Pathogenic Roles of CD14, Galectin-3, and OX40 during Experimental Cerebral Malaria in Mice

An in-depth knowledge of the host molecules and biological pathways that contribute towards the pathogenesis of cerebral malaria would help guide the development of novel prognostics and therapeutics. Genome-wide transcriptional profiling of the brain tissue during experimental cerebral malaria (ECM ) caused by Plasmodium berghei ANKA parasites in mice, a well established surrogate of human cerebral malaria, has been useful in predicting the functional classes of genes involved and pathways altered during the course of disease. To further understand the contribution of individual genes to the pathogenesis of ECM, we examined the biological relevance of three molecules – CD14, galectin-3, and OX40 that were previously shown to be overexpressed during ECM. We find that CD14 plays a predominant role in the induction of ECM and regulation of parasite density; deletion of the CD14 gene not only prevented the onset of disease in a majority of susceptible mice (only 21% of CD14-deficient compared to 80% of wildtype mice developed ECM, p<0.0004) but also had an ameliorating effect on parasitemia (a 2 fold reduction during the cerebral phase). Furthermore, deletion of the galectin-3 gene in susceptible C57BL/6 mice resulted in partial protection from ECM (47% of galectin-3-deficient versus 93% of wildtype mice developed ECM, p<0.0073). Subsequent adherence assays suggest that galectin-3 induced pathogenesis of ECM is not mediated by the recognition and binding of galectin-3 to P. berghei ANKA parasites. A previous study of ECM has demonstrated that brain infiltrating T cells are strongly activated and are CD44+CD62L− differentiated memory T cells [1]. We find that OX40, a marker of both T cell activation and memory, is selectively upregulated in the brain during ECM and its distribution among CD4+ and CD8+ T cells accumulated in the brain vasculature is approximately equal

Design, baseline characteristics, and retention of African American light smokers into a randomized trial involving biological data

<p>Abstract</p> <p>Background</p> <p>African Americans experience significant tobacco-related health disparities despite the fact that over half of African American smokers are light smokers (use ≤10 cigarettes per day). African Americans have been under-represented in smoking cessation research, and few studies have evaluated treatment for light smokers. This paper describes the study design, measures, and baseline characteristics from <it>Kick It at Swope III </it>(KIS-III), the first treatment study of bupropion for African American light smokers.</p> <p>Methods</p> <p>Five hundred forty African American light smokers were randomly assigned to receive bupropion (150mg bid) (n = 270) or placebo (n = 270) for 7 weeks. All participants received written materials and health education counseling. Participants responded to survey items and provided blood samples for evaluation of phenotype and genotype of CYP2A6 and CYP2B6 enzymes involved in nicotine and bupropion metabolism. Primary outcome was cotinine-verified 7-day point prevalence smoking abstinence at Week 26 follow-up.</p> <p>Results</p> <p>Of 2,628 individuals screened, 540 were eligible, consented, and randomized to treatment. Participants had a mean age of 46.5 years and 66.1% were women. Participants smoked an average of 8.0 cigarettes per day, had a mean exhaled carbon monoxide of 16.4ppm (range 1-55) and a mean serum cotinine of 275.8ng/ml. The mean Fagerström Test for Nicotine Dependence was 3.2, and 72.2% of participants smoked within 30 minutes of waking. The average number of quit attempts in the past year was 3.7 and 24.2% reported using pharmacotherapy in their most recent quit attempt. Motivation and confidence to quit were high.</p> <p>Conclusion</p> <p>KIS-III is the first study designed to examine both nicotine and bupropion metabolism, evaluating CYP2A6 and CYP2B6 phenotype and genotype in conjunction with psychosocial factors, in the context of treatment of African American light smokers. Of 1629 smokers screened for study participation, only 18 (1.1%) were ineligible to participate in the study because they refused blood draws, demonstrating the feasibility of recruiting and enrolling African American light smokers into a clinical treatment trial involving biological data collection and genetic analyses. Future evaluation of individual factors associated with treatment outcome will contribute to advancing tailored tobacco use treatment with the goal of enhancing treatment and reducing health disparities for African American light smokers.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="URL">NCT00666978</a></p

Cellular Inflammatory Response to Flaviviruses in the Central Nervous System of a Primate Host

Flaviviruses such as tick-borne encephalitis virus, Japanese encephalitis virus, West Nile virus, and St. Louis encephalitis virus are important neurotropic human pathogens, typically causing a devastating and often fatal neuroinfection. Flaviviruses induce neuroinflammation with typical features of viral encephalitides, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. Development of safe and effective live-virus vaccines against neurotropic flavivirus infections demands a detailed knowledge of their neuropathogenesis in a primate host that is evolutionarily close to humans. Here, we used computerized morphometric analysis to quantitatively assess the cellular inflammatory responses in the central nervous system (CNS) of rhesus monkeys infected with three antigenically divergent attenuated flaviviruses. The kinetics, spatial pattern, and magnitude of microglial activation, trafficking of T and B cells, and changes in T cell subsets within the CNS define unique phenotypic signatures for each of the three viruses. Our results provide a benchmark for investigation of cellular inflammatory responses induced by attenuated flaviviruses in the CNS of primate hosts and provide insight into the neuropathogenesis of flavivirus encephalitis that might guide the development of safe and effective live-virus vaccines. (J Histochem Cytochem 57:973–989, 2009

<i>Pb-A</i> parasites collected from whole blood at approximately 10% parasitemia were cultured overnight to obtain schizont stage parasites.

<p>Parasites were then incubated with <i>A:</i> no galectin-3 <i>B:</i> galectin-3 <i>C:</i> galectin-3 and sucrose and <i>D:</i> galectin-3 and lactose and subsequently stained with a goat antibody specific for galectin-3 and a fluorescent donkey antibody specific for goat IgG. Right panels show galectin-3 adherence to mouse lymphocytes. Images were collected on an epifluorescence microscope.</p

Expression of OX40 in the brain of mice with experimental cerebral malaria.

<p>Brain sections from <i>A:</i> moribund and <i>B:</i> non-moribund <i>Pb-A</i> infected mice were stained with a goat antibody specific for mouse OX40 and visualized at 40×magnification. Immunostaining demonstrated strong but highly specific staining of a subset of lymphoid cells within and adjacent to blood vessels in the brains of moribund mice (A) but much fewer positive cells were observed in non-moribund (B) mice.</p

OX40 expression on T cell subsets present in the brain of moribund mice.

<p>Total brain leukocytes (sequestered and nonsequestered) were stained with fluorescent-labeled antibodies against TCRβ, CD4, CD8, and OX40. The proportion of <i>A:</i> CD4⁺ and CD8⁺<i>B:</i> OX40⁺CD4⁺ and <i>C:</i> OX40⁺ CD8⁺ T cells was determined by flow cytometry. <i>D:</i> Although OX40 is preferentially expressed on CD4⁺ T cells, due to the comparatively higher number of CD8⁺ T cells in the brain, the absolute number of OX40⁺CD4⁺ and OX40⁺CD8⁺ T cells accumulated in the brain vasculature is approximately the same.</p

Galectin-3 deficient mice are partially protected against experimental cerebral malaria and developed higher peripheral parasitemia.

<p><i>A:</i> Fourteen of 15 (93%) WT mice versus 8 of 17 (47%) galectin-3-KO mice succumbed to ECM by day 8 post-infection. Data shown is cumulated from two independent experiments. <i>B:</i> In galectin-3-KO (n = 5) and WT (n = 9) mice that developed ECM, galectin-3-KO mice had moderately higher parasitemia than WT mice (p<0.0137, two-way ANOVA). <i>C:</i> Among mice that did not develop ECM, although parasitemia did not differ markedly between days 4 and 12, parasitemia was higher on day 20 in the galectin-3-KO (53.3±8.29%, n = 4) versus WT (36%, n = 1) group.</p

Deletion of CD14 confers protection against ECM and has a negative effect on parasite growth <i>in vivo</i>.

<p>C57BL/6 WT and CD14-KO mice were challenged with 10⁶<i>Pb-A</i> parasites and susceptibility to ECM was determined based on symptoms exhibited and ability of mice to survive beyond day 10 post-infection. <i>A:</i> 16 of 20 (80%) WT mice versus 4 of 19 (21%) CD14-KO mice succumbed to ECM by day 10 post-challenge. <i>B:</i> CD14-KO mice that did not develop ECM had approximately 2-fold lower parasite burden between days 6 to 8 and 3.6-fold lower parasite burden between days 9 to 13 post-<i>Pb-A</i> infection. <i>C:</i> In contrast, WT and CD14-KO mice that developed ECM had no significant differences in parasite burden at onset of ECM.</p

Induction of galectin-3 during experimental cerebral malaria.

<p>Expression of galectin-3 was measured in brain tissue samples in individual moribund (n = 5), non-moribund (n = 5), CD8-KO ( = 3), and normal (non-infected, n = 3) C57BL/6 mice by ECL-based western blot analysis. Expression levels were determined based on the intensity of protein bands using Meta1 Morph 6.1 software and are represented as average integrated optical densities (IOD) units. The IOD units shown are values×1000. Details of antibodies and western blot reagents used can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006793#s4" target="_blank">Materials and Methods</a>.</p

Maternal immunization : collaborating with Mother Nature

Maternal immunization offers much hope to substantially reduce morbidity and mortality from infectious diseases after birth. The success of tetanus, influenza and pertussis immunization during pregnancy has led to consideration of additional maternal immunization strategies to prevent Group B Streptococcus (GBS) and respiratory syncytial virus (RSV) infections, among others. However, there remain multiple gaps in our knowledge regarding the immunobiology of maternal immunization that prevent optimal design and application of this successful public health intervention. An innovative landscape analysis was therefore undertaken to identify research priorities. Key topics were delineated through review of the published literature, consultation with vaccine developers and regulatory agencies, and a collaborative workshop gathering experts across several current maternal immunization initiatives - GBS, RSV, pertussis, and influenza. Finally, a global online survey prioritized the identified knowledge gaps based on expert opinion regarding their importance and relevance. This article presents the results of this worldwide landscape analysis and discusses the identified research gaps.Medicine, Faculty ofNon UBCInfectious Diseases, Division ofMedicine, Department ofPediatrics, Department ofReviewedFacult