Robert M. Anthony

A proteomic view of the Plasmodium falciparum life cycle.

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.

Memory TH2 cells induce alternatively activated macrophages to mediate protection against nematode parasites

Although primary and memory responses against bacteria and viruses have been studied extensively, T helper type 2 (TH2) effector mechanisms leading to host protection against helminthic parasites remain elusive. Examination of the intestinal epithelial submucosa of mice after primary and secondary infections by a natural gastrointestinal parasite revealed a distinct immune-cell infiltrate after challenge, featuring interleukin-4–expressing memory CD4+ T cells that induced IL-4 receptorhi (IL-4Rhi) CD206+ alternatively activated macrophages. In turn, these alternatively activated macrophages (AAMacs) functioned as important effector cells of the protective memory response contributing to parasite elimination, demonstrating a previously unknown mechanism for host protection against intestinal helminths.

Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy

The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.

Peripheral CD4 T Cells Rapidly Accumulate at the Host:Parasite Interface during an Inflammatory Th2 Memory Response

Memory peripheral Th2 immune responses to infectious pathogens are not well studied due to the lack of suitable models and the difficulty of assessing Th2 cytokine expression at sites of inflammation. We have examined the localized immune response to a nematode parasite that encysts in the small intestine. An unexpected architecture was observed on day 4 of the memory response, with granulocytes and macrophages infiltrating the cyst and CD4+, TCR-αβ+ T cells surrounding the cyst. Laser capture microdissection analysis showed a pronounced CD4-dependent Th2 cytokine pattern at the cyst region only during the memory response, demonstrating that the Th2 memory response is readily distinguished from the primary response by the rapid accumulation of Th2 effector cells at the host:parasite interface.

Regional distribution of methamphetamine in autopsied brain of chronic human methamphetamine users

We measured levels of methamphetamine and those of its metabolite amphetamine in 15 autopsied brain regions of 14 human methamphetamine users. Only slight regional differences were observed in drug concentrations among the brain areas. Although, some redistribution of the drugs probably occurred postmortem, these data suggest that methamphetamine might not be preferentially retained in dopamine-rich brain areas but is heterogenously distributed in brain of chronic human users of the drug. The possible pharmacological actions of methamphetamine in both dopamine-rich and poor brain areas of chronic drug users need to be considered.

Requirements for the development of IL‐4‐producing T cells during intestinal nematode infections: what it takes to make a Th2 cell in vivo

Summary:  Components of the type 2 immune response may mediate host protection against both helminthic parasites and harmful allergic responses. A central player in this response is the T‐helper 2 (Th2) effector cell, which produces interleukin (IL)‐4, IL‐5, IL‐13, and other Th2 cytokines during the primary and memory response. Specific aspects of the parasite that trigger Th2‐cell differentiation are not yet defined. Furthermore, the cell types and cell surface and secreted molecules that provide the immune milieu required for the development of Th2 effector cells and also Th2 memory cells are not well understood. They will probably vary with the particular helminth or other antigen inducing the Th2 response. We have used third stage larvae of intestinal nematode parasites as adjuvants to promote naïve nonparasite antigen‐specific T cells to differentiate into Th2 cells. This model system avoids possible parasite antigen‐specific T‐cell clones or cross‐reactive memory T cells that may preferentially differentiate into Th2 effector cells during the course of infection and confound the stereotypical components of parasite‐induced Th2 cell differentiation. We have found that these parasites have a potent adjuvant effect and have used our model system to begin to investigate the events that lead to the development of polarized Th2 cells in vivo.

IL-2 and Autocrine IL-4 Drive the In Vivo Development of Antigen-Specific Th2 T Cells Elicited by Nematode Parasites

The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4−/− mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Rα-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4−/− recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.

Suicide by pipe bomb: a case report.

While lying down, a 23-year-old man detonated an improvised explosive device placed behind his head. The posterior neck and shoulders were singed, and much of the brain was avulsed. Death was due to laceration and partial avulsion of the cerebrum, midbrain, and brain stem. The injuries had a directional nature. Facts derived from the scene investigation and gross dissection, including nature, distribution, and extent of the wounds, in conjunction with preceding medical and social history, allowed for a reasonable reconstruction of the circumstances.

Plasmodium falciparum DNA microarrays and interpretation of data

Publisher Summary Microarray analysis is a powerful technique that can examine the gene expression of thousands of genes simultaneously. The completion of the P. falciparum genome allows a targeted approach to the analysis of global gene expression, by allowing the design of gene-specific polymerase chain reaction (PCR) product arrays. Such arrays can be used in a multitude of experimental designs, including comparing gene expression between different stages of the parasite life cycle, the examination of the effect of drugs on gene expression, as a single-dose event, or by following a time course of parasite growth in the presence of drug, and genomic analysis, can be performed by comparing the hybridization patterns of fluorescently labelled DNA from different strains of the parasite. Such an approach has been shown to be an effective way to identify gene deletions in Campylobacter jejuni .