Christopher D. Benjamin

Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plaques. Implications for the mode of progression of advanced coronary atherosclerosis.

Endothelial attachment is the initial step in leukocyte recruitment into developing atherosclerotic lesions. To determine whether vascular cell adhesion molecule-1 (VCAM-1) expression may play a role in inflammatory cell recruitment into human atherosclerotic lesions, immunohistochemistry was performed with a polyclonal rabbit antisera, raised against recombinant human VCAM-1, on 24 atherosclerotic coronary plaques and 11 control coronary segments with nonatherosclerotic diffuse intimal thickening from 10 patients. Immunophenotyping was performed on adjacent sections to identify smooth muscle cells, macrophages, and endothelial cells. To confirm VCAM-1-expressing cell types, double immunostaining with VCAM-1 antisera and each of the cell-specific markers and in situ hybridization were performed. All atherosclerotic plaques contained some VCAM-1, compared to 45% of control segments. VCAM-1 was found infrequently on endothelial cells at the arterial lumen din both plaques (21%) and in control segments (27%), but was prevalent in areas of neovascularization and inflammatory infiltrate in the base of plaques. Double immunostaining and in situ hybridization confirmed that most VCAM-1 was expressed by subsets of plaque smooth muscle cells and macrophages. The results document the presence of VCAM-1 in human atherosclerosis, demonstrate VCAM-1 expression by human smooth muscle cells in vivo, and suggest that intimal neovasculature may be an important site of inflammatory cell recruitment into advanced coronary lesions.

Attenuation of colitis in the cotton-top tamarin by anti-alpha 4 integrin monoclonal antibody.

Recent studies have demonstrated the induced expression of endothelial adhesion molecules including E-selectin (also called endothelial leukocyte adhesion molecule-1), vascular cell adhesion molecule and intercellular adhesion molecule in actively involved mucosa of patients with ulcerative colitis and Crohns disease. Similar induction has been demonstrated in the colon of the Cotton-top tamarin (CTT), a New World primate that experiences a spontaneous acute and chronic colitis resembling ulcerative colitis. To assess the potential importance of leukocyte adhesion as a necessary step in acute colitis, the effect of parenteral mAb directed against adhesion molecules on CTT colitis was evaluated in placebo-controlled blinded trials. Serial administration of either of two anti-E-selectin mAb designated BB11 and EH8 effectively coated endothelial surfaces expressing this vascular adhesion molecule. Although colitis activity was slightly diminished after the 10-d treatment period in CTT receiving either BB11 or EH8, this reduction was not significantly different than that seen in animals given a placebo control when assessed by a previously validated standardized scale of inflammatory activity: mean histologic activity grade 2.2 +/- 0.2 pretreatment vs 1.5 +/- 0.5 posttreatment in group receiving mAb and 2.1 +/- 0.1 pretreatment vs 1.3 +/- 0.5 posttreatment in the placebo group (P > 0.2). In contrast, administration of an anti-alpha 4 integrin mAb designated HP1/2 that binds VLA4 (alpha 4 beta 1) and presumably alpha 4 beta 7 integrins resulted in significant attenuation of acute colitis when compared to both pretreatment activity index (P = 0.005) and the placebo control group (P < 0.01): mean histologic activity grade 1.6 +/- 0.3 pretreatment vs 0.2 +/- 0.1 posttreatment in the group receiving HP1/2 and 1.8 +/- 0.5 pretreatment and 1.2 +/- 0.2 posttreatment in the placebo control group. These studies using a model of spontaneous colitis in the CTT demonstrate the feasibility of modulation of leukocyte-vascular adhesion and/or other integrin-mediated events possibly including T cell aggregation and T cell-stromal interactions, as well as lymphocyte homing. These results suggest both that these processes are important and possibly essential elements in sustaining acute colitis and that their disruption may result in therapeutic benefit.

Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo

Cripto, a cell surface-associated protein belonging to the EGF-CFC family of growth factor-like molecules, is overexpressed in many human solid tumors, including 70-80% of breast and colon tumors, yet how it promotes cell transformation is unclear. During embryogenesis, Cripto complexes with Alk4 via its unique cysteine-rich CFC domain to facilitate signaling by the TGF-beta ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-beta ligand, Activin B, and that Cripto overexpression blocks Activin B growth inhibition of breast cancer cells. This result suggests a novel mechanism for antagonizing Activin signaling that could promote tumorigenesis by deregulating growth homeostasis. We show that an anti-CFC domain antibody, A8.G3.5, both disrupts Cripto-Nodal signaling and reverses Cripto blockade of Activin B-induced growth suppression by blocking Criptos association with either Alk4 or Activin B. In two xenograft models, testicular and colon cancer, A8.G3.5 inhibited tumor cell growth by up to 70%. Both Nodal and Activin B expression was found in the xenograft tumor, suggesting that either ligand could be promoting tumorigenesis. These data validate that functional blockade of Cripto inhibits tumor growth and highlight antibodies that block Cripto signaling mediated through its CFC domain as an important class of antibodies for further therapeutic development.

B Lymphocyte Memory Role of Stromal Cell Complement and FcγRIIB Receptors

To dissect the influence of CD21/CD35 and FcγRIIB in antigen retention and humoral memory, we used an adoptive transfer model in which antigen-primed B and T lymphocytes were given to sublethally irradiated wild-type mice or mice deficient in CD21/CD35 (Cr2−/−) or FcγRIIB receptors (FcγRIIB−/−). Cr2−/− chimeras showed impaired memory as characterized by a decrease in antibody titer, reduced frequency of antibody secreting cells, an absence of affinity maturation, and significantly reduced recall response. The impaired memory in Cr2−/− chimeras corresponded with the reduced frequency of antigen-specific memory B cells. Interestingly, FcγRIIB−/− chimeras showed a differential phenotype with impaired splenic but normal bone marrow responses. These data suggest that CD21/CD35 on stroma, including follicular dendritic cells, is critical to the maintenance of long-term B lymphocyte memory.

Comparison of Soluble Decoy IgG Fusion Proteins of BAFF-R and BCMA as Antagonists for BAFF

BAFF is considered a therapeutic target because dysregulated production of BAFF can induce systemic lupus erythematosus-like phenotype in mice, and elevated levels of BAFF are associated with disease severity in systemic lupus erythematosus and rheumatoid arthritis patients. Fc fusion decoy receptors, BCMA-Fc and BAFF-R-Fc, are therapeutic candidates for blocking BAFF. While studying their interactions with BAFF, we found that BAFF-R-Fc is more effective than BCMA-Fc for blocking BAFF binding to its receptors. We also found that a trimeric BAFF can bind more than one BAFF-R-Fc but only one BCMA-Fc. Moreover, we show that, in contrast to monovalent BAFF-R-Fc, monovalent BCMA does not form stable complexes with BAFF. Differences in their interaction with BAFF predict BAFF-R-Fc would be a better inhibitor. Indeed, we show BAFF-R-Fc is 10-fold more efficacious than BCMA-Fc for blocking BAFF-induced B cell proliferation in vitro and for blocking BAFF-mediated survival of mouse splenic B lymphocytes in vivo.

Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1

Vascular cell adhesion molecule 1 (VCAM1) is a leukocyte adhesion molecule induced on human endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for VCAM1 was constructed, stably expressed in Chinese Hamster Ovary (CHO) cells, and the secreted recombinant soluble form of VCAM1 (rsVCAM1) purified to homogeneity by immunoaffinity chromatography. Immobilized rsVCAM1 is a functional adhesion protein, and selectively binds only VLA4-expressing cells, including human B and T lymphocytes, NK cells, and certain lymphoblastoid cell lines. T cell subset analyses indicate preferential binding of CD8+ memory cells. rsVCAM1 should prove valuable for the further study of the role of VCAM1 during inflammatory and immune responses in vivo.

Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody.

BACKGROUND CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor (TNF) family, plays a critical role in both humoral and cellular immune responses and has been implicated in biological pathways involving epithelial cells, fibroblasts, and platelets. Such a pathway is T cell-mediated B cell activation, a process that occurs through the interaction of CD40L with CD40 receptor expressed on B cells. It results in various B cell responses, including immunoglobulin isotype switching and B cell differentiation and proliferation. These responses can be inhibited by the monoclonal antibody 5c8, which binds with high affinity to CD40L. RESULTS To understand the structural basis of the inhibition, we determined the crystal structure of the complex of the extracellular domain of CD40L and the Fab fragment of humanized 5c8 antibody. The structure shows that the complex has the shape of a three-bladed propeller with three Fab fragments bound symmetrically to a CD40L homotrimer. To further study the nature of the antibody-antigen interface, we assessed the ability of 23 site-directed mutants of CD40L to bind to 5c8 and CD40 and analyzed the results in the context of the crystal structure. Finally, we observed via confocal microscopy that 5c8 binding to CD40L on the cell surface results in the formation of patches of clustered complexes. CONCLUSIONS The structure reveals that 5c8 neutralizes CD40L function by sterically blocking CD40 binding. The antigenic epitope is localized in a region of the surface that is likely to be structurally perturbed as a result of genetic mutations that cause hyper-IgM syndrome. The symmetric trimeric arrangement of the Fab fragments in the complex results in a geometry that facilitates the formation of large clusters of complexes on the cell surface.

Characterization of Antihuman IFNAR-1 Monoclonal Antibodies: Epitope Localization and Functional Analysis

The type I interferon receptor (IFNAR) is composed of two subunits, IFNAR-1 and IFNAR-2, encoding transmembrane polypeptides. IFNAR-2 has a dominant role in ligand binding, but IFNAR-1 contributes to binding affinity and to differential ligand recognition. A panel of five monoclonal antibodies (mAb) to human IFNAR-1 (HuIFNAR-1) was produced and characterized. The reactivity of each mAb toward HuIFNAR-1 on native and transfected cells and in Western blot and ELISA formats was determined. In functional assays, one mAb, EA12, blocked IFN-a2 binding to human cells and interfered with Stat activation and antiviral activity. Epitopes for the mAb were localized to subdomains of the HuIFNAR-1 extracellular domain by differential reactivity of the mAb to a series of human/bovine IFNAR-1 chimeras. The antibody EA12 seems to require native HuIFNAR-1 for reactivity and does not map to a single subdomain, perhaps recognizing an epitope containing noncontiguous sequences in at least two subdomains. In contrast, the epitopes of the non-neutralizing mAb FB2, AA3, and GB8 mapped, respectively, to the first, second, and third subdomains of HuIFNAR-1. The mAb DB2 primarily maps to the fourth subdomain, although its reactivity may be affected by other determinants.

A blocking monoclonal antibody to endothelial-leukocyte adhesion molecule-1 (ELAM1)

ELAM1 is a leukocyte adhesion molecule induced on human umbilical vein endothelial cells (HUVECs) by inflammatory cytokines. Balb/C mice were immunized with COS cells transiently expressing cell-surface ELAM1 after transfection with ELAM1 cDNA. After fusion, ELAM1-specific monoclonal antibodies (Mabs) were identified by selective adhesion to ELAM1-expressing, but not control, CHO cells, and to cytokine-treated but not untreated HUVECs. One Mab, designated BB11, binds to and immunoprecipitates ELAM1 expressed on HUVECs, COS and CHO cells. BB11 blocks the interaction of ELAM1 with human PMN, the human myelomonocytic cell line HL60, and the human colon carcinoma line HT29.

Are events after endotoxemia related to circulating phospholipase A2

ObjectiveThe authors sought to determine whether the signs and symptoms of endotoxemia were related to the endotoxin–stimulated increase in circulating phospholipase A2 (PLA2) activity. BackgroundBecause hypotension and pulmonary injury have been associated with elevated PLA2 activity in septic shock and PLA2 levels are reduced with the administration of glucocorticoids, the PLA2 response to endotoxin was investigated in volunteers pretreated with and without hydrocortisone. MethodsCarefully screened human subjects were studied under four conditions: (1) saline, (2) hydrocortisone, (3) endotoxin, and (4) hydrocortisone administration before endotoxin exposure. Pulse rate, blood pressure, temperature, and symptoms of endotoxemia were serially measured. Plasma for tumor necrosis factor concentrations and PLA2 activity was obtained. ResultsAfter lipopolysaccharide, pulse rate and tumor necrosis factor concentrations rose at 1 to 2 hours; temperature increased maximally at 4 hours. PLA2 activity reached peak levels at 24 hours. With hydrocortisone pretreatment, a 50% reduction in the concentrations of tumor necrosis factor and PIAa occurred. Significant correlations between other variables and PLA2 activity were not observed. The enzyme identified by monoclonal antibody was the secreted nonpancreatic PLA2 (SNP-PLA2). ConclusionsThe results of this study suggest that elevations in circulating SNP-PLA2 activity and systemic events associated with intravenous endotoxin administration are unrelated.