Post-traumatic anxiety associates with failure of the innate immune receptor TLR9 to evade the pro-inflammatory NFκB pathway.

Post-traumatic anxiety notably involves inflammation, but its causes and functional significance are yet unclear. Here, we report that failure of the innate immune system Toll-like receptor 9 (TLR9) to limit inflammation is causally involved with anxiety-associated inflammation and that peripheral administration of specific oligonucleotide activators of TLR9 may prevent post-traumatic consequences in stressed mice. Suggesting involvement of NFκB-mediated enhancement of inflammatory reactions in the post-traumatic phenotype, we found association of serum interleukin-1ß increases with symptoms severity and volumetric brain changes in post-traumatic stress disorder patients. In predator scent-stressed mice, the moderate NFκB-activating oligonucleotides mEN101 and its human ortholog BL-7040, but not the canonic NFκB activator oligonucleotide ODN1826, induced anxiolytic effects. In stressed mice, peripherally administered mEN101 prevented delayed stress-inducible serum interleukin-1ß increases while limiting stress-characteristic hippocampal transcript modifications and the anxiety-induced EGR1-mediated neuronal activation. Attesting to the TLR9 specificity of this response, BL-7040 suppressed NFκB-mediated luciferase in transfected cells co-expressing TLR9, but not other TLRs. Furthermore, TLR9-/- mice were mEN101 and BL-7040 resistant and presented unprovoked anxiety-like behavior and anxiety-characteristic hippocampal transcripts. Our findings demonstrate functional relevance of TLR9 in protecting stressed mammals from overreacting to traumatic experiences and suggest using oligonucleotide-mediated peripheral TLR9 activation to potentiate the innate immune system and prevent post-traumatic inflammation and anxiety.

Retinal artery occlusion in a patient with factor V Leiden and prothrombin G20210A mutations.

Retinal artery occlusion is rare in young adults, and may be associated with hereditary thrombophilia. We present a 19-year-old male who was evaluated for central retinal artery occlusion and found to be homozygous for the factor V Leiden mutation and heterozygous for the prothrombin G20210A mutation. Anterior chamber paracenthesis resulted in dramatic improvement, but recurring loss of vision necessitated repeated paracenthesis and the addition of aspirin to standard anticoagulation treatment. The literature concerning hereditary thrombophilia and retinal artery occlusion is reviewed, and the synergistic effect of multiple risk factors is emphasized. Screening for hereditary thrombophilia should be considered for young people presenting with unexplained retinal artery occlusion.

Multiple epitope specificity of monoclonal antibodies to a single synthetic peptide: use in the characterization of the GP IIb-IIIa binding domain of von Willebrand factor.

Monoclonal antibodies have been induced against the synthetic peptide with sequence Tyr-Glu-Val-Val-Thr-Gly-Ser-Pro-Arg-Gly-Asp-Ser-Gln-Ser-Ser. This peptide represents residues Glu1737-Ser1750 of the mature von Willebrand factor (vWF) subunit and contains the sequence Arg-Gly-Asp, thought to be important in mediating binding to the platelet receptor glycoprotein (GP) IIb-IIIa complex. Twelve antibodies were obtained, eight of which bound to native vWF as well as to the peptide immunogen insolubilized onto agarose beads. These antibodies defined at least three distinct epitopes, as demonstrated by antibody interaction with peptides having a single phenylalanine substitution at each position in the sequence. In particular, two antibodies bound to epitopes on vWF that included one or more of the three residues (arginine, glycine, aspartic acid) thought to be involved in binding to GP IIb-IIIa, whereas one antibody bound to an epitope that did not include any of those residues. Nevertheless, the three antibodies cross-reacted with each other, a finding explained by the fact that the corresponding epitopes had at least two residues in common, namely Gly1741 and Ser1742. In spite of the cross-reactivity for binding to vWF, only the two antibodies whose epitopes included residues in the Arg-Gly-Asp sequence inhibited vWF interaction with GP IIb-IIIa. The third antibody had no inhibitory effect even though it was bound to an epitope located at a distance of only few residues on the amino terminal side of Arg-Gly-Asp. These results demonstrate that monoclonal antibodies raised against a single synthetic peptide with sequence limited to fifteen residues may exhibit distinct epitope specificity and may be used to define functional domains in macromolecules with a high degree of resolution.

Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells.

Endothelial cells and activated platelets express integrin-type receptors responsible for adhesion to fibrinogen. We have located distinct integrin-directed endothelial cell and platelet attachment sites on immobilized fibrinogen using a combination of synthetic peptides, fibrinogen fragments, and specific anti-peptide monoclonal antibodies. Endothelial cells exclusively recognize an Arg-Gly-Asp-containing site near the C-terminus of the alpha chain (alpha residues 572-574) but fail to recognize the Arg-Gly-Asp sequence in the N-terminal region of the same chain (alpha residues 95-97). In contrast, platelets do not require either Arg-Gly-Asp sequence for binding to intact fibrinogen and are capable of recognizing, in addition to the alpha 572-574 sequence, a site at the C-terminus of the gamma chain (gamma residues 400-411). These data suggest a molecular mechanism whereby platelets and endothelial cells interact with distinct sites on the fibrinogen molecule during hemostasis and wound healing.

Generation and characterization of specific antibodies directed to the binding domain of adhesive molecules.

Antibodies were selected in rabbits using as immunogens five synthetic peptides representing the putative cell-binding domains of von Willebrand factor (vWF), fibrinogen (Fn), fibronectin (Fn) and vitronectin. All peptide immunogens contained the sequence Arg-Gly-Asp, thought to be involved in ligand binding to platelet glycoprotein (GP) IIb-IIIa; two of them corresponded to two distinct regions in the alpha-chain of fibrinogen. All anti-peptide antibodies recognized the corresponding immunogen, and four of them, i.e. all except the anti-vitronectin antibody, reacted with the native macromolecule containing the cognate sequence. Each antibody was specific for the corresponding adhesive protein and did not cross-react with any of the others in spite of the presence of the common sequence Arg-Gly-Asp. Two murine monoclonal antibodies reacting with the vWF peptide and with native vWF were used to define the amino acid residues involved in the antigen-antibody interaction. These residues included the glycine and aspartic acid residues of the common Arg-Gly-Asp sequence, as well as other residues specific to the vWF sequence. The antibodies were potent inhibitors of vWF binding to GP IIb-IIIa. The present studies demonstrate that the putative cell binding domains of four adhesive proteins may be part of distinct antigenic epitopes with unique conformations in each of the four GP IIb-IIIa ligands. Moreover, they provide evidence that the Arg-Gly-Asp sequence is the only, or essential, GP IIb-IIIa binding site in the vWF molecule.

A relatively high frequency of severe (type III) von Willebrand's disease in Israel.

Sixty patients with von Willebrand's disease belonging to 34 unrelated families were classified into the various types of the disease by analysis of the multimer patterns of von Willebrand factor. Type I disease was observed in 62% of the families, a finding similar to the recently published British and Swedish series of patients. Type III (severe) von Willebrand's disease was observed more frequently in the Israeli group (29%) than in the other two series. Of the 16 patients with type III disease eight were Arabs living in Israel, the West Bank and the Gaza Strip (total population about 1.5 X 10(6). Thus, it appears that the frequency of type III disease is very high among Arabs (5.3/10(6]. Sibship analysis of all families affected by the type III mutant gene was compatible with an autosomal recessive mode of inheritance. An attempt was made to define carriers of type III disease and to distinguish them from type I patients and from healthy subjects. In 15 obligate carriers of type III disease mean levels of factor VIII clotting activity, of von Willebrand factor and of ristocetin cofactor were significantly higher than the corresponding mean values observed in 31 symptomatic and 12 asymptomatic type I patients, and in turn significantly lower than the corresponding mean values observed in 30 healthy subjects. Ristocetin cofactor was the best criterion for discrimination among carriers of type III disease, normal subjects and type I patients.