Platelet integrin αIIbβ3 plays a key role in venous thrombogenesis in a mouse model.

Venous thrombosis (VT) is a common vascular disease associated with reduced survival and a high recurrence rate. Previous studies have shown that the accumulation of platelets and neutrophils at sites of endothelial cell activation is a primary event in VT, but a role for platelet αIIbβ3 in the initiation of venous thrombosis has not been established. This task has been complicated by the increased bleeding linked to partial agonism of current αIIbβ3 inhibitory drugs such as tirofiban (Aggrastat ).

Structure-guided design of pure orthosteric inhibitors of αIIbβ3 that prevent thrombosis but preserve hemostasis.

A prevailing dogma is that inhibition of vascular thrombosis by antagonizing platelet integrin αIIbβ3 cannot be achieved without compromising hemostasis, thus causing serious bleeding and increased morbidity and mortality. It is speculated that these adverse outcomes result from drug-induced activating conformational changes in αIIbβ3 but direct proof is lacking. Here, we report the structure-guided design of peptide Hr10 and a modified form of the partial agonist drug tirofiban that act as "pure" antagonists of αIIbβ3, i.

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1.

Targeting both integrins αVβ3 and α5β1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.

uPAR isoform 2 forms a dimer and induces severe kidney disease in mice.

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis.

Atomic basis for the species-specific inhibition of αV integrins by monoclonal antibody 17E6 is revealed by the crystal structure of αVβ3 ectodomain-17E6 Fab complex.

The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6.

Structural basis for pure antagonism of integrin αVβ3 by a high-affinity form of fibronectin.

Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVβ3 bound to a physiologic ligand, the tenth type III RGD domain of wild-type fibronectin (wtFN10), or to a high-affinity mutant (hFN10) shown here to act as a pure antagonist.

Structure of the kidney slit diaphragm adapter protein CD2-associated protein as determined with electron microscopy.

CD2-associated protein (CD2AP) is a multidomain scaffolding protein that has a critical role in renal function. CD2AP is expressed in glomerular podocytes at the slit diaphragm, a modified adherens junction that comprises the protein filtration barrier of the kidney, and interacts with a number of protein ligands involved in cytoskeletal remodeling, membrane trafficking, cell motility, and cell survival. The structure of CD2AP is unknown.

Immunogenetic responses in calves to intranasal delivery of bovine respiratory syncytial virus (BRSV) epitopes encapsulated in poly (DL-lactide-co-glycolide) microparticles.

Bovine respiratory syncytial virus (BRSV) is the principal aetiological agent of the bovine respiratory disease complex. A BRSV subunit vaccine candidate consisting of two synthetic peptides representing putative protective epitopes on BRSV surface glycoproteins in soluble form or encapsulated in poly(lactide-co-glycolide) (PLG) microparticles were prepared. Calves (10 weeks old) with diminishing levels of BRSV-specific maternal antibody were intranasally administered a single dose of the different peptide formulations.

EM structure of the ectodomain of integrin CD11b/CD18 and localization of its ligand-binding site relative to the plasma membrane.

One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS). We used electron microscopy to determine the 3D structure of the αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMβ2) in its inactive state. A well defined density for αA was observed within a bent ectodomain conformation, while the structure of the ectodomain in complex with the Fab fragment of mAb107, which binds at the MIDAS face of CD11b and stabilizes the inactive state, further revealed that αA is restricted to a relatively small range of orientations relative to the Propeller domain.

Local and systemic immune responses in mice to intranasal delivery of peptides representing bovine respiratory syncytial virus epitopes encapsulated in poly (DL-lactide-co-glycolide) microparticles.

The potential of a microparticulate vaccine delivery system in eliciting a specific mucosal antibody response in the respiratory tract of mice was evaluated. Two vaccine candidate peptides representing epitopes from the G attachment and F fusion antigens from bovine respiratory syncytial virus (BRSV) were encapsulated into poly(DL-lactide co-glycolide) biodegradable microparticles. The encapsulation process did not denature the entrapped peptides as verified by detection of peptide-specific antibodies in mucosal secretions by ELISA using peptide as antigen.

Stable coordination of the inhibitory Ca2+ ion at the metal ion-dependent adhesion site in integrin CD11b/CD18 by an antibody-derived ligand aspartate: implications for integrin regulation and structure-based drug design.

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg(2+) ion hexacoordinated at the metal ion-dependent adhesion site (MIDAS) in the integrin A domain. This interaction stabilizes the A domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials.

Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland.

We report the isolation in cell cultures of two novel bocavirus species in pigs from farms in Northern Ireland with clinical postweaning multisystemic wasting syndrome (PMWS). We have designated the isolates as porcine bocavirus-3 (PBoV3) and porcine bocavirus-4 (PBoV4). To date 5082 and 4125 bps of PBoV3 and PBoV4 have been sequenced, respectively.

Specialized roles for cysteine cathepsins in health and disease.

Cathepsins were originally identified as proteases that act in the lysosome. Recent work has uncovered nontraditional roles for cathepsins in the extracellular space as well as in the cytosol and nucleus. There is strong evidence that subspecialized and compartmentalized cathepsins participate in many physiologic and pathophysiologic cellular processes, in which they can act as both digestive and regulatory proteases.

Sensitive detection of African swine fever virus using real-time PCR with a 5' conjugated minor groove binder probe.

The design of a 5' conjugated minor groove binder (MGB) probe real-time PCR assay is described for the rapid, sensitive and specific detection of African swine fever virus (ASFV) DNA. The assay is designed against the 9GL region and is capable of detecting 20 copies of a DNA standard. It does not detect any of the other common swine DNA viruses tested in this study.

Nanoparticle vaccines against respiratory viruses.

Influenza virus, respiratory syncytial virus (RSV), and parainfluenza type 3 virus (PI-3V) are the major viral agents which are consistently involved in causing lower respiratory tract disease in humans and animals. The virus infection begins in the upper respiratory tract, where immune responses are initiated, and then progresses to the lower respiratory tract where destruction of cells and tissues leads to bronchitis, bronchiolitis, and pneumonia, which is occasionally fatal. Nanoparticle vaccines, incorporating antigenic components from influenza, RSV, or PI-3V have been shown to be capable of stimulating mucosal and systemic immune responses, which can prevent the spread of infection to the lower respiratory tract.

Purification and electron cryomicroscopy of coronavirus particles.

Intact, enveloped coronavirus particles vary widely in size and contour, and are thus refractory to study by traditional structural means such as X-ray crystallography. Electron microscopy (EM) overcomes some problems associated with particle variability and has been an important tool for investigating coronavirus ultrastructure. However, EM sample preparation requires that the specimen be dried onto a carbon support film before imaging, collapsing internal particle structure in the case of coronaviruses.

Single particle image reconstruction of the human recombinant Kv2.1 channel.

Kv2.1 channels are widely expressed in neuronal and endocrine cells and generate slowly activating K+ currents, which contribute to repolarization in these cells. Kv2.

T lymphocyte epitope mapping of porcine circovirus type 2.

Immunoreactive T lymphocyte epitopes within the ORF1, ORF2, and ORF 3 products of porcine circovirus type 2 (PCV2) were mapped. For this, overlapping linear 20-mer peptides were synthesized and tested for their ability to induce T lymphocyte proliferation in porcine peripheral blood mononuclear cells (PBMCs) isolated from experimentally PCV2-infected pigs. After a preliminary screening of 31 (ORF1), 23 (ORF2), and 10 (ORF3) peptides using PBMCs from 4 PCV2-infected pigs, none of the peptides appeared to be immunoreactive (stimulation index [SI] : 2) in all four pigs.

Electron microscopy of integrins.

Integrins are a family of heterodimeric, cell-surface receptors that mediate interactions between the cytoskeleton and the extracellular matrix. We have used electron microscopy and single-particle image analysis combined with molecular modeling to investigate the structures of the full-length integrin alpha(IIb)beta(3) and the ectodomain of alpha(V)beta(3) in a complex with fibronectin. The full-length integrin alpha(IIb)beta(3) is purified from human platelets by ion exchange and gel filtration chromatography in buffers containing the detergent octyl-beta-D-glucopyranoside, whereas the recombinant ectodomain of alpha(V)beta(3) is soluble in aqueous buffer.

Molecular beacon real-time PCR detection of swine viruses.

Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described.

Supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy.

Coronavirus particles are enveloped and pleomorphic and are thus refractory to crystallization and symmetry-assisted reconstruction. A novel methodology of single-particle image analysis was applied to selected virus features to obtain a detailed model of the oligomeric state and spatial relationships among viral structural proteins. Two-dimensional images of the S, M, and N structural proteins of severe acute respiratory syndrome coronavirus and two other coronaviruses were refined to a resolution of approximately 4 nm.

Cytokine and C-reactive protein profiles induced by porcine circovirus type 2 experimental infection in 3-week-old piglets.

The purpose of this study was to determine serum profiles of cytokines at a protein level and Creactive protein (CRP) during the development of postweaning multisystemic wasting syndrome (PMWS) in experimentally inoculated pigs. Levels of serum IFN-alpha, IL-6, IL-10, and CRP were examined for a 35-day period in 10 piglets experimentally infected with PCV2 at 3 weeks of age. Four of the infected piglets developed severe PMWS at 14 to 21 days post-infection (d.