Human properdin modulates macrophage: Mycobacterium bovis BCG interaction via thrombospondin repeats (TSR) 4 and 5

Mycobacterium tuberculosis can proficiently enter macrophages and diminish complement activation on its cell surface. Within macrophages, the mycobacterium can suppress macrophage apoptosis and survive within the intracellular environment. Previously, we have shown that complement regulatory proteins such as factor H may interfere with pathogen–macrophage interactions during tuberculosis infection. In this study, we show that Mycobacterium bovis BCG binds properdin, an upregulator of the complement alternative pathway. TSR4+5, a recombinant form of thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an inhibitor of the alternative pathway, was also able to bind to M. bovis BCG. Properdin and TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Quantitative real-time PCR revealed elevated pro-inflammatory responses (TNF-α, IL-1β, and IL-6) in the presence of properdin or TSR4+5, which gradually decreased over 6 h. Correspondingly, anti-inflammatory responses (IL-10 and TGF-β) showed suppressed levels of expression in the presence of properdin, which gradually increased over 6 h. Multiplex cytokine array analysis also revealed that properdin and TSR4+5 significantly enhanced the pro-inflammatory response (TNF-α, IL-1β, and IL-1α) at 24 h, which declined at 48 h, whereas the anti-inflammatory response (IL-10) was suppressed. Our results suggest that properdin may interfere with mycobacterial entry into macrophages via TSR4 and TSR5, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. This study offers novel insights into the non-complement related functions of properdin during host–pathogen interactions in tuberculosis.MA-A has been supported by the Ministry of Higher Education, Malaysia and the Universiti Sains Malaysia

Surfactant protein D inhibits HIV-1 infection of target cells via interference with gp120-CD4 interaction and modulates pro-inflammatory cytokine production

© 2014 Pandit et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SPD against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection. © 2014 Pandit et al.The work (Project no. 2011-16850) was supported by Medical Innovation Fund of Indian Council of Medical Research, New Delhi, India (www.icmr.nic.in/)

Epstein-Barr virus BART gene products

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Macromolecular Structure Underlying Recognition in Innate Immunity

Immune molecules have evolved to distinguish “self “molecules from “non-self”, “altered self” and “danger” molecules. Recognition is mediated via interactions between pattern recognition receptor molecules (PPRs) and their ligands, which include hydrophobic and electrostatic interactions between amino acid residues on the PPRs and uncharged or charged groups on amino acid residues, sugar rings or DNA/RNA molecules. Recognition in innate immunity range from cases (C1q, mannin-binding protein etc) where recognition is orchestrated by interaction between many ligands with one receptor molecule, and density of interaction is necessary for strong specific recognition, distinct from weak non-specific binding, and cases such as TLRs and NLRs where recognition involves complexation of single receptor and ligand, followed by oligomerisation of the receptor molecule. The majority of PPR molecules bind and recognise a wide variety of ligands, e.g TLR4 recognises LPS (gram negative bacteria), Lipotechoic acid (gram positive bacteria), heat shock protein hsp60, respiratory syncytial virus fusion protein etc, molecules that are structurally dissimilar to each other. This indicates considerable flexibility in their binding domains (amino acid residue variations) and modes (hydrophobic and charged, direct or mediated via an adaptor molecule). However, in many cases there is a dearth of structural and molecular data available, required to delineate the mechanism of ligand binding underlining recognition in pathogen receptors in innate immunity. Insights into requirements of conformation, charge, surface etc in the recognition and function of innate immunity receptors and their activation pathways, based on current data can suggest valuable avenues for future work

Updates on Measles Incidence and Eradication: Emphasis on the Immunological Aspects of Measles Infection

Measles is an RNA virus infectious disease mainly seen in children. Despite the avail-ability of an effective vaccine against measles, it remains a health issue in children. Although it is a self-limiting disease, it becomes severe in undernourished and immune-compromised individuals. Measles infection is associated with secondary infections by opportunistic bacteria due to the immunosuppressive effects of the measles virus. Recent reports highlight that measles infection erases the already existing immune memory of various pathogens. This review covers the incidence, pathogenesis, measles variants, clinical presentations, secondary infections, elimination of measles virus on a global scale, and especially the immune responses related to measles infection

Test-based De-isolation in COVID-19 Immunocompromised patients: Ct value versus SARS-CoV-2 viral cultures

Background Immunocompromised patients with coronavirus disease 2019 (COVID-19) have prolonged infectious viral shedding for more than 20 days. A test-based approach is suggested for de-isolation of these patients. Methods The strategy was evaluated by comparing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load (cycle threshold (Ct) values) and viral culture at the time of hospital discharge in a series of 13 COVID-19 patients: six immunocompetent and seven immunocompromised (five solid organ transplant patients, one lymphoma patient, and one hepatocellular carcinoma patient). Results Three of the 13 (23%) patients had positive viral cultures: one patient with lymphoma (on day 16) and two immunocompetent patients (on day 7 and day 11). Eighty percent of the patients had negative viral cultures and had a mean Ct value of 20.5. None of the solid organ transplant recipients had positive viral cultures. Conclusions The mean Ct value for negative viral cultures was 20.5 in this case series of immunocompromised patients. Unlike those with hematological malignancies, none of the solid organ transplant patients had positive viral cultures. Adopting the test-based approach for all immunocompromised patients may lead to prolonged quarantine. Large-scale studies in disease-specific populations are needed to determine whether a test-based approach versus a symptom-based approach or a combination is applicable for the de-isolation of various immunocompromised patients

SP-D and rhSP-D recognize HIV-1 gp41, gp120 and gp160.

<p>(<b>A</b>) A representative image of a ligand blot analysis (n = 3) using a ready-to-use HIV-1 antigen PVDF strip, probed with SP-D or rhSP-D (1 µg/ml) in presence of 2 mM CaCl₂ and developed using a monoclonal antibody against human SP-D and a 1∶1000 anti-mouse IgG-peroxidase. SP-D and rhSP-D specifically bind to HIV-1 glycoproteins gp41, gp120 and gp 160. (<b>B</b>) ELISA assay showing the binding of different concentrations of SP-D (solid line) and rhSP-D (dotted line) to immobilized recombinant r-gp120 (2 µg/mL). SP-D or rhSP-D bind to gp120 in the presence of 5 mM CaCl₂, and the interaction is inhibited by 1 mM EDTA. Each data point represents the mean ± S.D. (n = 4). †, * shows a statistically significant increase in the binding of SP-D or rhSP-D respectively to HIV-1 gp120 in the presence of calcium compared to EDTA (p<0.05) (<b>C</b>) A representative histogram of FITC labeled gp120 on HL2/3 cells (n = 3). HIV-1 gp120 expressing HL2/3 cells were incubated with rhSP-D (2 µg/mL) or no rhSP-D and further probed with a polyclonal FITC-tagged anti-gp120 antibody. In presence of rhSP-D, significantly lesser FITC signal was detected by flow cytometry (gray tinted histogram) comparing to no rhSP-D (black lined histogram).</p

SP-D and rhSP-D interfere with binding of gp120 to CD4.

<p>(<b>A</b>) Docked solution displaying SP-D timer docked with glycosylated model of HIV-1 glycoprotein gp120. (<b>i</b>) SP-D trimer (light green, dark green and orange cartoon) interacting with HIV-1 gp120 (grey cartoon) (<b>ii</b>) SP-D trimer (green) bound proximal to the CD4 binding region of gp120 (PDB ID: 1GC1). The CD4 (red cartoon) binding regions C2 (D loop), C4 and C5 of gp120 are depicted as light red, purple and cyan surfaces, respectively. Asn234 is depicted in blue and Asn276, a part of C2 domain is depicted in light red. (<b>iii</b>) Enlarged image of site where SP-D and CD4 interact on gp120 (<b>B</b>) To assess whether SP-D or rhSP-D interfered with gp120 and CD4 binding, PBMCs were incubated together with r-gp120 (2 µg/mL) and SP-D or rhSP-D (1 and 2 µg/ml) in 5 mM CaCl₂. The interaction was probed using a polyclonal FITC-tagged anti-gp120 antibody. Cells were acquired on a flow cytometer. (<b>i</b>) Lymphocyte population was gated. Differences in the FITC positive gated lymphocytes were calculated in terms of % inhibition. (<b>ii</b>) Mean fluorescence intensity was converted to % FITC positive cells. rhSP-D (at 1 µg 74.32±0.5%, at 2 µg 48.32±3.2%) showed a significant reduction in FITC positive cells as compared to untreated control. (iii) rhSP-D (at 10 µg–28.73±2.8%) showed a significant reduction in FITC positive cells as compared to untreated control. The data is a representative image of 4 independent experiments. A similar experiment was conducted using SP-D (data not shown) (<b>C</b>) Fusion experiment where HL2/3 cells that express gp120 were incubated with indicated concentrations rhSP-D or SP-D in presence of 5 mM CaCl₂. These cells were fused with TZM-bl cells that express CD4. The interaction of gp120 and CD4 is directly proportional to the blue color foci observed under a light microscope in TZM-bl cells. Each data point represents the mean ± S.D. (n = 3). Presence of SP-D and rhSP-D significantly decreased gp120 and CD4 binding and further β-gal positive TZM-bl cells.</p

rhSP-D modulates the phosphorylation of Akt and MAPKs upon HIV-1 challenge.

<p>5×10⁵ Jurkat T cells or U937 monocytic cells were treated with rhSP-D (10 µg/ml) in presence of 5 mM CaCl₂ and HIV-1USNG31. After 30 min, cell were harvested, lysed, and SDS-PAGE and Western blots were performed using pAKT, p-p38 and p-Erk1/2 or β-actin (house-keeping control) antibodies. Representative Western blots (<b>A</b>) <b>and</b> (<b>C</b>) and bar graphs (<b>B</b>) <b>and</b> (<b>D</b>), generated by densitometric analysis for Jurkat and U937 cells, respectively. Each bar represents the mean ± S.D. (n = 3). *, ** shows statistical significant % inhibition cytokine levels of HIV-1 infection by rhSP-D in each cell type as compared to control (*p<0.05, **p<0.01) represent cumulative data.</p

rhSP-D inhibits pro-inflammatory cytokine production of HIV-1 challenged Jurkat T cells, U937 monocytic cells and activated PBMCs.

<p>Jurkat T cells, U937 monocytes and activated PBMCs were treated with indicated concentrations of rhSP-D, 20 min prior to HIV-1 challenge. 24 h post viral challenge, culture supernatant were collected and assayed for cytokine levels by a multiplex system. (<b>A</b>) Dose-dependent % inhibition of rhSP-D-treated HIV-1 challenged cytokines IL-2, TNF-α, IFN-γ, IL-1α, VEGF as compared to HIV alone in Jurkat T cells. Each bar represents the mean ± S.D. (n = 3). rhSP-D showed significant (p<0.05) reduction in the levels of these pro-inflammatory cytokines. (<b>B</b>) Dose-dependent % inhibition of rhSP-D-treated HIV-1 challenged cytokines IL-2, IL-6, IFN-γ, MCP-1, VEGF as compared to HIV alone in U937 monocytes. Each bar represents the mean ± S.D. (n = 3). rhSP-D showed significant (p<0.05) reduction in the levels of these pro-inflammatory cytokines. (<b>C</b>) Dose-dependent % inhibition of rhSP-D treated HIV challenged cytokines IL-2, IFN-γ, TNF-α, IL-1α, IL-1β, VEGF as compared to HIV alone. Each bar represents the mean ± S.D. (n = 3). rhSP-D showed significant (p<0.05) reduction in the levels of these pro-inflammatory cytokines.</p