Malaria parasites within red blood cells digest host hemoglobin into a hydrophobic heme polymer, known as hemozoin (HZ), which is subsequently released into the blood stream and then captured by and concentrated in the reticulo-endothelial system. Accumulating evidence suggests that HZ is immunologically active, but the molecular mechanism(s) through which HZ modulates the innate immune system has not been elucidated. This work demonstrates that HZ purified from Plasmodium falciparum is a novel non-DNA ligand for Toll-like receptor (TLR)9. HZ activated innate immune responses in vivo and in vitro, resulting in the production of cytokines, chemokines, and up-regulation of costimulatory molecules. Such responses were severely impaired in TLR9−/− and myeloid differentiation factor 88 (MyD88)−/−, but not in TLR2, TLR4, TLR7, or Toll/interleukin 1 receptor domain–containing adaptor-inducing interferon β−/− mice. Synthetic HZ, which is free of the other contaminants, also activated innate immune responses in vivo in a TLR9-dependent manner. Chloroquine (CQ), an antimalarial drug, abrogated HZ-induced cytokine production. These data suggest that TLR9-mediated, MyD88-dependent, and CQ-sensitive innate immune activation by HZ may play an important role in malaria parasite–host interactions.

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CD40-deficient mice are susceptible to Leishmania major infection while their wild-type littermates can resolve the infection. Upon stimulation with L. major antigens, draining lymph node T cells of the mutant mice and the susceptible mice, BALB/c, secrete comparable amounts of IL-4. The mutant mice produce less IFN gamma than wild-type mice. The expression of IL-12 p40 mRNA was significantly lower in L. major antigen-stimulated cells of mutant mice than those of wild-type or BALB/c mice. In normal mice, engagement of CD40 activates macrophages to a leishmanicidal state in vitro in the presence of IFN gamma. The results suggest that the CD40-CD40 ligand interaction plays an important role in two critical steps of cell-mediated immunity to L. major infection: the generation of a Th1 response and activation of macrophages to a leishmanicidal state.

Plasmodium cynomolgi, a malaria parasite of Asian Old World monkeys, is the sister taxon of Plasmodium vivax, the most prevalent human malaria species outside Africa. Since P. cynomolgi shares many phenotypic, biologic and genetic characteristics of P. vivax, we generated draft genome sequences of three P. cynomolgi strains and performed comparative genomic analysis between them and P. vivax, as well as a third previously sequenced simian parasite, Plasmodium knowlesi. Here we show that genomes of the monkey malaria clade can be characterized by CNVs in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites, and CNVs in the P. cynomolgi genome, providing a map of genetic variation for mapping parasite traits and studying parasite populations. The P. cynomolgi genome is a critical step in developing a model system for P. vivax research, and to counteract the neglect of P. vivax.

With the severe acute respiratory syndrome epidemic of 2003 and renewed attention on avian influenza viral pandemics, new surveillance systems are needed for the earlier detection of emerging infectious diseases. We applied a “next-generation” parallel sequencing platform for viral detection in nasopharyngeal and fecal samples collected during seasonal influenza virus (Flu) infections and norovirus outbreaks from 2005 to 2007 in Osaka, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.1–0.25 ml of nasopharyngeal aspirates (N = 3) and fecal specimens (N = 5), and more than 10 µg of cDNA was synthesized. Unbiased high-throughput sequencing of these 8 samples yielded 15,298–32,335 (average 24,738) reads in a single 7.5 h run. In nasopharyngeal samples, although whole genome analysis was not available because the majority (>90%) of reads were host genome–derived, 20–460 Flu-reads were detected, which was sufficient for subtype identification. In fecal samples, bacteria and host cells were removed by centrifugation, resulting in gain of 484–15,260 reads of norovirus sequence (78–98% of the whole genome was covered), except for one specimen that was under-detectable by RT-PCR. These results suggest that our unbiased high-throughput sequencing approach is useful for directly detecting pathogenic viruses without advance genetic information. Although its cost and technological availability make it unlikely that this system will very soon be the diagnostic standard worldwide, this system could be useful for the earlier discovery of novel emerging viruses and bioterrorism, which are difficult to detect with conventional procedures.

Poor health and malnutrition in preschool children are longstanding problems in Bangladesh. Gut microbiota plays a tremendous role in nutrient absorption and determining the state of health. In this study, metagenomic tool was employed to assess the gut microbiota composition of healthy and malnourished children. DNA was extracted from fecal samples of seven healthy and seven malnourished children (n = 14; age 2–3 years) were analyzed for the variable region of 16S rRNA genes by universal primer PCR followed by high-throughput 454 parallel sequencing to identify the bacterial phyla and genera. Our results reveal that the healthy children had a significantly higher number of operational taxonomic unit in their gut than that of the malnourished children (healthy vs. malnourished: 546 vs. 310). In malnourished children, bacterial population of the phyla Proteobacteria and Bacteroidetes accounted for 46 and 18%, respectively. Conversely, in healthy children, Proteobacteria and Bacteroidetes accounted for 5% and 44, respectively (p < 0.001). In malnourished children, the phylum Proteobacteria included pathogenic genera, namely Klebsiella and Escherichia, which were 174-fold and 9-fold higher, respectively, than their healthy counterpart. The predominance of potentially pathogenic Proteobacteria and minimal level of Bacteroidetes as commensal microbiota might be associated to the ill health of malnourished children in Bangladesh.

Malaria is among the most serious infectious diseases affecting humans, accounting for approximately half a million deaths annually1. Plasmodium falciparum is the causative agent of most life-threatening malaria cases. Acquired immunity to malaria is inefficient, even after repeated exposures to P. falciparum2; immune regulatory mechanisms employed by P. falciparum remain largely unclear. Here, we show that P. falciparum uses immune inhibitory receptors for immune evasion. RIFINs, products of a polymorphic multigene family comprising approximately 150–200 genes per parasite genome3, are expressed on the surface of infected erythrocytes. We found that a subset of RIFINs binds to either leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associated immunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs inhibited activation of LILRB1-expressing B cells and NK cells. Furthermore, interactions between LILRB1 and P. falciparum-infected erythrocytes isolated from malaria patients were associated with severe malaria, although an extended study with larger sample sizes is required to confirm the findings. These results suggest that P. falciparum has acquired multiple RIFINs to evade the host immune system by targeting immune inhibitory receptors.

Although whole-parasite vaccine strategies for malaria infection have regained attention, their immunological mechanisms of action remain unclear. We find that immunization of mice with a crude blood stage extract of the malaria parasite Plasmodium falciparum elicits parasite antigen-specific immune responses via Toll-like receptor (TLR) 9 and that the malarial heme-detoxification byproduct, hemozoin (HZ), but not malarial DNA, produces a potent adjuvant effect. Malarial and synthetic (s)HZ bound TLR9 directly to induce conformational changes in the receptor. The adjuvant effect of sHZ depended on its method of synthesis and particle size. Although natural HZ acts as a TLR9 ligand, the adjuvant effects of synthetic HZ are independent of TLR9 or the NLRP3-inflammasome but are dependent on MyD88. The adjuvant function of sHZ was further validated in a canine antiallergen vaccine model. Thus, HZ can influence adaptive immune responses to malaria infection and may have therapeutic value in vaccine adjuvant development.