Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity

<p>Abstract</p> <p>Background</p> <p>The presence of cholesterol in the Human Immunodeficiency Virus (HIV) lipid envelop is important for viral function as cholesterol depleted viral particles show reduced infectivity. However, it is less well established whether other viral membrane lipids are also important for HIV infection.</p> <p>The ABCB4 protein is a phosphatidyl choline (PC) floppase that mediates transport of PC from the inner to the outer membrane leaflet. This property enabled us to modulate the lipid composition of HIV vectors and study the effects on membrane composition and infection efficiency.</p> <p>Results</p> <p>Virus generated in the presence of ABCB4 was enriched in PC and cholesterol but contained less sphingomyelin (SM). Viral titers were reduced 5.9 fold. These effects were not observed with an inactive ABCB4 mutant. The presence of the ABC transport inhibitor verapamil abolished the effect of ABCB4 expression on viral titers.</p> <p>The ABCB4 mediated reduction in infectivity was caused by changes in the viral particles and not by components co purified with the virus because virus made in the presence of ABCB4 did not inhibit virus made without ABCB4 in a competition assay.</p> <p>Incorporation of the envelope protein was not affected by the expression of ABCB4. The inhibitory effect of ABCB4 was independent of the viral envelope as the effect was observed with two different envelope proteins.</p> <p>Conclusion</p> <p>Our data indicate that increasing the PC content of HIV particles reduces infectivity.</p

Communion by extension : discrepancies between policy and practice

The growing practice of Communion by Extension was given formal authorisation by the Church of England General Synod in 2000 with the expectation that it would be used in particular circumstances, including explicitly the rural multi-church benefice. This paper reviews the historical origins of the practice of Communion by Extension and clarifies the intentions of the authorisation given in 2000. Then the intentions of the 2000 authorisation are compared and contrasted with current parochial practice within one English diocese. Considerable divergence is found. Five main themes are identified and discussed: the relationship between worship and mission; the pressures on clerical time; sacramental self-sufficiency; the value given to familiarity; and the choice between reservation and congregationalism

Triggering of the dsRNA Sensors TLR3, MDA5, and RIG-I Induces CD55 Expression in Synovial Fibroblasts

Background: CD55 (decay-accelerating factor) is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS. Methods: FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR) ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C)) and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (ds)RNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry. Results: CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1 beta and especially by the TLR3 ligand poly(I:C). Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55. Conclusions: Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocyte

Serine proteases of the human immune system in health and disease

Serine proteases form a large family of protein-cleaving enzymes that play an essential role in processes like blood coagulation, apoptosis and inflammation. Immune cells express a wide variety of serine proteases such as granzymes in cytotoxic lymphocytes, neutrophil elastase, cathepsin G and proteinase 3 in neutrophils and chymase and tryptase in mast cells. Regulation of proteolysis induced by these serine proteases is essential to prevent self-induced damage Hence, there are specialized serine protease inhibitors, serpins, which are broadly distributed Here, we discuss the function of human serine proteases in inflammation, apoptosis and tissue remodeling. Furthermore, we address their Impact on development and progression of immune mediated-diseases Understanding the mode of action of senile proteases will help to unravel molecular processes involved in immunological disorders and will facilitate the identification of new therapeutic targets. (C) 2010 Elsevier Ltd All rights reserve

Expression of complement regulatory proteins on cultured FLS of patients with different forms of arthritis.

<p>CD55, CD46, and CD59 expression was measured by flow cytometry on cultured FLS from patients with RA, OA, PsA, and SpA. Indicated is the fold difference in mean fluorescence intensity (MFI) over respective isotype control Ig (cMFI) (mean, n = 4−5).</p

CD55 is upregulated by poly(I:C) and IL-1β on synovial fibroblasts.

<p>RA-derived synovial fibroblasts (<b>A, C-F</b>) and dermal fibroblasts (<b>B</b>) were starved overnight and subsequently stimulated for 2 days with 100 ng/ml TNFα, 100 ng/ml IFNγ, 100 ng/ml IL-1β, 1 ng/ml IL-6, 100 U/ml IFNα, 100 µg/ml LTA (TLR2 ligand), 100 µg/ml poly(I:C) (TLR3 ligand), 10 µg/ml LPS (TLR4 ligand), 100 µg/ml imiquimod (TLR7 ligand), or 10 µg/ml CpG oligonucleotides (TLR9 ligand). Expression of CD55 (<b>A</b> and <b>B</b>), CD46 (<b>C</b>) and CD59 (<b>D</b>) was studied by flow cytometry. <b>E,</b> Upregulation of CD55 in response to increasing concentrations of poly(I:C). <b>F,</b> Inhibition of CD55 upregulation by chloroquine (HCQ), an inhibitor of endosomal acidification, added prior to poly(I:C) stimulation. Indicated is the relative protein expression as percentage of the medium control (mean ± SD, n = 6 (<b>A</b>) and 3–5 (<b>B-F</b>)). *, p<0.05; **, p<0.005.</p

Poly(I:C)-induced upregulation of CD55 on synovial fibroblasts increases the binding capacity for CD97.

<p>Synovial fibroblasts were stimulated for 2 days with 100 µg/ml poly(I:C). Affinity for CD97 was measured with multivalent fluorescent probes loaded with recombinant CD97-3EGF or EMR2-2EGF (control). To confirm specificity, cells were preincubated with mAb CLB-CD97L/1, directed against the first SCR of CD55. On top, representative histogram plots are shown. The bars represent the fold difference in mean fluorescence intensity (MFI) for CD97-3EGF over EMR2-2EGF (mean ± SD, n = 3). *, p<0.05.</p

Stimulation of cytoplasmic dsRNA receptors in FLS upregulates CD55 expression and, through MDA5, induces cell death.

<p>RA-derived synovial fibroblasts were stimulated for 2 days with the indicated concentrations (µg/ml) of poly(I:C), poly(I:C) with fugene, or 3pRNA with fugene to trigger, respectively, TLR3, MDA5, and RIG-I. <b>A,</b> Expression of CD55 analyzed by flow cytometry (mean ± SD, n = 6). <b>B,</b> Representative flow cytometry plots of annexin V and propidium iodide staining. <b>C,</b> Percentages of annexin V and/or propidium iodide-positive cells analyzed by flow cytometry (mean ± SD, n = 6). <b>D, E,</b> Effect of the pan-caspase inhibitor QVD on cell death and CD55 expression induced by intracellular delivery of poly(I:C) (mean ± SD, n = 3). *, p<0.05; **, p<0.005; ***, p<0.001.</p

FLS express functional cytoplasmic dsRNA sensors.

<p>RA-derived synovial fibroblasts were stimulated for 16 h with the indicated concentrations (µg/ml) of poly(I:C), poly(I:C) with fugene, or 3pRNA with fugene to trigger, respectively, TLR3, MDA5, and RIG-I. Transcription levels of (<b>A</b>) TLR3, MDA5, and RIG-I, and (<b>B</b>) the anti-viral/pro-inflammatory response genes IFN β, IP-10, and TNFα was measured by quantitative and semiquantitative PCR, respectively. Depicted is the fold change gene expression compared to medium control (mean ± SD, n = 4) (<b>A</b>) or representative photographs (<b>B</b>). *, p<0.05, **, p<0.005.</p