Isolation of Plasmodium berghei ookinetes in culture using Nycodenz density gradient columns and magnetic isolation

BACKGROUND: Large scale in vitro production of the mosquito stages of malaria parasites remains elusive, with only limited success for complete sporogonic development and only one report of development through to infective sporozoites. The initial step in this process is the production, in vitro, of ookinetes from gametocytaemic blood. Methods for isolation of these ookinetes from blood cells have been described; however, in addition to yield often being low, processing time and potential for contamination by erythrocytes remain high. METHODS: This study compares two procedures for retaining mature ookinetes from blood stage cultures, whilst removing red blood cells and other contaminants prior to further culture of the parasite. The well established method of isolation on Nycodenz cushions is compared with a novel method utilizing the innate magnetic properties of the haem pigment crystals found in the cytoplasm of ookinetes. RESULTS: Yield and viability of ookinetes were similar with both isolation methods. However, in our hands magnetic isolation produced a cleaner ookinete preparation much more quickly. Moreover, decreasing the flow rate through the magnetic column could further enhance the yield. CONCLUSION: We recommend the enrichment of an ookinete preparation prior to further culture being performed using the magnetic properties of Plasmodium berghei ookinetes as an alternative to their density. The former technique is faster, removes more erythrocytes, but day-to-day costs are greater

Characterisation of Innate Fungal Recognition in the Lung

The innate recognition of fungi by leukocytes is mediated by pattern recognition receptors (PRR), such as Dectin-1, and is thought to occur at the cell surface triggering intracellular signalling cascades which lead to the induction of protective host responses. In the lung, this recognition is aided by surfactant which also serves to maintain the balance between inflammation and pulmonary function, although the underlying mechanisms are unknown. Here we have explored pulmonary innate recognition of a variety of fungal particles, including zymosan, Candida albicans and Aspergillus fumigatus, and demonstrate that opsonisation with surfactant components can limit inflammation by reducing host-cell fungal interactions. However, we found that this opsonisation does not contribute directly to innate fungal recognition and that this process is mediated through non-opsonic PRRs, including Dectin-1. Moreover, we found that pulmonary inflammatory responses to resting Aspergillus conidia were initiated by these PRRs in acidified phagolysosomes, following the uptake of fungal particles by leukocytes. Our data therefore provides crucial new insights into the mechanisms by which surfactant can maintain pulmonary function in the face of microbial challenge, and defines the phagolysosome as a novel intracellular compartment involved in the innate sensing of extracellular pathogens in the lung

Dectin-1: a role in antifungal defense and consequences of genetic polymorphisms in humans

The clinical relevance of fungal infections has increased dramatically in recent decades as a consequence of the rise of immunocompromised populations, and efforts to understand the underlying mechanisms of protective immunity have attracted renewed interest. Here we review Dectin-1, a pattern recognition receptor involved in antifungal immunity, and discuss recent discoveries of polymorphisms in the gene encoding this receptor which result in human disease

Effect of antimicrobial peptides against <i>P. berghei</i> infections in <i>Anopheles stephensi</i>.

<p>Mosquitoes were provided with a gametocytaemic blood meal mixed with 50 µM of peptide, performed in triplicate. Prevalence (the proportion of infected mosquitoes with total numbers in parentheses) and intensity (mean number of oocysts with the range in parentheses) of infections with paired controls are shown. N/S indicates non-significance. Significant differences are indicated by probability values with (▴) representing oocyst numbers significantly higher than control and (▾) representing oocyst numbers lower than control.</p

Antimicrobial peptide used in the current study: origin and size.

<p>A description of all antimicrobial peptides tested in the current study. AMPs were sourced from a variety of organisms displaying antibacterial, antifungal and/or anti-parasitic properties. Custom peptides were also included.</p

Effect of Melittin on <i>Plasmodium</i> development in mosquitoes.

<p>Mosquitoes were fed blood containing gametocytes of rodent malaria (fed to <i>An. stephensi</i>) or human malaria (fed to <i>An. gambiae</i>) supplemented with the AMP Melittin. Fully engorged females were maintained in standardized conditions for 7–8 days prior to dissection for oocyst burdens. Each experiment was performed in triplicate with control feeds containing no AMP. Individual value plots for each dissected midgut are shown. Black diamonds represent the median oocyst burden for each group. Approximately 40–50 individuals were dissected for <i>P. berghei</i> infections and 30 individuals for <i>P. falciparum</i> infections (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003790#ppat-1003790-t005" target="_blank">Tables 5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003790#ppat-1003790-t006" target="_blank">6</a> for full details). A. 50 µM of Melittin added to blood containing <i>P. berghei</i> gametocytes. B. 50 µM of Melittin added to blood containing <i>P. falciparum</i> gametocytes.</p

Effect of AMPs on <i>P. berghei</i> ookinetes after 30 minutes.

<p>Peptides were incubated with ookinetes for 30 min to assess their speed of action and efficacy. This table summarizes peptides with significant effects on ookinete viability in these conditions (n = 150 ookinetes per treatment in each replicate). Where 100% mortality was not achieved at 50 µM, peptides were doubled in concentration (100 µM) or added in combination with another peptide to total 50 µM (25 µM of each peptide). All results were significant for Wilcoxon Rank sign test at p<0.009.</p

Effect of antimicrobial peptides on field parasites and mosquitoes.

<p>Peptides (final concentration of 50 µM) were mixed with human blood containing <i>P. falciparum</i> parasites from gametocyte carriers in the village of Nyaganabougou. This was membrane-fed to the progeny of <i>An. gambiae</i> mosquitoes collected from neighbouring areas. For each replicate, oocyst prevalence (number of oocyst positive mosquitoes, total number in parentheses) and oocyst intensity (mean number of oocysts present per gut, range in parentheses) were recorded. An equivalent volume of water without peptide was used for the control. N/D indicates not determined. N/S indicates non-significance.</p