Prophylactic and therapeutic use of antibodies for protection against respiratory infection with Francisella tularensis

The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcgammaR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-gamma. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-gamma. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-gamma. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24-48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.     

Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.     

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.     

Genetic risk score analysis indicates migraine with and without comorbid depression are genetically different disorders

Migraine and major depressive disorder (MDD) are comorbid, moderately heritable and to some extent influenced by the same genes. In a previous paper, we suggested the possibility of causality (one trait causing the other) underlying this comorbidity. We present a new application of polygenic (genetic risk) score analysis to investigate the mechanisms underlying the genetic overlap of migraine and MDD. Genetic risk scores were constructed based on data from two discovery samples in which genome-wide association analyses (GWA) were performed for migraine and MDD, respectively. The Australian Twin Migraine GWA study (N = 6,350) included 2,825 migraine cases and 3,525 controls, 805 of whom met the diagnostic criteria for MDD. The RADIANT GWA study (N = 3,230) included 1,636 MDD cases and 1,594 controls. Genetic risk scores for migraine and for MDD were used to predict pure and comorbid forms of migraine and MDD in an independent Dutch target sample (NTR–NESDA, N = 2,966), which included 1,476 MDD cases and 1,058 migraine cases (723 of these individuals had both disorders concurrently). The observed patterns of prediction suggest that the ‘pure’ forms of migraine and MDD are genetically distinct disorders. The subgroup of individuals with comorbid MDD and migraine were genetically most similar to MDD patients. These results indicate that in at least a subset of migraine patients with MDD, migraine may be a symptom or consequence of MDD.     

Significant Role for p16 in p53-Independent Telomere-Directed Senescence

Telomere attrition in primary human fibroblasts induces replicative senescence accompanied by activation of the p53 and p16(INK4a)/RB tumor suppressor pathways. Although the contribution of p53 and its target, p21, to telomere-driven senescence have been well established, the role of p16(INK4a) is controversial. Attempts to dissect the significance of p16(INK4a) in response to telomere shortening have been hampered by the concomitant induction of p16(INK4a) by cell culture conditions. To circumvent this problem, we studied the role of p16(INK4a) in the cellular response to acute telomere damage induced by a dominant negative allele of TRF2, TRF2(Delta B Delta M). This approach avoids the confounding aspects of culture stress because parallel cultures with and without telomere damage can be compared. Telomere damage generated with TRF2(Delta B Delta M) resulted in induction of p16(INK4a) in the majority of cells as detected by immunohistochemistry. Inhibition of p16(INK4a) with shRNA or overexpression of BMI1 had a significant effect on the telomere damage response in p53-deficient cells. While p53 deficiency alone only partially abrogated the telomere damage-induced cell cycle arrest, combined inhibition of p16(INK4a) and p53 led to nearly complete bypass of telomere-directed senescence. We conclude that p16(INK4a) contributes to the p53-independent response to telomere damage.     

羊草(Leymus chinensis)草地雨天放牧与非雨天放牧的绵羊采食特征

通过受控实验,比较了雨天和非雨天放牧在采食方式、采食损失量、践踏折损率以及土壤含水量和容重的差异.得出雨天放牧条件下,羊草各种采食方式的比例降低,去顶量依旧随放牧压升高而增加,但与非雨天放牧不同的是不再占主要比例.羊草采食损失量与非雨天差异不大,对于适口性更高的植物,如芦苇和全叶马兰,雨天放牧时损失更高.绵羊在冷湿环境中活动明显减少,羊草的践踏折损率各放牧压下差异不大,但芦苇的折损率依旧表现为随放牧压增强而增加.放牧后土壤15cm的含水量都低于对照小区,表明绵羊践踏加速了土壤水分的蒸发.放牧后土壤容重与放牧前和非牧区差异显著,并且容重随放牧压增强而增加.     

Arylphthalazines: identification of a new phthalazine chemotype as inhibitors of VEGFR kinase

A novel class of 4-arylamino-phthalazin-1-yl-benzamides is described as inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). Several compounds display potent VEGFR-2 inhibitory activity with an IC50 as low as 0.078 microM in an HTRF enzymatic assay. These compounds are relatively selective against a small kinase panel.     

DNA Barcodes of Rosy Tetras and Allied Species (Characiformes: Characidae: Hyphessobrycon) from the Brazilian Amazon Basin

DNA barcoding can be an effective tool for fast and accurate species-level identification based on sequencing of the mitochondrial cytochrome c oxidase subunit (COI) gene. The diversity of this fragment can be used to estimate the richness of the respective species. In this study, we explored the use of DNA barcoding in a group of ornamental freshwater fish of the genus Hyphessobrycon. We sequenced the COI from 10 species of Hyphessobrycon belonging to the “Rosy Tetra Clade” collected from the Amazon and Negro River basins and combined our results with published data. The average conspecific and congeneric Kimura 2-parameter distances were 2.3% and 19.3%, respectively. Six of the 10 species were easily distinguishable by DNA barcoding (H. bentosi, H. copelandi, H. eques, H. epicharis, H. pulchrippinis, and H. sweglesi), whereas the remaining species (H. erythrostigma, H. pyrrhonotus, H. rosaceus and H. socolofi) lacked reciprocal monophyly. Although the COI gene was not fully diagnostic, the discovery of distinct evolutionary units in certain Hyphessobrycon species under the same specific epithet as well as haplotype sharing between different species suggest that DNA barcoding is useful for species identification in this speciose genus.