Infection with Rhinovirus Facilitates Allergen Penetration Across a Respiratory Epithelial Cell Layer.

BACKGROUND: Rhinovirus infections are a major risk factor for asthma exacerbations. We sought to investigate in an in vitro system whether infection with human rhinovirus reduces the integrity and barrier function of a respiratory epithelial cell layer and thus may influence allergen penetration. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate of respiratory epithelium. The cell monolayer was infected with human rhinovirus 14 at 2 different doses. The extent and effects of transepithelial allergen penetration were assessed using transepithelial resistance measurements and a panel of (125)I-labeled purified recombinant respiratory allergens (rBet v 1, rBet v 2, and rPhl p 5). RESULTS: Infection of respiratory cell monolayers with human rhinovirus decreased transepithelial resistance and induced a pronounced increase in allergen penetration. CONCLUSIONS: Our results indicate that infection with rhinovirus damages the respiratory epithelial barrier and allows allergens to penetrate more efficiently into the subepithelial tissues where they may cause increased allergic inflammation.

ADAMTS13 Antibody and Inhibitor Assays.

A finding of an ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) activity level of <10% of normal is usually sufficient to distinguish thrombotic thrombocytopenic purpura (TTP) from other thrombotic microangiopathies. TTP can be congenital or acquired, the most common form being acquired immune-mediated TTP caused by autoantibodies than inhibit ADAMTS13 function and/or increase its clearance. Basic 1 + 1 mixing tests can detect the presence of inhibitory antibodies, and quantification can be achieved with Bethesda-type assays that measure loss of function in a series of mixtures of test plasma and normal plasma. Not all patients present with inhibitory antibodies, and here the ADAMTS13 deficiency may be caused by clearing antibodies alone, which are not detectable in functional assays. ELISA assays are commonly used to detect clearing antibodies via capture with recombinant ADAMTS13. Since they also detect inhibitory antibodies, they are the preferred assay, although they cannot distinguish between inhibitory and clearing antibodies. The present chapter describes principles, performance, and practical aspects of a commercial ADAMTS13 antibody ELISA and a generic approach to Bethesda-type assays for detecting inhibitory ADAMTS13 antibodies.

Engagement of ICAM-1 by major group rhinoviruses activates the LFA-1/ICAM-3 cell adhesion pathway in mononuclear phagocytes.

Cell-cell interactions are critical at key points of immune responses and are mediated by a complex array of adhesion receptors. One of the most important adhesion molecules on leukocytes is intercellular adhesion molecule 1 (ICAM-1, CD54). Here we demonstrate that engagement of ICAM-1 with human major group rhinoviruses (HRV) enhances adhesiveness and homotypic aggregation of human monocytes and monocyte-derived dendritic cells (DC). Cluster formation upon engagement of ICAM-1 with HRV14 represents an active process. It is temperature and energy dependent, requires divalent cations, an intact cytoskeleton and protein de novo synthesis. Homotypic interaction between monocytes induced by HRV14 can be inhibited with blocking mAbs against LFA-1 (CD11a/CD18) and ICAM-3 (CD50) as well as with a mAb against the first immunoglobulin (Ig)-domain of PECAM-1 (CD31). Induction of enhanced cytoadhesiveness by HRV14 was not accompanied with an upregulation of LFA-1, ICAM-3 or PECAM-1 expression. Binding studies with recombinant PECAM-1 proteins indicated, however, that monocyte clustering upon engagement of ICAM-1 with HRV was accompanied with increased homophilic PECAM-1 interactions. Taken together the results of our study demonstrate that signalling via ICAM-1 induces adhesiveness of mononuclear phagocytes, which critically involves PECAM-1 and is mediated via LFA-1/ICAM-3.

Expression of recombinant human factor VIII in milk of several generations of transgenic rabbits.

Transgenic founder rabbits carrying a gene construct consisting of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb of the human clotting factor VIII (hFVIII) cDNA and 4.6 kb of 3' flanking sequences of mWAP gene were crossed for three generations. All transgenic animals showed stable transgene transmission. Transgenic females showed high level of recombinant hFVIII (rhFVIII) mRNA expression in biopsed mammary gland tissues, while marginal expression of rhFVIII mRNA was observed in the spleen, lung and brain. No adverse effects of ectopic expression on the physiology of the rabbits were observed. Expression was not detected in the liver, kidney, heart and skeletal muscle. In transgenic females derived from three generations, rhFVIII protein was secreted from the mammary gland of lactating females, as shown by Western blotting. Biological activity of rhFVIII protein, as revealed in clotting assays was ranged from 0.012 to 0.599 IU/ml corresponding to 1.2% and 59.9% of the hFVIII level in normal human plasma. No apparent effect of secreted rhFVIII on the milk performance of rabbits was observed. Our results confirm the possibility of producing a significant amount of a biologically active rhFVIII in the mammary gland of established transgenic rabbit lines.