Posttransplant lymphoproliferative disorders in neuronal xenotransplanted macaques

Posttransplant lymphoproliferative disorders (PTLDs) are a heterogeneous group of lymphoid proliferations that occur in the setting of depressed T-cell function due to immunosuppressive therapy used following solid organ transplantation, hematopoietic stem cell transplantation, and also xenotransplantation. In the present study, 28 immunosuppressed parkinsonian Macaca fascicularis were intracerebrally injected with wild-type or CTLA4-Ig transgenic porcine xenografts to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Nine of 28 (32%) immunosuppressed primates developed masses compatible with PTLD, located mainly in the gastrointestinal tract and/or nasal cavity. The masses were classified as monomorphic PTLD according to the World Health Organization classification. Immunohistochemistry and polymerase chain reaction (PCR) analyses revealed that the PTLDs were associated with macaca lymphocryptovirus as confirmed by double-labeling immunohistochemistry for CD20 and Epstein-Barr nuclear antigen 2 (EBNA-2), where the viral protein was located within the CD20+ neoplastic B cells. In sera from 3 distinct phases of the experimental life of the primates, testing by quantitative PCR revealed a progression of the viral load that paralleled the PTLD progression and no evidence of zoonotic transmission of porcine lymphotropic herpesvirus through xenoneuronal grafts. These data suggest that monitoring the variation of macaca lymphocryptovirus DNA in primates could be used as a possible early diagnostic tool for PTLD progression, allowing preemptive treatment such as immunosuppression therapy reduction

Anthrax Toxin Receptor 2 Determinants that Dictate the pH Threshold of Toxin Pore Formation

The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH ∼6.0 or below when it is bound to ANTXR1, but only at pH ∼5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation

Delayed Toxicity Associated with Soluble Anthrax Toxin Receptor Decoy-Ig Fusion Protein Treatment

Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream

Sheep Updates 2003 - Husbandry

This session covers seven papers from different authors:1. Setting up a successful, low input feedlot Paul Barrett, ‘Bimberdong’ Jerramungup 2. Effective mineral supplementation of sheep Kevin Bell, Sheep Management and Production Consultants, Kojonup, WA 3. Genetic benchmarking for WA sheep producers J. Greeff, L. Butler, S. Brown, K. Hart and A. Gray Department of Agriculture Western Australia 4. Does selecting sheep for low WEC reduce scouring? John Karlsson, Johan Greeff and Paula Coombe, Department of Agriculture Western Australia 5. Summer quarters for sheep - stubbles Ron McTaggart, Department of Agriculture Western Australia, Albany 6. Thinking about breeding Easy Care Sheep? David Scobie, AgResearch PO Box 60 Lincoln, 8152, New Zealand 7. Increasing lambing percentages and lamb survival Sandy White, Department of Agriculture Western Australia, Jerramungu

The Structure of Tumor Endothelial Marker 8 (TEM8) Extracellular Domain and Implications for Its Receptor Function for Recognizing Anthrax Toxin

Anthrax toxin, which is released from the Gram-positive bacterium Bacillus anthracis, is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds a receptor on the surface of the target cell and further assembles into a homo-heptameric pore through which EF and LF translocate into the cytosol. Two distinct cellular receptors for anthrax toxin, TEM8/ANTXR1 and CMG2/ANTXR2, have been identified, and it is known that their extracellular domains bind PA with low and high affinities, respectively. Here, we report the crystal structure of the TEM8 extracellular vWA domain at 1.7 Å resolution. The overall structure has a typical integrin fold and is similar to that of the previously published CMG2 structure. In addition, using structure-based mutagenesis, we demonstrate that the putative interface region of TEM8 with PA (consisting of residues 56, 57, and 154–160) is responsible for the PA-binding affinity differences between the two receptors. In particular, Leu56 was shown to be a key factor for the lower affinity of TEM8 towards PA compared with CMG2. Because of its high affinity for PA and low expression in normal tissues, an isolated extracellular vWA domain of the L56A TEM8 variant may serve as a potent antitoxin and a potential therapeutic treatment for anthrax infection. Moreover, as TEM8 is often over-expressed in tumor cells, our TEM8 crystal structure may provide new insights into how to design PA mutants that preferentially target tumor cells

Uptake of synthetic low density lipoprotein by leukemic stem cells — a potential stem cell targeted drug delivery strategy

Chronic Myeloid Leukemia (CML) stem/progenitor cells, which over-express Bcr-Abl, respond to imatinib by a reversible block in proliferation without significant apoptosis. As a result, patients are unlikely to be cured owing to the persistence of leukemic quiescent stem cells (QSC) capable of initiating relapse. Previously, we have reported that intracellular levels of imatinib in primary primitive CML cells (CD34<sup>+</sup>38<sup>lo/−</sup>), are significantly lower than in CML progenitor cells (total CD34<sup>+</sup>) and leukemic cell lines. The aim of this study was to determine if potentially sub-therapeutic intracellular drug concentrations in persistent leukemic QSC may be overcome by targeted drug delivery using synthetic Low Density Lipoprotein (sLDL) particles. As a first step towards this goal, however, the extent of uptake of sLDL by leukemic cell lines and CML patient stem/progenitor cells was investigated. Results with non-drug loaded particles have shown an increased and preferential uptake of sLDL by Bcr-Abl positive cell lines in comparison to Bcr-Abl negative. Furthermore, CML CD34<sup>+</sup> and primitive CD34<sup>+</sup>38<sup>lo/−</sup> cells accumulated significantly higher levels of sLDL when compared with non-CML CD34<sup>+</sup> cells. Thus, drug-loading the sLDL nanoparticles could potentially enhance intracellular drug concentrations in primitive CML cells and thus aid their eradication

Tumor Endothelium Marker-8 Based Decoys Exhibit Superiority over Capillary Morphogenesis Protein-2 Based Decoys as Anthrax Toxin Inhibitors

Anthrax toxin is the major virulence factor produced by Bacillus anthracis. The toxin consists of three protein subunits: protective antigen (PA), lethal factor, and edema factor. Inhibition of PA binding to its receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) can effectively block anthrax intoxication, which is particularly valuable when the toxin has already been overproduced at the late stage of anthrax infection, thus rendering antibiotics ineffectual. Receptor-like agonists, such as the mammalian cell-expressed von Willebrand factor type A (vWA) domain of CMG2 (sCMG2), have demonstrated potency against the anthrax toxin. However, the soluble vWA domain of TEM8 (sTEM8) was ruled out as an anthrax toxin inhibitor candidate due to its inferior affinity to PA. In the present study, we report that L56A, a PA-binding-affinity-elevated mutant of sTEM8, could inhibit anthrax intoxication as effectively as sCMG2 in Fisher 344 rats. Additionally, pharmacokinetics showed that L56A and sTEM8 exhibit advantages over sCMG2 with better lung-targeting and longer plasma retention time, which may contribute to their enhanced protective ability in vivo. Our results suggest that receptor decoys based on TEM8 are promising anthrax toxin inhibitors and, together with the pharmacokinetic studies in this report, may contribute to the development of novel anthrax drugs