Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

BACKGROUND: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored. METHODS: Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes. RESULTS: Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge. CONCLUSIONS: DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

Animal models of efficacy to accelerate drug discovery in malaria.

The emergence of resistance to artemisinins and the renewed efforts to eradicate malaria demand the urgent development of new drugs. In this endeavour, the evaluation of efficacy in animal models is often a go/no go decision assay in drug discovery. This important role relies on the capability of animal models to assess the disposition, toxicology and efficacy of drugs in a single test. Although the relative merits of each efficacy model of malaria as human surrogate have been extensively discussed, there are no critical analyses on the use of such models in current drug discovery. In this article, we intend to analyse how efficacy models are used to discover new antimalarial drugs. Our analysis indicates that testing drug efficacy is often the last assay in each discovery stage and the experimental designs utilized are not optimized to expedite decision-making and inform clinical development. In light of this analysis, we propose new ways to accelerate drug discovery using efficacy models.

Eosinophilia in transgenic mice expressing interleukin 5.

Experiments in vitro suggest that although interleukin 5 (IL-5) stimulates the late stages of eosinophil differentiation, other cytokines are required for the generation of eosinophil progenitor cells. In this study transgenic mice constitutively expressing the IL-5 gene were established using a genomic fragment of the IL-5 gene coupled to the dominant control region from the gene encoding human CD2. Four independent eosinophilic transgenic lines have thus far been established, two of which with 8 and 49 transgene copies, are described in detail. These mice appeared macroscopically normal apart from splenomegaly. Eosinophils were at least 65- and 265-fold higher in blood from transgenics, relative to normal littermates, and approximately two- or sevenfold more numerous relative to blood from mice infected with the helminth Mesocestoides corti. Much more modest increases in blood neutrophil, lymphocyte, and monocyte numbers were noted in transgenics, relative to normal littermates (less than threefold). Thus IL-5 in vivo is relatively specific for the eosinophil lineage. Large numbers of eosinophils were present in spleen, bone marrow, and peritoneal exudate, and were highest in the line with the greatest transgene copy number. Eosinophilia was also noted in histological sections of transgenic lungs, Peyer's patches, mesenteric lymph nodes, and gut lamina propria but not in other tissues examined. IL-5 was detected in the sera of transgenics at levels comparable to those seen in sera from parasite-infected animals. IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were not found. IL-5 mRNA was detected in transgenic thymus, Peyer's patches, and superficial lymph nodes, but not in heart, liver, brain, or skeletal muscle or in any tissues from nontransgenics. Bone marrow from transgenic mice was rich in IL-5-dependent eosinophil precursors. These data indicate that induction of the IL-5 gene is sufficient for production of eosinophilia, and that IL-5 can induce the full pathway of eosinophil differentiation. IL-5 may therefore not be restricted in action to the later stages of eosinophil differentiation, as suggested by earlier in vitro studies.

Infection of IL5 transgenic mice with Mesocestoides corti induces very high levels of IL5 but depressed production of eosinophils.

Transgenic mice expressing interleukin 5 (IL5) have been demonstrated to show a lifelong high level eosinophilia. These mice were produced using a construct in which the dominant control region (DCR) of the human CD2 gene was ligated to a 10-kb fragment containing the mouse IL5 gene. The construct allows the expression of the IL5 gene under the control of its own promoter, but the DCR ensures constitutive expression by all T cells. Infection of these transgenic mice with Mesocestoides corti, which is itself a potent inducer of eosinophilia, increases serum IL5 to very high levels. This demonstrates that the transgenes retain inducibility, which is a feature of the endogenous gene. However, despite the high levels of IL5, the numbers of eosinophils in the blood, marrow, and spleen decrease during the period 1-4 weeks after infection. Furthermore, there is a decrease in eosinophil precursors, as assessed by the capacity of bone marrow to produce eosinophils in culture. After this decrease eosinophils return to their previous high levels, although the levels of IL5 remain high. These results suggest that a control mechanism is operating to limit the numbers of eosinophils produced. This control appears to act at the level of the precursor production and may not be directly related to the high levels of IL5.

Generation of Transgenic Rodent Malaria Parasites Expressing Human Malaria Parasite Proteins.

We describe methods for the rapid generation of transgenic rodent Plasmodium berghei (Pb) parasites that express human malaria parasite (HMP) proteins, using the recently developed GIMO-based transfection methodology. Three different genetic modifications are described resulting in three types of transgenic parasites. (1) Additional Gene (AG) mutants. In these mutants the HMP gene is introduced as an "additional gene" into a silent/neutral locus of the Pb genome under the control of either a constitutive or stage-specific Pb promoter. This method uses the GIMO-transfection protocol and AG mutants are generated by replacing the positive-negative selection marker (SM) hdhfr::yfcu cassette in a neutral locus of a standard GIMO mother line with the HMP gene expression cassette, resulting in SM free transgenic parasites. (2) Double-step Replacement (DsR) mutants. In these mutants the coding sequence (CDS) of the Pb gene is replaced with the CDS of the HMP ortholog in a two-step GIMO-transfection procedure. This process involves first the replacement of the Pb CDS with the hdhfr::yfcu SM, followed by insertion of the HMP ortholog at the same locus thereby replacing hdhfr::yfcu with the HMP CDS. These steps use the GIMO-transfection protocol, which exploits both positive selection for Pb orthologous gene-deletion and negative selection for HMP gene-insertion, resulting in SM free transgenic parasites. (3) Double-step Insertion (DsI) mutants. When a Pb gene is essential for blood stage development the DsR strategy is not possible. In these mutants the HMP expression cassette is first introduced into the neutral locus in a standard GIMO mother line as described for AG mutants but under the control elements of the Pb orthologous gene; subsequently, the Pb ortholog CDS is targeted for deletion through replacement of the Pb CDS with the hdhfr::yfcu SM, resulting in transgenic parasites with a new GIMO locus permissive for additional gene-insertion modifications.The different types of transgenic parasites can be exploited to examine interactions of drugs/inhibitors or immune factors with HMP molecules in vivo. Mice either immunized with HMP-vaccines or treated with specific drugs can be infected/challenged with these transgenic mutants to evaluate drug or vaccine efficacy in vivo.

[Regulation of the immune response to intestinal nematode infections in mice]. / Regulacja odpowiedzi immunologicznej na inwazje nicieni jelitowych u myszy.

Control of parasitic infections is dependent on mechanisms that limit invasion, reproduction or survival of the parasite, including elevated serum IgE, eosinophilia and intestinal mast cell hyperplasia. Studies with mice infected with Heligmosomoides polygyrus, Trichuris muris, Nippostrongylus brasiliensis and Trichinella spiralis have provided considerable information about immune mechanisms correlated with resistance and susceptibility. Activation and cytokine secretion of distinct Th cell subset leads to the generation of effective or ineffective responses resulting in clearance of the parasite load or maintenance of chronic infection. The induction of differential responses remains to be determined but is likely to be influenced at a number of levels including the host genetic background, involvement of accessory cells, activation of co-stimulatory molecules on antigen presenting cells. The regulation of responses to intestinal nematode infections is discussed.

Malaria in beta-thalassemic mice and the effects of the transgenic human beta-globin gene and splenectomy.

To investigate the protective effects of beta-thalassemia against malaria, rodent malaria parasites were studied in C57BL/6J mice with beta-thalassemia, in mice in which the thalassemia had been transgenically corrected with the human beta A-globin gene, and in hematologically normal mice. In thalassemic mice, Plasmodium chabaudi adami infection was inhibited and peak parasitemia was variably delayed. In transgenically corrected mice, infection proceeded as in normal mice. Plasmodium berghei infection proceeded more rapidly in thalassemic mice, but survival was not different. Splenectomized normal mice displayed high-level parasitemia that peaked twice and persisted as a low-level parasitemia for more than 20 days after normal intact mice were free of all parasites. Splenectomized thalassemic mice showed a delay of 5 days in attaining peak parasitemia, but the parasitemia persisted as in normal splenectomized mice. Thus, for P. chabaudi, which displayed no preference for immature erythrocytes, beta-thalassemia offers enhanced resistance for the host. However, for P. berghei, which preferentially invades reticulocytes, thalassemia is not protective. The protective effects of the normal mouse spleen were observed, but the paradoxical facilitation of parasite growth by the thalassemic spleen is a new finding that will require further experimentation to explain. This new in vivo laboratory documentation of thalassemic protection against some rodent malaria parasites may serve as a useful model in further efforts to control this major infectious disease.

Interleukin-4 transgenic mice of resistant background are susceptible to Leishmania major infection.

The outcome of cutaneous leishmaniasis is dependent on the balance of Th1 and Th2 cells. In the murine model, Th1 cells are host-protective whereas the Th2 cells are disease-promoting. However, the in vivo role of interleukin-4 (IL-4), a signature product of Th2 cells, is uncertain. We compared the course of Leishmania major infection in the genetically resistant 129/Sv mice and the mutant 129/Sv mice transgenic for the murine IL-4 gene under the control of the immunoglobulin heavy chain enhancer and promoter. We report here that in contrast to their wild-type parents, the IL-4 transgenic mice are susceptible to L. major infection. This is associated with the development of inexorably progressive lesions and parasite loads. Spleen cells from infected transgenic mice produced significantly higher levels of IL-4 but lower amounts of interferon-gamma when stimulated in vitro with leishmanial antigens compared to those from infected normal 129/Sv mice. Furthermore, sera from the infected transgenic mice contained higher levels of IL-4 and IgE than the sera of infected normal 129/Sv mice. These results, therefore, establish in a new animal model that IL-4 promotes disease development in murine cutaneous leishmaniasis.

Th2 immune responses in GATA-3-transgenic mice infected with Heligmosomoides polygyrus.

To examine the participation of transcription factor GATA-3 in Th2 immune responses in vivo, we generated transgenic mice having several copies of GATA-3 with LCK promotor. Mice were infected with the intestinal nematode Heligmosomoides polygyrus to induce Th2 immune responses. Upon antigen stimulation, IL-5 and IL-13 production of mesenteric lymph node cells from H. polygyrus-infected mice, was significantly enhanced in GATA-3-transgenic mice compared with nontransgenic control mice. However, IL-4 production was the same in GATA-3-transgenic and control mice. H. polygyrus-infected GATA-3-transgenic mice exhibited significantly more peripheral blood eosinophils and total IgE levels compared with control mice. These results suggest that GATA-3 promotes IL-5 and IL-13 production, and that the function of these cytokines results in eosinophilia and hyper-IgE, respectively.