Macrophage Receptors for Influenza A Virus: Role of the Macrophage Galactose-Type Lectin and Mannose Receptor in Viral Entry

Although sialic acid has long been recognized as the primary receptor determinant for attachment of influenza virus to host cells, the specific receptor molecules that mediate viral entry are not known for any cell type. For the infection of murine macrophages by influenza virus, our earlier study indicated involvement of a C-type lectin, the macrophage mannose receptor (MMR), in this process. Here, we have used direct binding techniques to confirm and characterize the interaction of influenza virus with the MMR and to seek additional macrophage surface molecules that may have potential as receptors for viral entry. We identified the macrophage galactose-type lectin (MGL) as a second macrophage membrane C-type lectin that binds influenza virus and is known to be endocytic. Binding of influenza virus to MMR and MGL occurred independently of sialic acid through Ca²⁺-dependent recognition of viral glycans by the carbohydrate recognition domains of the two lectins; influenza virus also bound to the sialic acid on the MMR. Multivalent ligands of the MMR and MGL inhibited influenza virus infection of macrophages in a manner that correlated with expression of these receptors on different macrophage populations. Influenza virus strain A/PR/8/34, which is poorly glycosylated and infects macrophages poorly, was not recognized by the C-type lectin activity of either the MMR or the MGL. We conclude that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.Infection of host cells by influenza virus is initiated by attachment of virus to sialic acid residues on the host cell surface through the receptor-binding site at the distal tip of the viral hemagglutinin (HA) (43). After attachment, the virus is internalized by endocytosis, and acidification of the endosome triggers a conformational change in viral HA that results in fusion of the viral envelope and host cell membrane (34). At the cell surface, sialic acid residues are commonly found at the termini of oligosaccharide chains that are attached in O or N linkage to cell surface proteins; they are also an essential component of acidic glycosphingolipids (gangliosides) that are present in all mammalian cell membranes. Although the abundance of sialic acid on mammalian cells provides influenza virus with multiple potential receptors, virus attachment does not always lead to virus entry (5, 8, 46). Furthermore, sialic acid-independent infection of Madin-Darby canine kidney (MDCK) cells by influenza virus has been reported (35). The specific host cell molecules that serve as functional receptors (or coreceptors) for the infectious entry of influenza virus have yet to be defined.We have studied the infectious entry of influenza virus into macrophages (Mφ), which represents an early event in recognition of the virus by the innate immune system (23, 44). After intranasal infection of mice, influenza virus replicates productively in cells of the respiratory epithelium. Mφ are also infected and viral proteins are produced, but replication is abortive and no live progeny are released (32); infection of Mφ is thus a dead-end for the virus leading to a reduction in viral load. In addition, influenza virus infection of Mφ stimulates production and release of proinflammatory cytokines and alpha/beta interferon (28), which may assist in further limiting viral replication and spread within the respiratory tract. Depletion of airway Mφ from mice prior to intranasal influenza virus infection leads to increased virus titers in the lung, attesting to the important role of Mφ in early host defense against the virus (38, 44).We observed in a previous study (30) that influenza A virus strains differed in their ability to infect murine Mφ, strains carrying a more highly glycosylated hemagglutinin (HA) molecule being more efficient at infecting Mφ than less glycosylated strains, although binding of viruses to the Mφ cell surface was equivalent. Our investigation of this phenomenon indicated involvement of the Mφ mannose receptor MMR (CD206), a C-type lectin, in infectious viral entry (29, 30). The involvement of other receptors was not excluded, and our subsequent observation that influenza virus can infect the RAW 264.7 Mφ cell line, which does not express the MMR, indeed points to the existence of other routes of infectious entry of the virus into Mφ.In the present study we used direct binding methods to confirm and characterize the interaction of influenza virus with the MMR and to seek additional Mφ surface molecules that may have potential as receptors for viral entry. We identify the Mφ galactose-type lectin (MGL) as a second Mφ membrane C-type lectin that binds influenza virus and investigate its involvement in the infectious process.     

Zebrafish: a model for the study of addiction genetics

Drug abuse and dependence are multifaceted disorders with complex genetic underpinnings. Identifying specific genetic correlates is challenging and may be more readily accomplished by defining endophenotypes specific for addictive disorders. Symptoms and syndromes, including acute drug response, consumption, preference, and withdrawal, are potential endophenotypes characterizing addiction that have been investigated using model organisms. We present a review of major genes involved in serotonergic, dopaminergic, GABAergic, and adrenoreceptor signaling that are considered to be directly involved in nicotine, opioid, cannabinoid, and ethanol use and dependence. The zebrafish genome encodes likely homologs of the vast majority of these loci. We also review the known expression patterns of these genes in zebrafish. The information presented in this review provides support for the use of zebrafish as a viable model for studying genetic factors related to drug addiction. Expansion of investigations into drug response using model organisms holds the potential to advance our understanding of drug response and addiction in humans.     

De-regulation of the RBBP6 isoform 3/DWNN in human cancers

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. The human RBBP6 gene codes for three protein isoforms and isoform 3 consists of the domain with no name domain only whilst the other two isoforms, 1 and 2 comprise of additional zinc, RING, retinoblastoma and p53 binding domains. In this study, the localization of RBBP6 using RBBP6 variant 3 mRNA-specific probe was performed to investigate the expression levels of the gene in different tumours and find a link between RBBP6 and human carcinogenesis. Using FISH, real-time PCR and Western blotting analysis our results show that RBBP6 isoform 3 is down-regulated in human cancers. RBBP6 isoform 3 knock-down resulted in reduced G2/M cell cycle arrest whilst its over-expression resulted in increased G2/M cell cycle arrest using propidium iodide DNA staining. The results further demonstrate that the RBBP6 isoform 3 may be the cell cycle regulator and involved in mitotic apoptosis not the isoform 1 as previously reported for mice. In conclusion, these findings suggest that RBBP6 isoform 3 is a cell cycle regulator and may be de-regulated in carcinogenesis.     

Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.     

Multiple forms of Spire-actin complexes and their functional consequences

Spire is a WH2 domain-containing actin nucleator essential for establishing an actin mesh during oogenesis. In vitro, in addition to nucleating filaments, Spire can sever them and sequester actin monomers. Understanding how Spire is capable of these disparate functions and which are physiologically relevant is an important goal. To study severing, we examined the effect of Drosophila Spire on preformed filaments in bulk and single filament assays. We observed rapid depolymerization of actin filaments by Spire, which we conclude is largely due to its sequestration activity and enhanced by its weak severing activity. We also studied the solution and crystal structures of Spire-actin complexes. We find structural and functional differences between constructs containing four WH2 domains (Spir-ABCD) and two WH2 domains (Spir-CD) that may provide insight into the mechanisms of nucleation and sequestration. Intriguingly, we observed lateral interactions between actin monomers associated with Spir-ABCD, suggesting that the structures built by these four tandem WH2 domains are more complex than originally imagined. Finally, we propose that Spire-actin mixtures contain both nuclei and sequestration structures.     

Candidate gene sequencing of SLC11A2 and TMPRSS6 in a family with severe anaemia: common SNPs, rare haplotypes, no causative mutation

Background

Iron-refractory iron deficiency anaemia (IRIDA) is a rare disorder which was linked to mutations in two genes (SLC11A2 and TMPRSS6). Common polymorphisms within these genes were associated with serum iron levels. We identified a family of Serbian origin with asymptomatic non-consanguineous parents with three of four children presenting with IRIDA not responding to oral but to intravenous iron supplementation. After excluding all known causes responsible for iron deficiency anaemia we searched for mutations in SLC11A2 and TMPRSS6 that could explain the severe anaemia in these children.

Methodology/Results

We sequenced the exons and exon–intron boundaries of SLC11A2 and TMPRSS6 in all six family members. Thereby, we found seven known and fairly common SNPs, but no new mutation. We then genotyped these seven SNPs in the population-based SAPHIR study (n = 1,726) and performed genetic association analysis on iron and ferritin levels. Only two SNPs, which were top-hits from recent GWAS on iron and ferritin, exhibited an effect on iron and ferritin levels in SAPHIR. Six SAPHIR participants carrying the same TMPRSS6 genotypes and haplotype-pairs as one anaemic son showed lower ferritin and iron levels than the average. One individual exhibiting the joint SLC11A2/TMPRSS6 profile of the anaemic son had iron and ferritin levels lying below the 5^th percentile of the population''s iron and ferritin level distribution. We then checked the genotype constellations in the Nijmegen Biomedical Study (n = 1,832), but the profile of the anaemic son did not occur in this population.

Conclusions

We cannot exclude a gene-gene interaction between SLC11A2 and TMPRSS6, but we can also not confirm it. As in this case candidate gene sequencing did not reveal causative rare mutations, the samples will be subjected to whole exome sequencing.     

Differential inflammatory response to inhaled lipopolysaccharide targeted either to the airways or the alveoli in man

Endotoxin (Lipopolysaccharide, LPS) is a potent inducer of inflammation and there is various LPS contamination in the environment, being a trigger of lung diseases and exacerbation. The objective of this study was to assess the time course of inflammation and the sensitivities of the airways and alveoli to targeted LPS inhalation in order to understand the role of LPS challenge in airway disease.In healthy volunteers without any bronchial hyperresponsiveness we targeted sequentially 1, 5 and 20 μg LPS to the airways and 5 μg LPS to the alveoli using controlled aerosol bolus inhalation. Inflammatory parameters were assessed during a 72 h time period. LPS deposited in the airways induced dose dependent systemic responses with increases of blood neutrophils (peaking at 6 h), Interleukin-6 (peaking at 6 h), body temperature (peaking at 12 h), and CRP (peaking at 24 h). 5 μg LPS targeted to the alveoli caused significantly stronger effects compared to 5 μg airway LPS deposition. Local responses were studied by measuring lung function (FEV(1)) and reactive oxygen production, assessed by hydrogen peroxide (H(2)O(2)) in fractionated exhaled breath condensate (EBC). FEV(1) showed a dose dependent decline, with lowest values at 12 h post LPS challenge. There was a significant 2-fold H(2)O(2) induction in airway-EBC at 2 h post LPS inhalation. Alveolar LPS targeting resulted in the induction of very low levels of EBC-H(2)O(2).Targeting LPS to the alveoli leads to stronger systemic responses compared to airway LPS targeting. Targeted LPS inhalation may provide a novel model of airway inflammation for studying the role of LPS contamination of air pollution in lung diseases, exacerbation and anti-inflammatory drugs.     

Experimental warming alters spring phenology of certain plant functional groups in an early successional forest community

Experimental study of the effects of projected climate change on plant phenology allows us to isolate effects of warming on life‐history events such as leaf out. We simulated a 2 °C temperature increase and 20% precipitation increase in a recently harvested temperate deciduous forest community in central Pennsylvania, USA, and observed the leaf out phenology of all species in 2009 and 2010. Over 130 plant species were monitored weekly in study plots, but due to high variability in species composition among plots, species were grouped into five functional groups: short forbs, tall forbs, shrubs, small trees, and large trees. Tall forbs and large trees, which usually emerge in the late spring, advanced leaf out 14–18 days in response to warming. Short forbs, shrubs, and small trees emerge early in spring and did not alter their phenology in response to warming or increased precipitation treatments. Earlier leaf out of tall forbs and large trees coincided with almost 3 weeks of increased community‐level leaf area index, indicating greater competition and a condensed spring green‐up period. While phenology of large trees and tall forbs appears to be strongly influenced by temperature‐based growth cues, our results suggest that photoperiod and chilling cues more strongly influence the leaf out of other functional groups. Reduced freeze events and warmer temperatures from predicted climate change will interact with nontemperature growth cues to have cascading consequences throughout the ecosystem.     

Loss of CCR7 Expression on CD56bright NK Cells Is Associated with a CD56dimCD16+ NK Cell-Like Phenotype and Correlates with HIV Viral Load

NK cells are pivotal sentinels of the innate immune system and distinct subpopulations in peripheral blood have been described. A number of studies addressed HIV-induced alterations of NK cell phenotype and functionality mainly focusing on CD56^dimCD16⁺ and CD56⁻CD16⁺ NK cells. However, the impact of HIV-infection on CD56^bright NK cells is less well understood. Here we report a rise of CD56^bright NK cells in HIV-infected individuals, which lack CCR7-expression and strongly correlate with HIV viral load. CCR7⁻CD56^bright NK cells were characterized by increased cytolytic potential, higher activation states and a more differentiated phenotype. These cells thus acquired a number of features of CD56^dimCD16⁺ NK cells. Furthermore, CD56^bright NK cells from HIV patients exhibited higher degranulation levels compared to uninfected individuals. Thus, chronic HIV-infection is associated with a phenotypic and functional shift of CD56^bright NK cells, which provides a novel aspect of HIV-associated pathogenesis within the NK cell compartment.     

Disruption of rolandic gamma-band functional connectivity by seizures is associated with motor impairments in children with epilepsy

Although children with epilepsy exhibit numerous neurological and cognitive deficits, the mechanisms underlying these impairments remain unclear. Synchronization of oscillatory neural activity in the gamma frequency range (>30 Hz) is purported to be a mechanism mediating functional integration within neuronal networks supporting cognition, perception and action. Here, we tested the hypothesis that seizure-induced alterations in gamma synchronization are associated with functional deficits. By calculating synchrony among electrodes and performing graph theoretical analysis, we assessed functional connectivity and local network structure of the hand motor area of children with focal epilepsy from intracranial electroencephalographic recordings. A local decrease in inter-electrode phase synchrony in the gamma bands during ictal periods, relative to interictal periods, within the motor cortex was strongly associated with clinical motor weakness. Gamma-band ictal desychronization was a stronger predictor of deficits than the presence of the seizure-onset zone or lesion within the motor cortex. There was a positive correlation between the magnitude of ictal desychronization and impairment of motor dexterity in the contralateral, but not ipsilateral hand. There was no association between ictal desynchronization within the hand motor area and non-motor deficits. This study uniquely demonstrates that seizure-induced disturbances in cortical functional connectivity are associated with network-specific neurological deficits.