Sherron Bullens

In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation

Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses.

A monoclonal antibody that inhibits mouse tissue factor function

The tissue factor (TF)/factor VIIa (FVIIa) complex not only controls hemostatic processes, but also contributes to thrombosis-related diseases and cancer. Genetic mouse models of human diseases (e.g. prostateand colorectal cancer, sickle cell anemia) could serve to investigate further the role of TF/FVIIa in disease progression. However, such attempts are hampered by the lack of specific antimouse TF reagents. Here, we describe the generation of the monoclonal rat antimouse TF antibody 1H1. The antibody was produced by immunizing rats with recombinant soluble mouse TF(1–219) (muTF) expressed in a baculovirus expression system. Using standard hybridoma producing techniques, the 1H1 antibody (rat IgG2a/kappa) was identified by its ability to bind to muTF in an enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining of mouse frozen tissue sections with 1H1-IgG purified from ascites demonstrated specific staining in various tissues including lung and kidney. Figure 1A illustrates mouse TF expression in pulmonary alveolar epithelial cells [1,2]. Similar to human TF expression [1,2], strong mouse TF staining was also found in vascular adventitia, perineural tissue and autonomic ganglia, with weaker expression in medial smooth muscle of the renal artery (Fig. 1B). In-vitro and in-vivo studies demonstrated that 1H1 inhibits mouse TF function. First, in clotting assays with the TFexpressingmousemelanoma cell line B16F10, we found 1H1 to prolong mouse plasma clotting in a concentration-dependent fashion (Fig. 1C). This was also observed in clotting assays with recombinant muTF and phospholipid vesicles. Similar to other function-blocking antibodies such as AP-1 [3,4], inhibitory activity was observed only when antibody and TF were allowed to preincubate before addition of plasma. Secondly, in a mouse lung metastasis model, B16F10 cells (0.1 · 10 cells) preincubated with 0.7 mg mL of 1H1 or phosphate-buffered saline (PBS) (control) were injected via tail vein to isogenic C57Bl6J mice (8–12 weeks of age). After 2 weeks, control animals developed 47 ± 6 (n 1⁄4 15, ± SEM) visible lung tumor foci. Treatment with 1H1 reduced the number of tumor foci by 68% (Fig. 1D) to 15 ± 2 (n 1⁄4 15, P < 0.0001). These results demonstrated that 1H1 efficiently inhibits TF function in vivo and indicate that TF activity is essential for B16F10 melanoma cell metastasis, consistent with studies using murine tissue factor pathway inhibitor [5] or the thrombin inhibitor desulfatohirudin [6]. In conclusion, the monoclonal 1H1 antibody represents a useful reagent to investigate further the biology and pathophysiology of TF in mouse models.

Effect of Selective or Combined Inhibition of Integrins αIIbβ3 and αvβ3 on Thrombosis and Neointima After Oversized Porcine Coronary Angioplasty

Background—Thrombosis and neointima formation limit the efficacy of coronary angioplasty (PTCA). Clinical trials have implicated the adhesion molecules integrin αIIbβ3 and integrin αvβ3 in these processes. The roles of these molecules in vascular smooth muscle cell adhesion, platelet aggregation, and the thrombotic and neointimal response to oversize porcine PTCA was investigated by use of a selective αIIbβ3 antagonist (lamifiban), a selective αvβ3 antagonist (VO514), and a combined αIIbβ3/αvβ3 antagonist (G3580). Methods and Results—In vitro, both αvβ3 inhibitors caused dose-dependent inhibition of porcine vascular smooth muscle cell adhesion to vitronectin but not to collagen type IV, fibronectin, or laminin, whereas selective αIIbβ3 inhibition had no effect. Intravenous infusions of either αIIbβ3 inhibitor in swine profoundly inhibited ex vivo platelet aggregation to ADP, whereas selective αvβ3 inhibition had no effect. In a porcine PTCA model, intravenous infusions of the integrin antagonists were adm...

Disulfide locked variants of factor VIIa with a restricted β‐strand conformation have enhanced enzymatic activity

Proteolytic processing of zymogen Factor VII to Factor VIIa (FVIIa) is necessary but not sufficient for maximal proteolytic activity, which requires an additional allosteric influence induced upon binding to its cofactor tissue factor (TF). A key conformational change affecting the zymogenicity of FVIIa involves a unique three‐residue shift in the position of β‐strand B2 in their zymogen and protease forms. By selectively introducing new disulfide bonds, we locked the conformation of these strands into an active TF•FVIIa‐like state. FVIIa mutants designated 136:160, 137:159, 138:160, and 139:157, reflecting the position of the new disulfide bond (chymotypsinogen numbering), were expressed and purified by TF affinity chromatography. Mass spectrometric analysis of tryptic peptides from the FVIIa mutants confirmed the new disulfide bond formation. Kinetic analysis of amidolytic activity revealed that all FVIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S‐2765, having 670‐ and 330‐fold increases, respectively. Notably, FVIIa disulfide‐locked variants no longer required TF as a cofactor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20‐ and 12‐fold for variants 136:160 and 138:160, respectively, compared to wild type. With relipidated TF, mutants 136:160 and 137:159 also had an approximate threefold increase in their Vmax/Km values for FX activation but no significant improvement in TF‐dependent clotting assays. Thus, while large rate enhancements were obtained for amidolytic substrates binding at the active site, macro‐molecular substrates that bind to FVIIa exosites entail more complex catalytic requirements.

Lung Gene Expression in a Rhesus Allergic Asthma Model Correlates with Physiologic Parameters of Disease and Exhibits Common and Distinct Pathways with Human Asthma and a Mouse Asthma Model

Experimental nonhuman primate models of asthma exhibit multiple features that are characteristic of an eosinophilic/T helper 2 (Th2)-high asthma subtype, characterized by the increased expression of Th2 cytokines and responsive genes, in humans. Here, we determine the molecular pathways that are present in a house dust mite-induced rhesus asthma model by analyzing the genomewide lung gene expression profile of the rhesus model and comparing it with that of human Th2-high asthma. We find that a prespecified human Th2 inflammation gene set from human Th2-high asthma is also present in rhesus asthma and that the expression of the genes comprising this gene set is positively correlated in human and rhesus asthma. In addition, as in human Th2-high asthma, the Th2 gene set correlates with physiologic markers of allergic inflammation and disease in rhesus asthma. Comparison of lung gene expression profiles from human Th2-high asthma, the rhesus asthma model, and a common mouse asthma model indicates that genes associated with Th2 inflammation are shared by all three species. However, some pathophysiologic aspects of human asthma (ie, subepithelial fibrosis, angiogenesis, neural biology, and immune host defense biology) are better represented in the gene expression profile of the rhesus model than in the mouse model. Further study of the rhesus asthma model may yield novel insights into the pathogenesis of human Th2-high asthma.