Mary Ann McDowell

Uninfected Mosquito Bites Confer Protection against Infection with Malaria Parasites

ABSTRACT Despite decades of research and multiple initiatives, malaria continues to be one of the worlds most debilitating infectious diseases. New insights for malaria control and vaccine development will be essential to thwart the staggering worldwide impact of this disease (A. Bjorkman and A. Bhattarai, Acta Trop. 94:163-169, 2005); ultimately successful vaccine strategies will undoubtedly be multifactorial, incorporating multiple antigens and targeting diverse aspects of the malaria parasites’ biology (M. F. Good et al., Immunol. Rev. 201:254-267, 2004). Using a murine model of malaria infection, we show here that exposure to bites from uninfected mosquitoes prior to Plasmodium yoelii infection influences the local and systemic immune responses and limits parasite development within the host. In hosts preexposed to bites from uninfected mosquitoes, reduced parasite burdens in the livers were detected early, and during the blood-stage of the life cycle, these burdens remained lower than those in hosts that received mosquito bites only at the time of infection. Repeated exposure to bites from uninfected mosquitoes skewed the immune response towards a T-helper 1 (Th1) phenotype as indicated by increased levels of interleukin-12, gamma interferon, and inducible nitric oxide synthase. These data suggest that the addition of mosquito salivary components to antimalaria vaccines may be a viable strategy for creating a Th1-biased environment known to be effective against malaria infection. Furthermore, this strategy may be important for the development of vaccines to combat other mosquito-transmitted pathogens.

The role of mannose receptor during experimental leishmaniasis

The primary host cells for Leishmania replication are macrophages (MP). Several molecules on the surface of professional phagocytic cells have been implicated in the initial process of parasite internalization and initiation of signaling pathways. These pattern recognition receptors distinguish molecular patterns on pathogen surfaces. Mannose receptor (MR), specifically, recognizes mannose residues on the surface of Leishmania parasites. We studied the role of MR in the pathogenesis of experimental cutaneous and visceral leishmaniasis using MR‐deficient [MR‐knockout (KO)] C57BL/6 mice. MR‐deficient MP exhibied a comparable infection rate and cytokine production. In the absence of MR, the clinical course of Leishmania major and Leishmania donovani infections was similar in MR‐KO and wild‐type mice (MR‐WT). Furthermore, immunohistochemistry of cutaneous lesions from MR‐KO and MR‐WT mice revealed no differences in lesion architecture or cell components. Inhibition of MP responses is a hallmark of Leishmania infection; our data demonstrate further that host MR is not essential for blocking IFN‐γ/LPS‐induced IL‐12 production and MAPK activation by Leishmania. Thus, we conclude that the MR is not essential for host defense against Leishmania infection or regulation of IL‐12 production.

Recent advances in phlebotomine sand fly research related to leishmaniasis control

Phlebotomine sand flies are the subject of much research because of the role of their females as the only proven natural vectors of Leishmania species, the parasitic protozoans that are the causative agents of the neglected tropical disease leishmaniasis. Activity in this field was highlighted by the eighth International Symposium on Phlebotomine Sand flies (ISOPS) held in September 2014, which prompted this review focusing on vector control. Topics reviewed include: Taxonomy and phylogenetics, Vector competence, Genetics, genomics and transcriptomics, Eco-epidemiology, and Vector control. Research on sand flies as leishmaniasis vectors has revealed a diverse array of zoonotic and anthroponotic transmission cycles, mostly in subtropical and tropical regions of Africa, Asia and Latin America, but also in Mediterranean Europe. The challenge is to progress beyond descriptive eco-epidemiology, in order to separate vectors of biomedical importance from the sand fly species that are competent vectors but lack the vectorial capacity to cause much human disease. Transmission modelling is required to identify the vectors that are a public health priority, the ones that must be controlled as part of the integrated control of leishmaniasis. Effective modelling of transmission will require the use of entomological indices more precise than those usually reported in the leishmaniasis literature.

Leishmania major Infection Activates NF-κB and Interferon Regulatory Factors 1 and 8 in Human Dendritic Cells

ABSTRACT The salient feature of dendritic cells (DC) is the initiation of appropriate adaptive immune responses by discriminating between pathogens. Using a prototypic model of intracellular infection, we previously showed that Leishmania major parasites prime human DC for efficient interleukin-12 (IL-12) secretion. L. major infection is associated with self-limiting cutaneous disease and powerful immunity. In stark contrast, the causative agent of visceral leishmaniasis, Leishmania donovani, does not prime human DC for IL-12 production. Here, we report that DC priming by L. major infection results in the early activation of NF-κB transcription factors and the up-regulation and nuclear translocation of interferon regulatory factor 1 (IRF-1) and IRF-8. The inhibition of NF-κB activation by the pretreatment of DC with caffeic acid phenethyl ester blocks L. major-induced IRF-1 and IRF-8 activation and IL-12 expression. We further demonstrate that IRF-1 and IRF-8 obtained from L. major-infected human DC specifically bind to their consensus binding sites on the IL-12p35 promoter, indicating that L. major infection either directly stimulates a signaling cascade or induces an autocrine pathway that activates IRF-1 and IRF-8, ultimately resulting in IL-12 transcription.

Leishmania infection inhibits cycloheximide-induced macrophage apoptosis in a strain-dependent manner

Activation of apoptosis is one of the most ancient mechanisms to eliminate intracellular infections; the capacity to subvert this programed cell death provides an adaptive advantage to pathogens that persist in an intracellular environment. Leishmania species are obligate intracellular parasites that primarily reside within host macrophages. We demonstrate here that Leishmania infection protects macrophages from cycloheximide-induced apoptosis in a species and strain specific manner. Our data further reveal that Leishmania phosphoglycans and direct contact between parasites and host cells are required for the inhibitory phenotype.

Inhibition of groups 1 and 2 CD1 molecules on human dendritic cells by Leishmania species

Dendritic cells are potent immune‐activating cells, which traditionally are thought of as presenters of protein antigen to lymphocytes to initiate an immune response. Recently, another mechanism of immune surveillance, the detection of lipid antigens, has been found to be mediated by the nonpolymorphic family of CD1 molecules. There are two different CD1 families, Group 1 consisting of CD1a, CD1b and CD1c, and Group 2 consisting only of CD1d. Leishmania donovani‐infected dendritic cells have previously been demonstrated to exhibit decreased surface levels of Group 1 CD1 molecules and are no longer able to initiate a CD1b‐restricted T cell response. In contrast to L. donovani, which disseminates to the visceral organs, L. major remains localized, forming a cutaneous lesion. We investigate here whether L. major, the aetiological agent of cutaneous leishmaniasis, also inhibits CD1 expression. We demonstrate that infection of human monocyte‐derived dendritic cells with either L. major or L. donovani results in transcriptional down‐regulation of both Groups 1 and 2 CD1 molecules. Furthermore, infection of monocytes during differentiation results in a cell phenotype similar to undifferentiated monocytes. Finally, we demonstrate that this down‐regulation is not mediated by lipophosphoglycan or other phosphoglycans.

Leishmaniasis: Middle East and North Africa research and development priorities.

The US-MENA Leishmaniasis conference provided an unprecedented opportunity for scientists to interact and focus on leishmaniasis research issues specific to the MENA region and resulted in several successful grant proposals funded through the Civilian Research and Development Foundation. The collaborations that arose from this conference will certainly influence leishmaniasis control in the MENA region specifically; moreover, the discussions and recommendations also have implications for the leishmaniasis field as a whole.

Characterization of the Anopheles gambiae octopamine receptor and discovery of potential agonists and antagonists using a combined computational-experimental approach

BackgroundOctopamine receptors (OARs) perform key functions in the biological pathways of primarily invertebrates, making this class of G-protein coupled receptors (GPCRs) a potentially good target for insecticides. However, the lack of structural and experimental data for this insect-essential GPCR family has promoted the development of homology models that are good representations of their biological equivalents for in silico screening of small molecules.MethodsTwo Anopheles gambiae OARs were cloned, analysed and functionally characterized using a heterologous cell reporter system. Four antagonist- and four agonist-binding homology models were generated and virtually screened by docking against compounds obtained from the ZINC database. Resulting compounds from the virtual screen were tested experimentally using an in vitro reporter assay and in a mosquito larvicide bioassay.ResultsSix An. gambiae OAR/tyramine receptor genes were identified. Phylogenetic analysis revealed that the OAR (AGAP000045) that encodes two open reading frames is an α-adrenergic-like receptor. Both splice variants signal through cAMP and calcium. Mutagenesis analysis revealed that D100 in the TM3 region and S206 and S210 in the TM5 region are important to the activation of the GPCR. Some 2,150 compounds from the virtual screen were structurally analysed and 70 compounds were experimentally tested against AgOAR45B expressed in the GloResponse™CRE-luc2P HEK293 reporter cell line, revealing 21 antagonists, 17 weak antagonists, 2 agonists, and 5 weak agonists.ConclusionReported here is the functional characterization of two An. gambiae OARs and the discovery of new OAR agonists and antagonists based on virtual screening and molecular dynamics simulations. Four compounds were identified that had activity in a mosquito larva bioassay, three of which are imidazole derivatives. This combined computational and experimental approach is appropriate for the discovery of new and effective insecticides.

Characterization of microRNA expression profiles in Leishmania-infected human phagocytes

Leishmania are intracellular protozoa that influence host immune responses eliciting parasite species‐specific pathologies. MicroRNAs (miRNAs) are short single‐stranded ribonucleic acids that complement gene transcripts to block protein translation and have been shown to regulate immune system molecular mechanisms. Human monocyte‐derived dendritic cells (DC) and macrophages (MP) were infected in vitro with Leishmania major or Leishmania donovani parasites. Small RNAs were isolated from total RNA and sequenced to identify mature miRNAs associated with leishmanial infections. Normalized sequence read count profiles revealed a global downregulation in miRNA expression among host cells following infection. Most identified miRNAs were expressed at higher levels in L. donovani‐infected cells relative to L. major‐infected cells. Pathway enrichments using in silico‐predicted gene targets of differentially expressed miRNAs showed evidence of potentially universal MAP kinase signalling pathway effects. Whereas JAK‐STAT and TGF‐β signalling pathways were more highly enriched using targets of miRNAs upregulated in L. donovani‐infected cells, these data provide evidence in support of a selective influence on host cell miRNA expression and regulation in response to differential Leishmania infections.

Elevated non-specific immunity and normal Listeria clearance in young and old vitamin D receptor knockout mice

1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and the vitamin D receptor (VDR) are important regulators of autoimmunity. The effect of the VDR on the ability of mice to fight a primary or secondary infection has not been determined. Young and old VDR knockout (KO) mice were able to clear both primary and secondary infections with Listeria monocytogenes. However, the kinetics of clearance was somewhat delayed in the absence of the VDR. Memory T cell development was not different in young VDR KO and wild-type (WT) mice; however, old VDR KO mice had significantly less memory T cells than their WT counterparts but still mounted an adequate immune response as determined by the complete clearance of L. monocytogenes. Although the primary and secondary immune responses were largely intact in the VDR KO mice, the old VDR KO mice had increased cytokines and antibody responses compared with the old WT mice. In particular, old VDR KO mice had elevated antigen non-specific antibodies; however, these magnified immune responses did not correspond to more effective Listeria clearance. The increased antibody and cytokine responses in the old VDR KO mice are consistent with the increased susceptibility of these mice to autoimmunity.