ALBERT

Description: The idea that noncrop habitat enhances pest control and represents a win–win opportunity to conserve biodiversity and bolster yields has emerged as an agroecological paradigm. However, while noncrop habitat in landscapes surrounding farms sometimes benefits pest predators, natural enemy responses remain heterogeneous across studies and effects on pests are inconclusive. The observed heterogeneity in species responses to noncrop habitat may be biological in origin or could result from variation in how habitat and biocontrol are measured. Here, we use a pest-control database encompassing 132 studies and 6,759 sites worldwide to model natural enemy and pest abundances, predation rates, and crop damage as a function of landscape composition. Our results showed that although landscape composition explained significant variation within studies, pest and enemy abundances, predation rates, crop damage, and yields each exhibited different responses across studies, sometimes increasing and sometimes decreasing in landscapes with more noncrop habitat but overall showing no consistent trend. Thus, models that used landscape-composition variables to predict pest-control dynamics demonstrated little potential to explain variation across studies, though prediction did improve when comparing studies with similar crop and landscape features. Overall, our work shows that surrounding noncrop habitat does not consistently improve pest management, meaning habitat conservation may bolster production in some systems and depress yields in others. Future efforts to develop tools that inform farmers when habitat conservation truly represents a win–win would benefit from increased understanding of how landscape effects are modulated by local farm management and the biology of pests and their enemies.

Description: Transfusion-related acute lung injury (TRALI), the leading cause of mortality associated with blood transfusion, usually results from passive transfer of antibodies present in donated blood to a patient. TRALI can be triggered by antibodies specific for Class I or Class II HLA antigens, human neutrophil antigens (HNA) and possibly other targets. For reasons not well understood, antibodies specific for the leukocyte antigen HNA-3a cause particularly severe, often fatal TRALI. It would be highly desirable therefore to be able to screen blood donors routinely for HNA-3a antibodies. HNA-3a/b antigens are carried on choline transporter-like protein 2 (CTL2), an apparent 10 membrane-spanning protein with 5 extracellular loops and N and C intracellular termini. The HNA-3a/b polymorphism is created by an R/Q substitution at position 154 in the first of these extracellular loops (Loop 1). In solid phase assays, about one-half of HNA-3a antibodies implicated in TRALI recognize Loop 1 peptides containing R154 (Type 1 antibodies). The remaining antibodies (Type 2) are non-reactive with peptides despite reacting well against full length CTL2. We studied reactions of Type 1 and Type 2 HNA-3a antibodies against soluble recombinant CTL2 fragments, human CTL2, mouse CTL2, and human/mouse CTL2 chimeras expressed in HEK293 cells to characterize the basis for HNA-3a antibody heterogeneity. The following observations were made:1) Only Type 1 antibodies react with detergent-solublized CTL2 in solid phase assays.2) A soluble recombinant fragment derived from the first extracellular (EC) loop (R154) of human CTL2 reacts only with Type 1 antibodies.3) Mouse CTL2 is 91% identical to human CTL2 and contains the R154 residue critical for HNA-3a expression. Type 1 antibodies recognize mouse CTL2, but Type 2 antibodies do not.4) Chimeric CTL2 containing human sequence in EC Loop 1 and mouse sequence in Loops 2-5 (H1M) reacts only with Type 1 antibodies. The reciprocal construct with mouse sequence in EC loop 1 and human sequence in Loops 2-5 (M1H) reacts with both Type 1 and Type 2 antibodies.5) Chimeric CTL2 containing human sequence in EC Loops 1-2 and mouse sequence in Loops 3-5 (H2M) reacts only with Type 1 antibodies.6) Chimeric CTL2 containing human sequence in EC Loops 1-3 and mouse sequence in Loops 4-5 (H3M) reacts with both Type 1 and Type 2 antibodies. These findings show that Loop 1 peptides containing R154 are sufficient for Type 1 antibodies to recognize CTL2 and the Type 1 epitope survives detergent solubilization of the protein. However, Type 2 antibodies require human sequence in EC Loops 1-3 for binding and the epitope they recognize does not survive detergent treatment. Moreover both Type 1 and Type 2 recognize the M1H chimera with the entire EC loop 1 sequence derived from mouse. The simplest explanation for these observations is that Type 2 HNA-3a antibodies need to contact human amino acid residues in EC Loop 3 in addition to Loop 1 for tight binding to CTL2. An alternative possibility is both Type 1 and 2 antibodies recognize only Loop1, but EC Loops 2 and 3 are required to hold Loop 1 in a configuration suitable for Type 2 antibody binding. In either case, it appears that at least the first 3 EC loops of CTL2 (R154) need to be in a configuration that closely mimics their natural state in the cell membrane in order to be recognized by Type 2 HNA-3a antibodies. Considerable ingenuity will be required to engineer a target capable of detecting both Type 1 and Type 2 HNA-3a antibodies in a format suitable for large-scale blood donor screening. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 266 Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal antibodies specific for fetal platelet antigens and is the most common cause of intracranial hemorrhage in full term infants. The antigen HPA-1a, carried on beta 3 integrin (GPIIIa), is the most common trigger for NAIT and the 2% of women who are HPA-1a-negative are at risk to produce such antibodies. Most HPA-1a antibodies causing NAIT can be easily detected but it is not rare for an HPA-1a negative mother who lacks detectable antibodies to give birth to an infant with thrombocytopenia. Several recent reports suggest that low avidity HPA-1a antibodies not detected by conventional serologic methods are responsible for some of these cases (Socher 2009, Bakchoul 2011). To examine this question, we retroactively analyzed a cohort of 3478 suspected NAIT cases referred for laboratory diagnosis. Among 677 HPA-1a-negative mothers, we identified 61 in whom HPA-1a-specific antibodies were not detected by conventional methods. Surface plasmon resonance (SPR) analysis enables ligand-receptor interaction to be studied in real time without washing the target. Using this approach, we studied reactions of IgG from the 61 archived serum samples against purified GPIIb/IIIa isolated from group O platelets and identified 18 samples that reacted preferentially with the HPA-1a-positive version of the integrin in comparison with HPA-1a-negative integrin. Information defining clinical status was obtained on 13 of these cases by follow-up communication. Seven cases had been referred because of neonatal thrombocytopenia. Platelet nadirs ranged from 8,000/ul to 141,000/ul (median 38,000/ul). Five of the 7 infants had bleeding and were given maternal platelet transfusions and/or IVIgG. Normal platelet counts were achieved after 4 to 70 days (median 7 days). One infant had a normal platelet count, however IVIgG had been given to its mother throughout pregnancy. The remaining 5 cases were referred in mid-pregnancy because an HPA-1a-negative sister previously gave birth to an infant with NAIT. Four of these infants had normal platelets at birth; one had mild TP (platelets 125,000/ul). Only 3 of 12 mothers typed for HLA were positive for HLA-DRB3*0101, a marker found in 〉95% of women who make “conventional” HPA-1a antibodies during pregnancy (p 〈 0.001). The ability of human antibodies to cause destruction of human platelets in vivo can be studied in the NOD/SCID mouse, which lacks xenoantibodies normally found in other species (Newman 2007). Serum from 4 mothers was available in quantities sufficient for mouse studies; three of the four maternal sera caused accelerated destruction of HPA-1a-positive, but not HPA-1a-negative platelets. These findings indicate that low-avidity HPA-1a antibodies not detectable by conventional serologic methods are made by a subset of women exposed to the HPA-1a antigen during pregnancy and that some, but not all of these antibodies are capable of causing NAIT, which is usually mild, but can be severe. Women negative for HLA-DRB3*0101 may be especially prone to produce antibodies of this type. Maternal-fetal incompatibility for platelet antigens other than HPA-1a is very common and many apparent NAIT cases not involving HPA-1a go unresolved. The possibility that low avidity antibodies specific for antigens other than HPA-1a are responsible for some of these cases deserves study. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factor and prothrombinase during early stages of the procoagulant response. It is produced in alternatively spliced isoforms. In humans, TFPIα is within platelets and released upon activation and also is present in plasma, while TFPIβ is bound to the endothelial surface. Mice differ from humans in that they do not have TFPIα in plasma, making them an optimal model system for study of the function of platelet TFPIα. Studies of murine hematopoietic cell TFPI using fetal liver transplantation models have found that platelet TFPI dampens platelet accumulation in a growing thrombus following vascular injury and alters bleeding severity in mice with hemophilia. However, results of these studies are potentially confounded by altered TFPI expression on other hematopoietic cells, such as monocytes. To better understand the function of platelet TFPI, murine models of total TFPIα deletion (TFPIα KO), of platelet-specific TFPIα deletion (pTFPIα KO) and platelet-specific TFPIα over expression (pTFPIα high) were produced and characterized. Methods: A TFPIα deleted mouse was generated by inserting a stop codon within exon 10 of the TFPI gene effectively blocking translation of the highly conserved basic C-terminal region of TFPIα, but not altering TFPIβ. The PF4-Cre transgene was bred into the floxed-TFPI(K1) mouse to produce mice specifically lacking platelet TFPIα. A transgenic mouse over expressing platelet TFPIα under control of the GP1b promoter was produced. Mice were characterized for platelet and plasma TFPI antigen by ELISA, for activity by TF-FVIIa inhibition and whole blood platelet aggregation, and for bleeding by tail clip. Results: All 3 strains were viable and reproduced. Platelet TFPI antigen was decreased approximately 25-fold in the TFPIα KO and pTFPIα KO mice and elevated approximately 5-fold in the pTFPIα high mice with corresponding changes in TF-FVIIa inhibitory activity. Plasma TFPI antigen was no different between pTFPIα KO (20.6±4.4 nM) and wild type (21.5±6.2 nM) littermates. Plasma thrombin-antithrombin complex levels were not elevated in any strain. In tail clip bleeding time assays, there was no difference between pTFPIα KO (0.23±0.16 mg/ml Hb) and wild type (0.23±0.37 mg/ml Hb), but there was a trend towards increased blood loss in pTFPIα high mice (1.25±1.8 mg/ml Hb). The pTFPIα high mice also had altered whole blood platelet aggregation that was evident primarily in a prolonged time for initiation of aggregation with 6.50uM ADP (81±12 sec vs. 6.1±5.6 sec for control) or 0.8ug/ml collagen (35±17 sec vs. 16±21 sec for control). Finally, the pTFPIα high transgene was bred into TFPI heterozygous mice which were then bred to determine if over expression of platelet TFPIα would rescue the TFPI null embryonic lethal phenotype. However, no live TFPI null pups were observed following genotyping of over 50 pups from these matings. Conclusions: Three new mouse models of altered TFPI expression have been made providing valuable tools to study the role of platelet TFPI in bleeding and clotting disorders. Plasma TFPI is unchanged in mice lacking platelet TFPI demonstrating that platelet TFPI does not contribute to plasma TFPI in mice. Platelet TFPIα antigen and activity correlate in all three models. Deletion of the basic C-terminal region in TFPIα in the TFPIα KO mice produces a TFPIα hypomorphic mouse, with only trace amounts of truncated TFPIα present in platelets. This lack of TFPIα does not produce a generalized procoagulant state. The 5-fold excess TFPIα antigen in the pTFPIα high mice substantially delays the onset of platelet aggregation in whole blood assays initiated with collagen or ADP. Results from tail clip bleeding assays suggest that over expression of platelet TFPIα may prolong bleeding in adult mice, but it is not sufficient to rescue the embryonic lethality of TFPI null mice. Disclosures Mast: Novo Nordisk: Honoraria, Research Funding.

Description: A major type of drug-induced immune thrombocytopenia (DITP), characterized by an acute, sometimes life-threatening drop in platelets following drug exposure, is caused by a unique type of antibody that recognizes its target on a platelet membrane glycoprotein, usually αIIb/β3 integrin [glycoprotein (GP) IIb/IIIa], only when the sensitizing drug is present in soluble form. Quinine (Qn) is the prototypic drug that causes this complication, but many other drugs have been implicated. It is widely thought that drug-dependent, platelet-reactive antibodies (DDAbs) characteristic of DITP recognize drug-induced structural modifications of platelet glycoproteins (GP), but this has not been confirmed experimentally. The mechanism responsible for DDAb binding is difficult to study with human DDAbs, which are often poly-specific and in short supply. We used newly-developed, Qn-dependent monoclonal antibodies (IgG1 mAbs 314.1, 314.3) that recognize the N-terminus of GPIIb and closely mimic the serologic behavior of antibodies from patients with Qn-induced immune thrombocytopenia (Blood 2009; 113;1105-11) as an alternative tool for studying the molecular basis of drug-dependent antibody binding. Previous studies failed to demonstrate a docking site for Qn in domains of GPIIb/IIIa that are known targets for the "314" mAbs and for human Qn-dependent antibodies. Therefore we examined an alternative possibility - that binding of drug to antibody might be the first step in DDAb binding. For this purpose, Qn was perfused over the "314" mAbs immobilized on Biacore chips and surface plasmon resonance (SPR) signals were recorded. Findings showed that Qn binds specifically to both mAbs with high affinity (Kd about 30 nM) and with 2:1 stoichiometry (Qn to mAb), consistent with recognition of Qn by complementarity determining regions (CDR) of the mAbs. To characterize monovalent binding of mAb to GPIIb/IIIa, purified integrin in 0.1% triton X-100 was perfused over immobilized mAb 314.1 and SPR signals recorded. Weak, but specific binding was observed in the absence of Qn (Kd 11 uM) that was enhanced 5-fold (Kd 2.2 uM) when Qn was present. Kds for Qn-dependent binding of mAb 314.1 (bivalent interaction) and its Fab fragment (monovalent interaction) to GPIIb/IIIa were determined by flow cytometry using labeled antibody and Fab under conditions that did not require washing prior to direct measurement of platelet bound IgG and Fab. Weak Fab binding was observed in the presence of Qn (≈19 uM) but with intact IgG the effective Kd was reduced to 0.15 nM, reflecting a 100,000-fold increase in avidity. Together with studies that have failed to demonstrate any docking site for Qn on GPIIb/IIIa, the findings support a model in which DDAb-GPIIb/IIIa interaction starts with binding of drug to the antibody CDR, leading to a structural change that markedly increases the avidity of antibody for a weak autoantigen. A requirement for bivalent antibody-target interaction to achieve tight binding could explain why DDAbs almost invariably recognize GPIIb/IIIa or GPIb/IX, the most highly expressed platelet glycoproteins. How this type of DDAb is induced by drug remains uncertain but the findings are consistent with a model in which sensitization starts with drug-induced modification of a B cell receptor that increases its affinity for a weak autoantigen. Disclosures Aster: BLOODCENTER OF WISCONSIN: Patents & Royalties: A patent application has been filed based partly on these findings (Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591).

Description: Drug-induced immune thrombocytopenia is caused by drug-dependent antibodies (DDAbs) that bind tightly to platelet glycoproteins only when drug is present. How drugs mediate this interaction is not yet resolved. Several studies indicate that sites recognized by DDAbs tend to cluster in specific structural domains, suggesting they may recognize a limited number of distinct epitopes. To address this issue, we characterized the binding sites for 16 quinine-dependent antibodies thought on the basis of preliminary studies to be possibly specific for a single epitope on glycoprotein IIIa (GPIIIa). Fourteen of the antibodies reacted with a 29-kDa GPIIIa fragment comprising only the GPIIIa hybrid and plextrin-semaphorin-integrin homology domains. However, studies with mutant GPIIIa and the blocking monoclonal antibody AP3 showed that the 14 DDAbs recognize at least 6 and possibly more distinct, but overlapping, structures involving GPIIIa residues 50 to 66. The findings suggest that even antibodies specific for restricted domains on a target glycoprotein may each have a slightly different fine specificity; ie, “unique” epitopes recognized by DDAbs may be rare or nonexistent. The observations are consistent with a recently proposed model in which drug reacts noncovalently with both target protein and antibody to promote binding of an otherwise nonreactive immunoglobulin.

Description: The IIb-IIIa glycoprotein complex is a favored target for allo-, auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large quantities and maintained in solution without detergent could provide a useful experimental tool for the study of platelet-reactive antibodies, but previous attempts to produce such a construct have yielded only small quantities of the end product. Using a baculovirus expression system and the dual-promoter transfer vector P2Bac, we were able to express soluble GPIIb-IIIa complex (srGPIIb-IIIa) lacking cytoplasmic and transmembrane domains in quantities of about 1,000 μg/L, about 40 times greater than reported previously. The high yield achieved may be related to inclusion of the entire extracellular region of the GPIIb light chain in the construct. srGPIIb-IIIa reacts spontaneously with fibrinogen, and this interaction is totally inhibited by the peptide RGDS. Reactions of 24 GPIIb-IIIa–specific antibodies evaluated (12 monoclonal, 3 allo-specific, 3 auto-specific, and 6 drug-dependent) with srGPIIb-IIIa were indistinguishable from reactions with platelet GPIIb-IIIa. Thus, srGPIIb-IIIa spontaneously assumes an active, ligand-binding conformation and contains epitopes for all monoclonal and human antibodies tested to date. srGPIIb-IIIa can be produced in large quantities, can readily be modified by site-directed mutagenesis, and should facilitate identification of epitopes recognized by GPIIb-IIIa–specific antibodies, study of the mechanism(s) by which certain drugs promote antibody binding to GPIIb-IIIa in drug-induced thrombocytopenia and structure-function relationships of GPIIb-IIIa. © 1998 by The American Society of Hematology.

Description: The IIb-IIIa glycoprotein complex is a favored target for allo-, auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large quantities and maintained in solution without detergent could provide a useful experimental tool for the study of platelet-reactive antibodies, but previous attempts to produce such a construct have yielded only small quantities of the end product. Using a baculovirus expression system and the dual-promoter transfer vector P2Bac, we were able to express soluble GPIIb-IIIa complex (srGPIIb-IIIa) lacking cytoplasmic and transmembrane domains in quantities of about 1,000 μg/L, about 40 times greater than reported previously. The high yield achieved may be related to inclusion of the entire extracellular region of the GPIIb light chain in the construct. srGPIIb-IIIa reacts spontaneously with fibrinogen, and this interaction is totally inhibited by the peptide RGDS. Reactions of 24 GPIIb-IIIa–specific antibodies evaluated (12 monoclonal, 3 allo-specific, 3 auto-specific, and 6 drug-dependent) with srGPIIb-IIIa were indistinguishable from reactions with platelet GPIIb-IIIa. Thus, srGPIIb-IIIa spontaneously assumes an active, ligand-binding conformation and contains epitopes for all monoclonal and human antibodies tested to date. srGPIIb-IIIa can be produced in large quantities, can readily be modified by site-directed mutagenesis, and should facilitate identification of epitopes recognized by GPIIb-IIIa–specific antibodies, study of the mechanism(s) by which certain drugs promote antibody binding to GPIIb-IIIa in drug-induced thrombocytopenia and structure-function relationships of GPIIb-IIIa. © 1998 by The American Society of Hematology.

Description: Striacosta albicosta is a crop pest that causes economic damage in the United States and Canada. Only maize and dry beans are shown to be suitable hosts, since larval development is incomplete on other hosts. The objective of this study was to describe the developmental parameters of immature stages of S. albicosta feeding on dry beans, non-Bt, Cry1F, and Vip3A maize. For Vip3A, mortality was 100% after 24 h. Larvae feeding on non-Bt maize had the highest larval survival (70.6%) compared to the other hosts. Maize expressing Cry1F had higher survival (31.3%) than dry beans (26.0%). Larvae feeding on dry beans had a significantly faster total development time (74.8 days), compared to 92.5 days for non-Bt and 96.2 days for Cry1F. All larvae developed through seven instars. Pupae from larvae that had fed on non-Bt maize were significantly heavier than pupae from other hosts. An understanding of S. albicosta immature development on various host plants is needed to improve recommendations for effective scouting, treatment timing, and economic thresholds. Differential development can result in an extended adult emergence period, and possibly result in assortative mating between Bt susceptible and resistant populations, which violates the assumption of random mating necessary for current resistance management strategies for Bt maize. Therefore, understanding the impact of host plant and transgenic traits on aspects of pest biology will aid in developing effective integrated pest management and insect resistance management strategies for this pest.