Differences in human macrophage receptor usage, lysosomal fusion kinetics and survival between logarithmic and metacyclic Leishmania infantum chagasi promastigotes

The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) undergoes receptor-mediated phagocytosis by macrophages followed by a transient delay in phagolysosome maturation. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). Both logarithmic and metacyclic promastigotes entered MDMs through a compartment lined by the third complement receptor (CR3). In contrast, many logarithmic promastigotes entered through vacuoles lined by mannose receptors (MR) whereas most metacyclic promastigotes did not ( P  < 0.005). CR3-positive vacuoles containing metacyclic promastigotes stained for caveolin-1 protein, suggesting CR3 localizes in caveolae during phagocytosis. Following entry, the kinetics of phagolysosomal maturation and intracellular survival also differed. Vacuoles containing metacyclic parasites did not accumulate lysosome-associated membrane protein-1 (LAMP-1) at early times after phagocytosis, whereas vacuoles with logarithmic promastigotes did. MDMs phagocytosed greater numbers of logarithmic than metacyclic promastigotes, yet metacyclics ultimately replicated intracellularly with greater efficiency. These data suggest that virulent metacyclic Leishmania promastigotes fail to ligate macrophage MR, and enter through a path that ultimately enhances intracellular survival. The relatively quiescent entry of virulent Leishmania spp. into macrophages may be accounted for by the ability of metacyclic promastigotes to selectively bypass deleterious entry pathways.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Cyclic hydrostatic pressure and cotton particles stimulate synthesis by human lung macrophages of cytokines in vitro

Background

Inhalation of particulates is a leading cause of the development of lung diseases and current understanding of the complex relationship between lung metabolism and airborne particulates is incomplete. It is well established that mechanical load is important in the development of the lung and in lung cell differentiation. The interaction between particle exposure and physical forces on alveolar macrophages is a physiologically relevant issue, but as yet understudied. This study examines the effect of cyclic hydrostatic pressure and cotton particles on synthesis of cytokines by human alveolar macrophages.

Methods

Alveolar macrophages were obtained from patients with lung disease, either from lavage samples or from lung tissue resection. The commonly used cell line THP-1 was included in the experiments. Cell cultures were exposed to cotton particles and/cyclic hydrostatic pressure (3 or 5 psi); control cultures were exposed to medium only. TNFα, IL-1β and IL-6 were assayed in the culture media using specific ELISAs. Cells were characterized using morphology and markers specific for macrophages (Jenner/Giemsa staining, CD14 and CD68).

Results

Exposure to cotton particles stimulated cytokine synthesis by macrophages from all three sources; exposure to cyclic hydrostatic pressure alone did not stimulate cytokine synthesis significantly. However, the combination of both particles and cyclic hydrostatic pressure increased the simulation of cytokine synthesis still further. Cell characterization demonstrated that the large majority of cells had a macrophage morphology and were positive for CD14 and CD68.

Conclusion

These data suggest an interaction between cyclic hydrostatic pressure and particulate exposure, which increases alveolar macrophage cytokine production. This interaction was only observed at the higher cyclic hydrostatic pressure. However, in patient samples, there was considerable variation in the amount by which secretion of an individual cytokine increased and there was also variation in the mechanosensitivity of cells from the three different sources. Cyclic hydrostatic pressure, therefore, may be an important modulator of the response of alveolar macrophages to cotton particles, but the source of the cells may be a confounding factor which demands further investigation.     

MouseCyc: a curated biochemical pathways database for the laboratory mouse

Linking biochemical genetic data to the reference genome for the laboratory mouse is important for comparative physiology and for developing mouse models of human biology and disease. We describe here a new database of curated metabolic pathways for the laboratory mouse called MouseCyc . MouseCyc has been integrated with genetic and genomic data for the laboratory mouse available from the Mouse Genome Informatics database and with pathway data from other organisms, including human.     

Species-specific primers for predation studies of the pollen beetle, Meligethes aeneus (Coleoptera, Nitidulidae)

Species‐specific primers were developed for the pollen beetle (a pest in oilseed rape) for studies of predation by natural insect predators. Two forward and three reverse primers were designed within the mitochondrial COI gene and used in combination to amplify fragments in the size range of 163–290 bp. Remains of pollen beetle DNA were consistently detected in Pardosa spiders up to 24 h after ingestion but dropped drastically at 48 h. These primers will facilitate studies on biological control of this oilseed rape pest. Detection time was not correlated with fragment length as might be expected as the DNA gradually degrades into progressively shorter fragments over time.     

Isoforms of U1-70k Control Subunit Dynamics in the Human Spliceosomal U1 snRNP

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B''. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B'' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.     

Assessment of Local Public Health Workers' Willingness to Respond to Pandemic Influenza through Application of the Extended Parallel Process Model

Background

Local public health agencies play a central role in response to an influenza pandemic, and understanding the willingness of their employees to report to work is therefore a critically relevant concern for pandemic influenza planning efforts. Witte''s Extended Parallel Process Model (EPPM) has been found useful for understanding adaptive behavior in the face of unknown risk, and thus offers a framework for examining scenario-specific willingness to respond among local public health workers. We thus aim to use the EPPM as a lens for examining the influences of perceived threat and efficacy on local public health workers'' response willingness to pandemic influenza.

Methodology/Principal Findings

We administered an online, EPPM-based survey about attitudes/beliefs toward emergency response (Johns Hopkins∼Public Health Infrastructure Response Survey Tool), to local public health employees in three states between November 2006 – December 2007. A total of 1835 responses were collected for an overall response rate of 83%. With some regional variation, overall 16% of the workers in 2006-7 were not willing to “respond to a pandemic flu emergency regardless of its severity”. Local health department employees with a perception of high threat and high efficacy – i.e., those fitting a ‘concerned and confident’ profile in the EPPM analysis – had the highest declared rates of willingness to respond to an influenza pandemic if required by their agency, which was 31.7 times higher than those fitting a ‘low threat/low efficacy’ EPPM profile.

Conclusions/Significance

In the context of pandemic influenza planning, the EPPM provides a useful framework to inform nuanced understanding of baseline levels of – and gaps in – local public health workers'' response willingness. Within local health departments, ‘concerned and confident’ employees are most likely to be willing to respond. This finding may allow public health agencies to design, implement, and evaluate training programs focused on emergency response attitudes in health departments.     

Epidemiological Pathology of Dementia: Attributable-Risks at Death in the Medical Research Council Cognitive Function and Ageing Study

Background

Dementia drug development aims to modulate pathological processes that cause clinical syndromes. Population data (epidemiological neuropathology) will help to model and predict the potential impact of such therapies on dementia burden in older people. Presently this can only be explored through post mortem findings. We report the attributable risks (ARs) for dementia at death for common age-related degenerative and vascular pathologies, and other factors, in the MRC Cognitive Function and Ageing Study (MRC CFAS).

Methods and Findings

A multicentre, prospective, longitudinal study of older people in the UK was linked to a brain donation programme. Neuropathology of 456 consecutive brain donations assessed degenerative and vascular pathologies. Logistic regression modelling, with bootstrapping and sensitivity analyses, was used to estimate AR at death for dementia for specific pathologies and other factors. The main contributors to AR at death for dementia in MRC CFAS were age (18%), small brain (12%), neocortical neuritic plaques (8%) and neurofibrillary tangles (11%), small vessel disease (12%), multiple vascular pathologies (9%), and hippocampal atrophy (10%). Other significant factors include cerebral amyloid angiopathy (7%) and Lewy bodies (3%).

Conclusions

Such AR estimates cannot be derived from the living population; rather they estimate the relative contribution of specific pathologies to dementia at death. We found that multiple pathologies determine the overall burden of dementia. The impact of therapy targeted to a specific pathology may be profound when the dementia is relatively “pure,” but may be less impressive for the majority with mixed disease, and in terms of the population. These data justify a range of strategies, and combination therapies, to combat the degenerative and vascular determinants of cognitive decline and dementia. Please see later in the article for the Editors'' Summary     

PIKK-dependent phosphorylation of Mre11 induces MRN complex inactivation by disassembly from chromatin

The role of Mre11 phosphorylation in the cellular response to DNA double-strand breaks (DSBs) is not well understood. Here, we show that phosphorylation of Mre11 at SQ/TQ motifs by PIKKs (PI3 Kinase-related Kinases) induces MRN (Mre11–Rad50–Nbs1) complex dissociation from chromatin by reducing Mre11 affinity for DNA. Whereas phosphorylation of Mre11 at these residues is not required for DSB-induced ATM (Ataxia-Telangiectasia mutated) activation, abrogation of Mre11 dephosphorylation impairs ATM signaling. Our study provides a functional characterization of the DNA damage-induced Mre11 phosphorylation, and suggests that MRN inactivation participates in the down-regulation of damage signaling during checkpoint recovery following DSB repair.     

Population genetic history of the dreissenid mussel invasions: expansion patterns across North America

This study tests population genetic patterns across the Eurasian dreissenid mussel invasions of North America—encompassing the zebra mussel Dreissena polymorpha (1986 detection) and the quagga mussel D. rostriformis bugensis (detected in 1990, which now has largely displaced the former in the Great Lakes). We evaluate their source-spread relationships and invasion genetics using 9–11 nuclear microsatellite loci for 583 zebra mussels (21 sites) and 269 quagga mussels (12 sites) from Eurasian and North American range locations, with the latter including the Great Lakes, Mississippi River basin, Atlantic coastal waterways, Colorado River system, and California reservoirs. Additionally, mtDNA cytochrome b gene sequences are used to verify species identity. Our results indicate that North American zebra mussels originate from multiple non-native northern European populations, whereas North American quagga mussels trace to native estuaries in the Southern Bug and Dnieper Rivers. Invasive populations of both species show considerable genetic diversity and structure (zebra F _ST = 0.006–0.263, quagga F _ST = 0.008–0.267), without founder effects. Most newer zebra mussel populations have appreciable genetic diversity, whereas quagga mussel populations from the Colorado River and California show some founder effects. The population genetic composition of both species changed over time at given sites; with some adding alleles from adjacent populations, some losing them, and all retaining closest similarity to their original composition. Zebra mussels from Kansas and California appear genetically similar and assign to a possible origin from the St. Lawrence River, whereas quagga mussels from Nevada and California assign to a possible origin from Lake Ontario. These assignments suggest that overland colonization pathways via recreational boats do not necessarily reflect the most proximate connections. In conclusion, our microsatellite results comprise a valuable baseline for resolving present and future dreissenid mussel invasion pathways.