Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.

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Fire accident victims, who sustain both thermal injury to skin and smoke inhalation, have gross evidence of systemic and pulmonary oxidant damage and acute lung injury. We hypothesized that gamma-Tocopherol (gT), a reactive O 2 and N 2 scavenger, when delivered into the airway will attenuate lung injury induced by burn and smoke inhalation. Acute lung injury was induced in chronically prepared, anesthetized sheep by 40% total burn surface area, 3rd degree skin burn and smoke insufflation (48 breaths of cotton smoke, <40°C). Study groups: 1) Sham (not injured, flax (FO)-nebulized, n=6); 2) SA-neb (injured, saline-nebulized, n=6); 3) FO-neb (injured, FO-nebulized, n=6); 4) gT+FO-neb (injured, FO+gT-nebulized, n=6). Nebulization was started 1 h post-injury and 24 ml of FO with or without gT (51 mg/ml) was delivered into airways over 47 h using our newly developed lipid aerosolization device (droplet size-2.5-5 μm). The burn and smoke inhalationinduced pathological changes seen in the saline group were attenuated by FO nebulization; gT addition further improved pulmonary function. Pulmonary gT delivery along with a FO source may be a novel effective treatment strategy in management of patients with acute lung injury.

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic-bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.Isolation of plasma membranes from cells or tissues is the first step in the characterization and purification of plasma membrane proteins. Current methods for plasma membrane purification depend on density gradient centrifugation to separate plasma membranes from other organelles in cell homogenates. Density gradient centrifugation used in isolating plasma membranes uses differences in sedimentation velocities to separate particles of different densities in lysed cell solutions [1]. This procedure is time-consuming because multiple steps of centrifugation are needed to obtain a crude plasma membrane preparation. It is also inaccurate because of the inconsistent nature of cell lysis and centrifugation settings. Often, much of the plasma membrane is lost in the early steps of centrifugation, and some organelles may remain in the plasma membrane fraction. As a result, these methods not only are lengthy but also yield only a small percentage of the plasma membranes [1][2][3][4]. The low recovery also presents difficulty Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Several methods have been developed to improve purification of plasma memb...

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This study describes a contribution of acetylcholine to the airway constrictive and lumenal obstructive response after inhalation injury and identifies low-dose nebulization of tiotropium bromide as a potentially efficacious therapy for burn patients with severe inhalation injury.

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Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.