The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity

<p>Abstract</p> <p>Background</p> <p>Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Herein we validate the use <it>in vitro </it>of recombinant 6 × His-tagged-PAI-2 lacking the intrahelical loop between C and D alpha-helices (PAI-2 ΔCD-loop) for these purposes.</p> <p>Results</p> <p>We show that PAI-2 ΔCD-loop expressed and purified from the pQE9 vector system presents an easier purification target than the previously used pET15b system. Additionally, PAI-2 ΔCD-loop gave both higher yield and purity than wild-type PAI-2 expressed and purified under identical conditions. Importantly, absence of the CD-loop had no impact on the inhibition of both solution phase and cell surface uPA or on the clearance of receptor bound uPA from the cell surface. Furthermore, uPA:PAI-2 ΔCD-loop complexes had similar binding kinetics (K_D~5 nM) with the endocytosis receptor Very Low Density Lipoprotein Receptor (VLDLR) to that previously published for uPA:PAI-2 complexes.</p> <p>Conclusion</p> <p>We demonstrate that the CD-loop is redundant for the purposes of cellular uPA inhibition and cell surface clearance (endocytosis) and is thus suitable for the development of anti-uPA targeted cancer therapeutics.</p

Novel immune genes associated with excessive inflammatory and antiviral responses to rhinovirus in COPD

Abstract Background Rhinovirus (RV) is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations, and primarily infects bronchial epithelial cells. Immune responses from BECs to RV infection are critical in limiting viral replication, and remain unclear in COPD. The objective of this study is to investigate innate immune responses to RV infection in COPD primary BECs (pBECs) in comparison to healthy controls. Methods Primary bronchial epithelial cells (pBECs) from subjects with COPD and healthy controls were infected with RV-1B. Cells and cell supernatant were collected and analysed using gene expression microarray, qPCR, ELISA, flow cytometry and titration assay for viral replication. Results COPD pBECs responded to RV-1B infection with an increased expression of antiviral and pro-inflammatory genes compared to healthy pBECs, including cytokines, chemokines, RNA helicases, and interferons (IFNs). Similar levels of viral replication were observed in both disease groups; however COPD pBECs were highly susceptible to apoptosis. COPD pBECs differed at baseline in the expression of 9 genes, including calgranulins S100A8/A9, and 22 genes after RV-1B infection including the signalling proteins pellino-1 and interleukin-1 receptor associated kinase 2. In COPD, IFN-β/λ1 pre-treatment did not change MDA-5/RIG-I and IFN-β expression, but resulted in higher levels IFN-λ1, CXCL-10 and CCL-5. This led to reduced viral replication, but did not increase pro-inflammatory cytokines. Conclusions COPD pBECs elicit an exaggerated pro-inflammatory and antiviral response to RV-1B infection, without changing viral replication. IFN pre-treatment reduced viral replication. This study identified novel genes and pathways involved in potentiating the inflammatory response to RV in COPD.</p

Differential DNA methylation profiles of infants exposed to maternal asthma during pregnancy

Background: Asthma is a complex disease that involves both genetic factors and environmental exposures. Aberrant epigenetic modifications, such as DNA methylation, may be important in asthma development. Fetal exposure to maternal asthma during critical periods of in utero development may lead to epigenetic alterations that predispose infants to a greater risk of developing asthma themselves. We investigated alterations in the DNA methylation profile of peripheral blood from infants exposed to maternal asthma during pregnancy. Methods: Peripheral blood was collected from 12-month-old infants born to women with (n = 25) and without (n = 15) doctor diagnosed asthma during pregnancy. Genomic DNA was extracted, bisulfite converted, and hybridized to Infinium Methylation 27 arrays (Illumina), containing over27,000 CpGs from 14,495 genes. CpG loci in only autosomal genes were classified as differentially methylated at the 99% level (P < 0.01, |DiffScore| > 22 and delta beta >0.06). Results: There were 70 CpG loci, corresponding to 67 genes that were significantly differentially methylated. Twelve CpG loci (11 genes) showed greater than 10% comparative difference in DNA methylation, including hyper-methylated loci of FAM181A, MRI1, PIWIL1, CHFR, DEFA1, MRPL28, AURKA, and hypo-methylated loci of NALP1L5, MAP8KIP3, ACAT2, and PM20D1 in maternal asthma. Methylation of MAPK8IP3 was significantly negatively correlated with maternal blood eosinophils (r = −0.38; P = 0.022), maternal eNO (r = −0.44; P = 0.005), and maternal serum total IgE (r = −0.39, P = 0.015). Methylation of AURKA negatively correlated with maternal hemoglobin (r = −0.43; P = 0.008), infants height (r = −0.51; P < 0.001) and weight (r = −0.36; P = 0.021). Methylation of PM20D1 was lower in infants born to mothers with asthma on inhaled corticosteroid treatment. Methylation of PM20D1 was lower and MRI1 was higher in infants born to atopic mothers without asthma. Conclusions: In an Australian study population, exposure to maternal asthma during pregnancy is associated with differential methylation profiles of infants' peripheral blood DNA, which may act as risk factors for future asthma development

Novel immune genes associated with excessive inflammatory and antiviral responses to rhinovirus in COPD

Background: Rhinovirus (RV) is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations, and primarily infects bronchial epithelial cells. Immune responses from BECs to RV infection are critical in limiting viral replication, and remain unclear in COPD. The objective of this study is to investigate innate immune responses to RV infection in COPD primary BECs (pBECs) in comparison to healthy controls. Methods: Primary bronchial epithelial cells (pBECs) from subjects with COPD and healthy controls were infected with RV-1B. Cells and cell supernatant were collected and analysed using gene expression microarray, qPCR, ELISA, flow cytometry and titration assay for viral replication. Results: COPD pBECs responded to RV-1B infection with an increased expression of antiviral and pro-inflammatory genes compared to healthy pBECs, including cytokines, chemokines, RNA helicases, and interferons (IFNs). Similar levels of viral replication were observed in both disease groups; however COPD pBECs were highly susceptible to apoptosis. COPD pBECs differed at baseline in the expression of 9 genes, including calgranulins S100A8/A9, and 22 genes after RV-1B infection including the signalling proteins pellino-1 and interleukin-1 receptor associated kinase 2. In COPD, IFN-β/λ1 pre-treatment did not change MDA-5/RIG-I and IFN-β expression, but resulted in higher levels IFN-λ1, CXCL-10 and CCL-5. This led to reduced viral replication, but did not increase pro-inflammatory cytokines. Conclusions: COPD pBECs elicit an exaggerated pro-inflammatory and antiviral response to RV-1B infection, without changing viral replication. IFN pre-treatment reduced viral replication. This study identified novel genes and pathways involved in potentiating the inflammatory response to RV in COPD