Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl3 models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.

Targeting of antioxidant enzymes to endothelium may improve management of vascular oxidative stress. To achieve this goal, we cross-linked PECAM-1 antibody with catalase using modifications of protein primary amines and following SH-maleimide conjugation. Polymerization-like conjugation yielded multivalent anti-PECAM-cat complexes with a size range of 50–500 nm. The conjugates did not bind to a PECAM-negative cell line. In contrast they readily bound to PECAM expressing endothelial cells and protected them against H2O2-induced oxidative stress. In vivo biodistribution studies demonstrated that anti-PECAM/[125I]cat conjugate accumulated in murine lung and heart within 1 h after intravenous injection in naïve C-57BL/65, but not in PECAM-1 KO mice. Further, we compared biodistribution of 100 and 300-nm anti-PECAM conjugates in C-57BL/65 vs. PECAM KO mice. Regardless of anti-PECAM/catalase size, there was no targeting to lungs in PECAM KO mice, while WT mice showed pulmonary accumulation of catalase, which was 3-fold higher with conjugate enlargement from 100 to 300 nm. Targeting and protective characteristics of the conjugates were similar or exceeded those observed for earlier described streptavidin-biotin based conjugates. Thus, characterized approach of antioxidant enzymes immunoconjugation may be an efficient tool in a treatment of oxidative component in a variety of vascular diseases. NHLBI RO1 HL/GM 71175-01.

Recent evidence suggests that CD38, an ectoenzyme that converts NAD(+) to cyclic ADP-ribose (cADPr), may play a role in cytokine-induced airway smooth muscle (ASM) cell hyper-responsiveness, a key feature associated with chronic asthma. In the present study, we investigated the major signaling pathways by which tumor necrosis factor-alpha (TNFalpha) induces CD38 expression and its role in regulating gene expression in human ASM cells. Using flow cytometry analyses, TNFalpha enhanced CD38 expression in a manner that was time-(0-24 h), concentration-(0.1-40 ng/ml), and protein synthesis-(cycloheximide blockade) dependent. A selective agonistic antibody against tumor necrosis factor receptor (TNFR) 1 also augmented CD38 expression, whereas anti-TNFR2 antagonistic antibody did not prevent the TNFalpha response. Inhibition of the Janus activated kinase/signal transducer and activator of transcription pathways using the soluble inhibitor 2-(1,1-dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz-[h]imidaz[4,5-f]isoquinolin-7-one (DBI) or with neutralizing antibody against interferon beta (IFNbeta) completely abrogated TNFalpha-induced CD38 expression at both protein and mRNA levels. Combining TNFalpha (0.1 and 1 ng/ml) and IFNbeta (100 IU/ml) at concentrations alone that had little effect on CD38 expression induced a robust synergistic induction of CD38 mRNA and protein levels. 8-Bromo-cADPr, a cADPr antagonist, significantly augmented TNFalpha-induced interleukin-6 secretion, whereas regulated on activation normal T cell expressed and secreted secretion was suppressed. 8-Bromo-cADPr, however, did not affect TNFalpha-induced cell surface expression of intercellular adhesion molecule-1. Our study is the first to demonstrate that IFNbeta-dependent activation of CD38 pathway is a novel component by which TNFalpha differentially regulates the expression of inflammatory genes in ASM cells.

Current evidence suggests that tumor necrosis factor alpha ( TNF alpha) and the family of interferons ( IFNs) synergistically regulate many cellular responses that are believed to be critical in chronic inflammatory diseases, although the underlying mechanisms of such interaction are complex, cell- specific, and not completely understood. In this study, TNF alpha in a time- dependent manner activated both janus tyrosine kinase 1 and Tyk2 tyrosine kinase and increased the nuclear translocation of interferon- regulatory factor- 1, STAT1, and STAT2 in human airway smooth muscle cells. In cells transfected with a luciferase reporter, TNF alpha stimulated gamma- activated site- dependent gene transcription in a time- and concentration- dependent manner. Using neutralizing antibodies to IFNbeta and TNFalpha receptor 1, we show that TNF alpha- induced secretion of IFN beta mediated gamma- activated site- dependent gene expression via activation of TNF alpha receptor 1. In addition, neutralizing antibody to IFN beta also completely abrogated the activation of interferon stimulation response element-dependent gene transcription induced by TNFalpha. Secreted IFN beta acted as a negative regulator of TNF alpha-induced interleukin- 6 expression, while IFN beta augmented TNF alpha- induced RANTES ( regulated on activation normal T cell expressed and secreted) secretion but had little effect on TNF alpha- induced intercellular adhesion molecule- 1 expression. Furthermore TNFalpha, a modest airway smooth muscle mitogen, markedly induced DNA synthesis when cells were treated with neutralizing anti-IFNbeta. Together these data show that TNFalpha, via the autocrine action of IFN beta, differentially regulates the expression of proinflammatory genes and DNA synthesis.