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.     

Loss of TBL1XR1 Disrupts Glucocorticoid Receptor Recruitment to Chromatin and Results in Glucocorticoid Resistance in a B-Lymphoblastic Leukemia Model

Although great advances have been made in the treatment of pediatric acute lymphoblastic leukemia, up to one of five patients will relapse, and their prognosis thereafter is dismal. We have previously identified recurrent deletions in TBL1XR1, which encodes for an F-box like protein responsible for regulating the nuclear hormone repressor complex stability. Here we model TBL1XR1 deletions in B-precursor ALL cell lines and show that TBL1XR1 knockdown results in reduced glucocorticoid receptor recruitment to glucocorticoid responsive genes and ultimately decreased glucocorticoid signaling caused by increased levels of nuclear hormone repressor 1 and HDAC3. Reduction in glucocorticoid signaling in TBL1XR1-depleted lines resulted in resistance to glucocorticoid agonists, but not to other chemotherapeutic agents. Importantly, we show that treatment with the HDAC inhibitor SAHA restores sensitivity to prednisolone in TBL1XR1-depleted cells. Altogether, our data indicate that loss of TBL1XR1 is a novel driver of glucocorticoid resistance in ALL and that epigenetic therapy may have future application in restoring drug sensitivity at relapse.     

Placozoa and the evolution of Metazoa and intrasomatic cell differentiation

The multicellular Metazoa evolved from single-celled organisms (Protozoa) and usually – but not necessarily – consist of more cells than Protozoa. In all cases, and thus by definition, Metazoa possess more than one somatic cell type, i.e. they show-in sharp contrast to protists–intrasomatic differentiation. Placozoa have the lowest degree of intrasomatic variation; the number of somatic cell types according to text books is four (but see also Jakob W, Sagasser S, Dellaporta S, Holland P, Kuhn K, and Schierwater B. The Trox-2 Hox/ParaHox gene of Trichoplax (Placozoa) marks an epithelial boundary. Dev Genes Evol 2004;214:170–5). For this and several other reasons Placozoa have been regarded by many as the most basal metazoan phylum. Thus, the morphologically most simply organized metazoan animal, the placozoan Trichoplax adhaerens, resembles a unique model system for cell differentiation studies and also an intriguing model for a prominent “urmetazoon” hypotheses—the placula hypothesis. A basal position of Placozoa would provide answers to several key issues of metazoan-specific inventions (including for example different lines of somatic cell differentiation leading to organ development and axis formation) and would determine a root for unraveling their evolution. However, the phylogenetic relationships at the base of Metazoa are controversial and a basal position of Placozoa is not generally accepted (e.g. Schierwater B, DeSalle R. Can we ever identify the Urmetazoan? Integr Comp Biol 2007;47:670–76; DeSalle R, Schierwater B. An even “newer” animal phylogeny. Bioessays 2008;30:1043–47). Here we review and discuss (i) long-standing morphological evidence for the simple placozoan bauplan resembling an ancestral metazoan stage, (ii) some rapidly changing alternative hypotheses derived from molecular analyses, (iii) the surprising idea that triploblasts (Bilateria) and diploblasts may be sister groups, and (iv) the presence of genes involved in cell differentiation and signaling pathways in the placozoan genome.     

In the limb AER Bmp2 and Bmp4 are required for dorsal-ventral patterning and interdigital cell death but not limb outgrowth

The apical ectodermal ridge (AER) in the vertebrate limb is required for limb outgrowth and patterning. To investigate the role BMP ligands expressed in the AER play in limb development we selectively inactivated both Bmp2 and Bmp4 in this tissue. The autopods of mice lacking both of these genes contained extra digits, digit bifurcations and interdigital webbing due to a decrease in programmed cell death and an increase in cell proliferation in the underlying mesoderm. Upon removal of Bmp2 and Bmp4 in the AER, no defects in proximal-distal patterning were observed. At the molecular level, removal of Bmp2 and Bmp4 in the AER caused an increase in Fgf expression, which correlated with an increase in both the width and length of the AER. Investigation of Engrailed-1 (En1) expression in the AER of limb buds in which Bmp2 and Bmp4 had been removed indicated that En1 expression was absent from this tissue. Our data suggests that AER expression of Bmp2 and Bmp4 is required for digit and dorsal-ventral patterning but surprisingly not for limb outgrowth.     

Brainstem Neurons Survive the Identical Ischemic Stress That Kills Higher Neurons: Insight to the Persistent Vegetative State

Global ischemia caused by heart attack, pulmonary failure, near-drowning or traumatic brain injury often damages the higher brain but not the brainstem, leading to a ‘persistent vegetative state’ where the patient is awake but not aware. Approximately 30,000 U.S. patients are held captive in this condition but not a single research study has addressed how the lower brain is preferentially protected in these people. In the higher brain, ischemia elicits a profound anoxic depolarization (AD) causing neuronal dysfunction and vasoconstriction within minutes. Might brainstem nuclei generate less damaging AD and so be more resilient? Here we compared resistance to acute injury induced from simulated ischemia by ‘higher’ hippocampal and striatal neurons versus brainstem neurons in live slices from rat and mouse. Light transmittance (LT) imaging in response to 10 minutes of oxygen/glucose deprivation (OGD) revealed immediate and acutely damaging AD propagating through gray matter of neocortex, hippocampus, striatum, thalamus and cerebellar cortex. In adjacent brainstem nuclei, OGD-evoked AD caused little tissue injury. Whole-cell patch recordings from hippocampal and striatal neurons under OGD revealed sudden membrane potential loss that did not recover. In contrast brainstem neurons from locus ceruleus and mesencephalic nucleus as well as from sensory and motor nuclei only slowly depolarized and then repolarized post-OGD. Two-photon microscopy confirmed non-recoverable swelling and dendritic beading of hippocampal neurons during OGD, while mesencephalic neurons in midbrain appeared uninjured. All of the above responses were mimicked by bath exposure to 100 µM ouabain which inhibits the Na⁺/K⁺ pump or to 1–10 nM palytoxin which converts the pump into an open cationic channel.Therefore during ischemia the Na⁺/K⁺ pump of higher neurons fails quickly and extensively compared to naturally resilient hypothalamic and brainstem neurons. The selective survival of lower brain regions that maintain vital functions will support the persistent vegetative state.     

Sperm contamination in archived and contemporary herring samples

Studies using archived scales and otoliths to examine ancient fish populations have become increasingly common, despite many methodological challenges in ancient DNA research. Here, we describe a case of DNA contamination in both modern and historical samples of Pacific herring (Clupea pallasii), where the source of the contamination is likely from milt spillage during collection. We describe a series of experiments to remove contamination using pre-extraction wash treatments. Though contamination was easily removed from contemporary fin clippings, no method was successful at removing contamination from historical scales. We discuss the implications of our findings to the genetic analysis of archived samples.     

Self-Reported Trait Mindfulness and Affective Reactivity: A Motivational Approach Using Multiple Psychophysiological Measures

As a form of attention, mindfulness is qualitatively receptive and non-reactive, and is thought to facilitate adaptive emotional responding. One suggested mechanism is that mindfulness facilitates disengagement from an affective stimulus and thereby decreases affective reactivity. However, mindfulness has been conceptualized as a state, intervention, and trait. Because evidence is mixed as to whether self-reported trait mindfulness decreases affective reactivity, we used a multi-method approach to study the relationship between individual differences in self-reported trait mindfulness and electrocortical, electrodermal, electromyographic, and self-reported responses to emotional pictures. Specifically, while participants (N = 51) passively viewed pleasant, neutral, and unpleasant IAPS pictures, we recorded high-density (128 channels) electrocortical, electrodermal, and electromyographic data to the pictures as well as to acoustic startle probes presented during the pictures. Afterwards, participants rated their subjective valence and arousal while viewing the pictures again. If trait mindfulness spontaneously reduces general emotional reactivity, then for individuals reporting high rather than low mindfulness, response differences between emotional and neutral pictures would show relatively decreased early posterior negativity (EPN) and late positive potential (LPP) amplitudes, decreased skin conductance responses, and decreased subjective ratings for valence and arousal. High mindfulness would also be associated with decreased emotional modulation of startle eyeblink and P3 amplitudes. Although results showed clear effects of emotion on the dependent measures, in general, mindfulness did not moderate these effects. For most measures, effect sizes were small with rather narrow confidence intervals. These data do not support the hypothesis that individual differences in self-reported trait mindfulness are related to spontaneous emotional responses during picture viewing.     

Novel 4,5-diaryl-3-hydroxy-2(5H)-furanones as anti-oxidants and anti-inflammatory agents

In order to study the effect of phenol moieties on biological activities of ascorbic acid derivatives, we synthesized 13 novel 4,5-diaryl-3-hydroxy-2(5H)-furanones 5a-m with various substitution patterns. Compound 5 g bearing a 2,3-dihydroxy phenyl ring on the 5-position of the heterocycle appeared to be the most powerful anti-oxidant furanone with reducing activity against DPPH (IC(50)=10.3 microM), superoxide anion quenching capacity (IC(50)=0.187 mM) and lipid peroxidation inhibitory effect (IC(50)=0.129 mM). To ascertain determinant molecular features for anti-oxidant activities, structure-activity relationships were studied. Lipophilicity and molecular parameters related to electron distribution and structure (difference in heats of formation between the compound and its radical or its cation radical, energy of the highest occupied molecular orbital, HOMO) were found to correlate with the anti-oxidant action of compounds 5 in the different tests used. Oxygen-derived free radicals are known to contribute to inflammatory disorders; therefore we have investigated effects of compounds 5 in two models of inflammation: phorbol ester-induced ear edema in mice (TPA-test) and carrageenan-induced paw edema in rat. At 100 mg/kg ip in the TPA-test, the anti-inflammatory activity of compounds 5 was potent compared with that of indomethacin and ketorolac and all the results suggested a cyclooxygenase inhibition in the emergence of such properties. The combined pharmacological actions of compounds 5 associated with a favorable therapeutic index prompt with interesting perspectives for their use in heart and brain disorders as well as in inflammatory diseases.