of a genetic cross between P. c. chabaudi lines AS-pyr1 and CB, a location on P. c. chabaudi chromosome 8 containing the gene for merozoite surface protein-1, a known candidate antigen for Strain Specific Protective Immunity, was strongly selected. P. c. chabaudi apical membrane antigen-1, another candidate for Strain Specific Protective Immunity, could not have been evaluated in this cross as AS-pyr1 and CB are identical within the cell surface domain of this protein. Here we use Linkage Group Selection analysis of Strain Specific Protective Immunity in a cross between P. c. chabaudi lines CB-pyr10 and AJ, in which merozoite surface protein-1 and apical membrane antigen-1 are both genetically distinct. In this analysis strain specific immune selection acted strongly on the region of P. c. chabaudi chromosome 8 encoding merozoite surface protein-1 and, less strongly, on the P. c. chabaudi chromosome 9 region encoding apical membrane antigen-1. The evidence from these two independent studies indicates that Strain Specific Protective Immunity in P. c. chabaudi in mice is mainly determined by a narrow region of the P. c. chabaudi genome containing the gene for the P. c. chabaudi merozoite surface protein-1 protein. Other regions, including that containing the gene for P. c. chabaudi apical membrane antigen-1, may be more weakly associated with Strain Specific Protective Immunity in these parasites.     

Rodent malaria parasites Plasmodium chabaudi and P. vinckei do not increase their rates of gametocytogenesis in response to mosquito probing

Several vector-borne infectious agents facultatively alter their life history strategies in response to local vector densities. Some evidence suggests that malaria parasites invest more heavily in transmission stage production (gametocytogenesis) when vectors are present. Such a strategy could rapidly increase malaria transmission rates, particularly when adult mosquitoes begin to appear after dry seasons. However, in contrast to a recent experiment with a rodent malaria (Plasmodium chabaudi), we found no change in gametocytogenesis in either P. chabaudi or in another rodent malaria, P. vinckei, when their mouse hosts were exposed to mosquitoes. Positive results in the earlier study may have been because mosquito-feeding caused anaemia in hosts, a known promoter of gametocytogenesis. The substantial evidence that malaria and a variety of other parasites facultatively alter transmission strategies in response to a variety of environmental influences makes our results surprising.     

Splenic and hepatic hemozoin in mice after malaria parasite clearance.

Hemozoin (malaria pigment) is found in many tissues during malaria infections. In mice that have self-cured from Plasmodium yoelii and Plasmodium chabaudi infections, liver hemozoin concentration and total content decreased for 6-9 mo after parasite clearance. However, both spleen hemozoin concentration and total hemozoin content increased dramatically during this time period. Thus, hemozoin or hemozoin-laden macrophages continue to accumulate in murine spleens for at least several months after malaria parasitemia becomes undetectable.