Imaging of the spleen in malaria

Splenomegaly, albeit variably, is a hallmark of malaria; yet, the role of the spleen in Plasmodium infections remains vastly unknown. The implementation of imaging to study the spleen is rapidly advancing our knowledge of this so-called "blackbox" of the abdominal cavity. Not only has ex vivo imaging revealed the complex functional compartmentalization of the organ and immune effector cells, but it has also allowed the observation of major structural remodeling during infections. In vivo imaging, on the other hand, has allowed quantitative measurements of the dynamic passage of the parasite at spatial and temporal resolution. Here, we review imaging techniques used for studying the malarious spleen, from optical microscopy to in vivo imaging, and discuss the bright perspectives of evolving technologies in our present understanding of the role of this organ in infections caused by Plasmodium.

Depletion of regulatory T cells augments a vaccine-induced T effector cell response against the liver-stage of malaria but fails to increase memory

Regulatory T cells (T(reg)) have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4(+)CD25(+) T(reg) were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3(+)CD25(-) T(reg). To obtain more insights in the specific function of T(reg) during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG). As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP) of Plasmodium berghei (Pb) was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC). Using this system we demonstrate here that the number of CSP-specific T cells increases when T(reg) are depleted during prime but also during boost immunization. Importantly, despite this increase of T effector cells no difference in the number of antigen-specific memory cells was observed.

Resisting infection by Plasmodium berghei increases the sensitivity of the malaria vector Anopheles gambiae to DDT

BACKGROUND The evolution of insecticide resistance threatens current malaria control methods, which rely heavily on chemical insecticides. The magnitude of the threat will be determined by the phenotypic expression of resistance in those mosquitoes that can transmit malaria. These differ from the majority of the mosquito population in two main ways; they carry sporozoites (the infectious stage of the Plasmodium parasite) and they are relatively old, as they need to survive the development period of the malaria parasite. This study examines the effects of infection by Plasmodium berghei and of mosquito age on the sensitivity to DDT in a DDT-resistant strain of Anopheles gambiae. METHODS DDT-resistant Anopheles gambiae (ZANU) mosquitoes received a blood meal from either a mouse infected with Plasmodium berghei or an uninfected mouse. 10 and 19 days post blood meal the mosquitoes were exposed to 2%, 1% or 0% DDT using WHO test kits. 24 hrs after exposure, mortality and Plasmodium infection status of the mosquitoes were recorded. RESULTS Sensitivity to DDT increased with the mosquitoes' age and was higher in mosquitoes that had fed on Plasmodium-infected mice than in those that had not been exposed to the parasite. The latter effect was mainly due to the high sensitivity of mosquitoes that had fed on an infected mouse but were not themselves infected, while the sensitivity to DDT was only slightly higher in mosquitoes infected by Plasmodium than in those that had fed on an uninfected mouse. CONCLUSIONS The observed pattern indicates a cost of parasite-resistance. It suggests that, in addition to the detrimental effect of insecticide-resistance on control, the continued use of insecticides in a population of insecticide-resistant mosquitoes could select mosquitoes to be more susceptible to Plasmodium infection, thus further decreasing the efficacy of the control.

The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.

Der Kampf gegen die Malaria

Max Jungmann

Die Malaria in Jerusalem

Aaron Sandler

Die Bekämpfung der Malaria

L. Sofer

Highly efficient subcloning of rodent malaria parasites by injection of single merosomes or detached cells

This protocol describes a method for obtaining rodent Plasmodium parasite clones with high efficiency, which takes advantage of the normal course of Plasmodium in vitro exoerythrocytic development. At the completion of development, detached cells/merosomes form, which contain hundreds to thousands of merozoites. As all parasites within a single detached cell/merosome derive from the same sporozoite, we predicted them to be genetically identical. To prove this, hepatoma cells were infected simultaneously with a mixture of Plasmodium berghei sporozoites expressing either GFP or mCherry. Subsequently, individual detached cells/merosomes from this mixed population were selected and injected into mice, resulting in clonal blood stage parasite infections. Importantly, as a large majority of mice become successfully infected using this protocol, significantly less mice are necessary than for the widely used technique of limiting dilution cloning. To produce a clonal P. berghei blood stage infection from a non-clonal infection using this procedure requires between 4 and 5 weeks.

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.

Protective efficacy and safety of liver stage attenuated malaria parasites

During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(-) or uis3(-) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(-) parasites protected better than uis3(-) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy.

Eine Vereinfachung der Malaria-Bekämpfung?

Aaron Sandler

Die Bekämpfung der Malaria, unter besonderer Berücksichtigung Palästinas

M. Hecker

Die Behandlung der Malaria mit Atoxyl

M. Georgopulos

Variables affecting the stability of multiple cloning sites and hairpin structures in retroviral vectors

Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate. The viral enzyme reverse transcriptase copies the retroviral genomic RNA into this DNA intermediate through the process of reverse transcription. Many variables can affect the fidelity of reverse transcriptase during reverse transcription, including specific sequences within the retroviral genome. ^ Previous studies have observed that multiple cloning sites (MCS) and sequences predicted to form stable hairpin structures are hotspots for deletion during retroviral replication. The studies described in this dissertation were performed to elucidate the variables that affect the stability of MCS and hairpin structures in retroviral vectors. Two series of retroviral vectors were constructed and characterized in these studies. ^ Spleen necrosis virus-based vectors were constructed containing separate MCS insertions of varying length, orientation, and symmetry. The only MCS that was a hotspot for deletion formed a stable hairpin structure. Upon more detailed study, the MCS previously reported as a hotspot for deletion was found to contain a tandem linker insertion that formed a hairpin structure. Murine leukemia virus-based vectors were constructed containing separate sequence insertions of either inverted repeat symmetry (122IR) that could form a hairpin structure, or little symmetry (122c) that would form a less stable structure. These insertions were made into either the neomycin resistance marker ( neo) or the hygromycin resistance marker (hyg) of the vector. 122c was stable in both neo and hyg, while 122IR was preferentially deleted in neo and was remarkably unstable in hyg. ^ These results suggest that MCS are hotspots for deletion in retroviral vectors if they can form hairpin structures, and that hairpin structures can be highly unstable at certain locations in retroviral vectors. This information may contribute to improved design of retroviral vectors for such uses as human gene therapy, and will contribute to a greater understanding of the basic science of retroviral reverse transcription. ^

Das Malaria-Fieber und sein Erreger

Von A. H. Krausse