The Human Antimicrobial Protein Bactericidal/Permeability-Increasing Protein (BPI) Inhibits the Infectivity of Influenza A Virus

In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPIpeptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely

Ectopic spinal calcification associated with diffuse idiopathic skeletal hyperostosis (DISH): A quantitative micro-ct analysis

Diffuse idiopathic skeletal hyperostosis (DISH) is a non-inflammatory spondyloarthropathy identified radiographically by calcification of the ligaments and/or entheses along the anterolateral aspect of the vertebral column. The etiology and pathogenesis of calcifications are unknown, and the diagnosis of DISH is currently based on radiographic criteria associated with advanced disease. To characterize the features of calcifications associated with DISH, we used micro-computed tomographic imaging to evaluate a cohort of 19 human cadaveric vertebral columns. Fifty-three percent of the cohort (n=10; 3 females, 7 males, mean age of death=81 years, range 67-94) met the radiographic criteria for DISH, with calcification of four or more contiguous vertebral segments. In almost all cases, the lower thoracic regions (T8-12) were affected by calcifications, consisting primarily of large, horizontal outgrowths of bony material. In contrast, calcifications localized to the upper thoracic regions demonstrated variability in their presentation and were categorized as either continuous vertical bands or discontinuous-patchy lesions. In addition to the variable morphology of the calcifications, our analysis demonstrated remarkable heterogeneity in the densities of calcifications, ranging from internal components below the density of cortical bone to regions of hyper-dense material that exceeded cortical bone. These findings establish that the current radiographic criteria for DISH capture heterogeneous presentations of ectopic spine calcification that can be differentiated based on morphology and density. These findings may indicate a naturally heterogenous disease, potential stage(s) in the natural progression of DISH, or distinct pathologies of ectopic calcifications. (c) 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Re

Mutant human BPI-peptide corresponding to the mouse peptide loose its activity and human BPI peptide inhibits the replication of VSV.

<p>BHK-cells were infected with 360 PFU/well of VSV-GFP virus. Prior to infection the VSV was incubated with 20 μg/mL of human BPI (huBPI) and mouse BPI-peptide (mBPI), respectively or remained untreated (control) for 1 h. After 1.5 h of the cells were overlaid with 1.25% Avicel medium and incubated for 42 h. Thereafter, the cells were fixed and stained with crystal violet for 1 h at 4°C. Finally the plaques were counted (A). MDCK(H) cells were infected with 500 PFU/well Influenza A virus strain A/PR/8/34 (H1N1) for 1 h. Before the infection the virus was incubated in the presence or absence of 20 μg/mL of either human BPI-peptide (huBPI), murine BPI-peptide (mBPI), human BPI-peptide ^N1^D (mutant 1), human BPI-peptide ^K11^M (mutant 2), human BPI-peptide ^N1^D and ^K11^M (mutant 3) or left untreated (control) for 1 h (B) or incubated in the presence or absence of 20 μg/mL of either human BPI-peptide (huBPI), murine BPI-peptide (mBPI), mouse BPI-peptide ^D1^N and ^M11^K (mutant 4) or left untreated (control) for 1 h (C). After the infection the virus containing supernatant was removed and the cells were grown for additional 13 h. Thereafter, the fixed and permeabilized cells were incubated with the mouse anti–nucleoprotein IAV monoclonal antibody. The binding of the antibody was detected by a donkey anti-mouse IgG-HRP antiserum and adding of the reagent TMB Super Sensitive One Component HRP Microwell Substrate. Substrate conversion was detected by 450 nm. One representative experiment out of 3 performed is shown. Statistically significant differences are given as p values (*** <0.001); n.s. is not significant; n = 5 ± SEM.</p

Human BPI-peptide specifically inhibits the replication of different IAV strains.

<p>Protease-deficient MDCK(H) cells were infected with 500 PFU/well of Influenza A virus strain A/PR/8/34 (H1N1) (A) and strain A/Aichi/2/68 (H3N2) (B) for 1 h. After the infection the virus containing supernatant was removed and the cells were grown for additional 13 h. Thereafter, the fixed and permeabilized cells were incubated with the mouse anti–nucleoprotein IAV monoclonal antibody. The binding of the antibody was detected by a donkey anti-mouse IgG-HRP antiserum and adding the reagent TMB Super Sensitive One Component HRP Microwell Substrate. Substrate conversion was detected by 450 nm. For the control sample is n = 8 ± SEM, all other samples n = 5 ± SEM. C) Calu-3 cells were infected with 300 PFU/well of Influenza A virus strain A/Aichi/2/68 (H3N2). Prior to the infection the virus was incubated in the presence or absences of the indicated amount of human or mouse BPI-peptide for 1 h. Thereafter, the virus solution was removed and the cells were incubated for 24 h. The virus amount in the supernatant was analysed by adding an aliquot of the supernatant to MDCK (H) cells for 1 h. After the infection the virus containing supernatant was removed and the cells were grown for additional 13 h. Thereafter, the fixed and permeabilized cells were incubated with the mouse anti–nucleoprotein IAV monoclonal antibody and detected as outlined above. (D) Furthermore, also the release of CCL5 into the supernatant of the infected Calu-3 cells was determined via a specific ELISA. One representative experiment out of 5 performed is displayed. Statistically significant differences are given as p values (** <0.01 and *** <0.001); n.s. is not significant; n = 3 ± SEM.</p

BPI peptides used.

<p>BPI peptides used.</p

Human BPI-peptide damages the IAV particles.

<p>Virus particles were incubated either with 100 (100) and 500 μg/mL (500) of human (A) or murine BPI-peptides (B) for 1 h or left untreated (C). After the incubation the virus particles were visualized by transmission electron microscopy. Therefore, the particles were negatively stained with 2% uranylacetate and transmission electron microscopy was carried out using a JEOL TEM 2100 at 120kV. Micrographs were recorded with a fast-scan 2k x 2k CCD camera F214. One representative experiment out of 3 performed is displayed.</p

Human BPI-peptide inhibits the activation of PBMCs by Influenza A virus.

<p>Human PBMCs were isolated by dense gradient centrifugation from buffy coats. Thereafter, the cells were seeded and stimulated with purified Influenza A virus (MOI 2) (H1N1) in the presence (black bars) of increasing concentrations and absence (white bar) of human and mouse BPI-peptides (50 μg/mL, (grey bar)), and left unstimulated (control). In addition, the cells were incubated in the presence of either human BPI-peptide (50 μg/mL, huBPI) or mouse BPI-peptide (50 μg/mL, mBPI), respectively. After 20 h the supernatant was collected and the release of IFNα (A) as well as IL-6 (B) was determined by a specific ELISA. One representative experiment out of three is displayed. Statistically significant differences are given as p values (* <0.05 and ** <0.01); n = 3 ± SEM.</p