EphrinB2 induces tyrosine phosphorylation of NR2B via Src-family kinases during inflammatory hyperalgesia.

In recent years a role for EphB receptor tyrosine kinases and their ephrinB ligands in activity-dependent synaptic plasticity in the CNS has been identified. The aim of the present study was to test the hypothesis that EphB receptor activation in the adult rat spinal cord is involved in synaptic plasticity and processing of nociceptive inputs, through modulation of the function of the glutamate ionotropic receptor NMDA (N-methyl-D-aspartate). In particular, EphB receptor activation would induce phosphorylation of the NR2B subunit of the NMDA receptor by a Src family non-receptor tyrosine kinase. Intrathecal administration of ephrinB2-Fc in adult rats, which can bind to and activate EphB receptors and induce behavioral thermal hyperalgesia, led to NR2B tyrosine phosphorylation, which could be blocked by the Src family kinase inhibitor PP2. Furthermore animals pre-treated with PP2 did not develop behavioral thermal hyperalgesia following EphrinB2-Fc administration, suggesting that this pathway is functionally significant. Indeed, EphB1-Fc administration, which competes with the endogenous receptor for ephrinB2 binding and prevents behavioral allodynia and hyperalgesia in the carrageenan model of inflammation, also inhibited NR2B phosphorylation in this model. Taken together these findings support the hypothesis that EphB-ephrinB interactions play an important role in NMDA-dependent, activity-dependent synaptic plasticity in the adult spinal cord, inducing the phosphorylation of the NR2B subunit of the receptor via Src family kinases, thus contributing to chronic pain states.

Cardiac troponin I levels and clinical outcomes in patients with acute coronary syndromes: the potential role of early percutaneous revascularization.

OBJECTIVES: To establish the role of early catheter-based coronary intervention among patients sustaining acute coronary syndromes (ACS) stratified according to admission plasma troponin I (Tn-I) levels. BACKGROUND: The impact of early revascularization strategy on the clinical outcomes in patients with ACS stratified by plasma Tn-I levels has not been established. METHODS: In-hospital complications and long-term outcomes were assessed in 1,321 consecutive patients with non-ST elevation ACS undergoing early (within 72 h) catheter-based coronary interventions. Patients were grouped according to admission Tn-I levels. Group I (n = 1,099) had no elevated plasma Tn-I (<0.15 ng/ml), Group II (n = 95) had Tn-I level between 0.15 to 0.45 ng/ml and Group III (n = 127) had Tn-I >0.45 ng/ml. In-hospital composite cardiac events (death, Q-wave MI, urgent in-hospital revascularization) and 8 months clinical outcomes (death, MI, repeat revascularization or any cardiac event) were compared between the three groups. RESULTS: The rate of in-hospital composite cardiac events was 6.1% among patients with Tn-I >0.45 ng/ml, 1.0% in patients with Tn-I between 0.15-0.45 ng/ml and 3.1% in patients without elevated admission Tn-I (p = 0.09 between groups). There was no difference in hospital mortality (p = 0.25). At eight months of follow-up, there was no difference in out-of-hospital death (3.5%, 3.8% and 1.8%, p = 0.17, respectively), MI (2.6%, 3.8% and 2.9%, p = 0.94) or target lesion revascularization (9.0%, 8.3% and 11.5%, p = 0.47), and cardiac event-free survival was also similar between groups (p = 0.66). By multivariate analysis, Tn-I >0.45 ng/ml was independently associated with in-hospital composite cardiac events [odds ratio (OR) = 2.4, p = 0.04] but not with out-of-hospital clinical events up to eight months. CONCLUSIONS: In patients with ACS, early (within 72 h) catheter-based coronary intervention may attenuate the adverse prognostic impact of admission Tn-I elevation during eight months of follow-up despite a trend towards increased in-hospital composite cardiac events.

Replicase-mediated resistance to potato virus Y in transgenic tobacco plants.

Nicotiana tabacum 'Turkish Samsun NN' plants were transformed with nuclear inclusion b (NIb) gene sequences of potato virus Y, O strain (PVYO). The full-length construct included an additional in-frame initiation codon contiguous to the putative N-terminal amino acid codon and a stop codon replacing the C-terminal amino acid codon. Of 13 independently transformed lines, four yielded 37 (out of 100) plants in the R1 generation that were resistant to PVYO infection. Progeny of 13 out of 15 of R1 plants tested expressed resistance in the R2 generation. Conversely, 30 independently transformed tobacco lines expressing essentially the same sequence but deleted for the Gly-Asp-Asp (GDD) motif were not resistant. Two other constructs encoding either the 5'-deleted or 3'-truncated NIb gene, but harboring the GDD motif, conferred resistance to PVYO in some tobacco plants. Despite the high level of nucleotide and amino acid identity shared by strains PVYO and PVYN for the NIb gene, PVYN replication was found in all PVYO-resistant plants. However, plants of one R2 line showed reduced PVYN replication.

Differential expression of a ligand induced binding site (LIBS) by GPIIb-IIIa ligand recognition peptides and parenteral antagonists.

The glycoprotein (GP) IIb-IIIa complex is an attractive anti-platelet target for the prevention of thrombotic events associated with coronary artery disease. Although GPIIb-IIIa antagonists inhibit GPIIb-IIIa binding to its ligands, the interactions have not been fully clarified, particularly with respect to their ability to induce structural changes in the complex that lead to exposure of neoantigenic epitopes or ligand-induced binding sites (LIBS). In this study we used the anti-LIBS monoclonal antibody (mAb) D3 to further define the activation states of purified active and inactive GPIIb-IIIa. We also compared the data obtained in the purified system to that observed with intact human platelets. Active GPIIb-IIIa expressed significantly greater high-affinity D3 LIBS sites compared to the inactive form. In addition, the ligand recognition peptides RGDS and H12 caused increased expression of the D3 epitope, with RGDS eliciting a much more potent response. The response of the purified GPIIb-IIIa to these peptides paralleled that observed with human platelets. To explore whether the platelet antagonists abciximab, eptifibatide and tirofiban induced expression of the D3 LIBS site, a modified competitive ELISA was developed. Our data indicate that the use of purified GPIIb-IIIa with this ELISA system provides a reproducible approach for exploring the interactions between GPIIb-IIIa and its antagonists. Whereas abciximab caused no detectable increase in the expression of the D3 epitope on purified GPIIb-IIIa, eptifibatide, tirofiban, RGDS, and H12 induced differential expression of the high-affinity LIBS. Studies with intact platelets suggested that abciximab blocked the binding of the D3 and LIBS6 mAbs, and that the pre bound anti-LIBS D3 sterically hindered abciximab binding.

A new gene, Ny (tbr), for hypersensitivity to Potato virus Y from Solanum tuberosumMaps to Chromosome IV.

A diploid backcross population derived from a cross between Solanum tuberosum and Solanum berthaultiisegregated for monogenic dominant hypersensitivity to Potato virus Y(PVY). We propose the symbol Ny (tbr) for this locus because plants carrying this gene develop necrosis after inoculation with PVY and the allele originated in S. tuberosum. The gene mapped to chromosome IV between TG316 and TG208 at LOD=2.72. This location does not correspond to any other mapped resistance genes in potato.

The effects of surface chemistry and coagulation factors on fibrinogen adsorption from plasma.

Fibrinogen adsorption from plasma exhibits an unusual displacement phenomenon (the Vroman effect) in that decreases in adsorption occur after longer contact time or as the plasma concentration increases. Fibrinogen adsorption and the displacement effect were found to depend markedly on the nature of the surface, the time and temperature of adsorption, and to a lesser extent on certain contact activation factors. Displacement still occurred from plasmas lacking kininogens, factor IX, and prekallikrein, and from plasma that had been treated with BaSO4 to remove factors II, VII, IX, and X. Furthermore, displacement was also observed from fibrinogen solutions to which either albumin or hemoglobin had been added. In addition, competitive adsorption of binary protein mixtures was also shown to depend strongly on surface type. It therefore appears that fibrinogen adsorption from plasma is subject to similar if not identical competitive processes that occur in simpler protein mixtures. The final adsorption then reflects the influence of all the proteins in plasma, each competing for the limited number of adsorption sites according to the fundamental physical properties of surface activity and mass concentration.

Tissue culture methods for the screening and analysis of putative virus-resistant transgenic potato plants.

ABSTRACT Following regeneration, putative virus-resistant transgenic plants are usually transferred from tissue culture to a greenhouse or growth chamber to screen for resistance to infection and disease development using mechanical, graft, or insect vector inoculation methods. To reduce initial screening costs and time, we developed mechanical and graft inoculation methods suitable for tissue culture use. The in vitro methods were validated by comparing them with similar greenhouse screens using putative potato virus Y strain o (PVY degrees ) replicase-mediated resistant regenerants of the potato cultivar Atlantic. Five transgenic lines were tested, with similar results obtained from in vitro and greenhouse experiments. Two of the transgenic lines, A1 and A3, showed the greatest resistance to PVY degrees infection, as indicated by low enzyme-linked immunosorbent assay values and infection rates. In vitro mechanical inoculation methods were also used to infect wild-type tomato and tobacco plants with cucumber mosaic virus and potato virus Y. Potato plants were also infected with the phloem-restricted potato leafroll virus, a low-titer virus, using in vitro graft inoculation methods. These results suggest the potential usefulness of these simple, effective, and economical techniques for screening large numbers of putative virus-resistant plants.

Effects of fibrinogen residence time and shear rate on the morphology and procoagulant activity of human platelets adherent to polymeric biomaterials.

Fibrinogen readily adsorbs to the surface of biomaterials and, because of its demonstrated ability to support platelet adhesion and aggregation, plays a role in thrombotic events associated with the implantation of synthetic materials in the human body. Thus, understanding the factors influencing the interactions of fibrinogen with biomaterials, and how platelet responses are affected, is crucial for the development of synthetic materials exhibiting improved blood compatibility. In this study, the effects of fibrinogen residence time and shear rate on the procoagulant activity of adherent platelets, along with their morphologic status, as deduced from scanning electron microscopy, were investigated. To examine whether adherent platelets promoted the generation of thrombin, polymeric materials (polytetrafluoroethylene, polyethylene, and silicone rubber) preadsorbed with fibrinogen were exposed to platelet suspensions at different wall shear rates and then incubated with clotting factors for 5 minutes under static conditions. The amount of thrombin generated per platelet was calculated from the optical density of the color developed by adding substrate S-2238. Scanning electron microscopy images of the platelets revealed that the platelets exhibited different morphologies, depending on the shear rate and residence time of the adsorbed fibrinogen. Platelets ranged from their normal discoid shape observed primarily under static conditions, to that of fully spread platelets. Results from this study show that platelets, in the presence of shear forces, undergo activation on exposure to surfaces on which adsorbed fibrinogen has resided for short residence times rather than long residence times. Interestingly, studies examining the procoagulant responses of such adherent platelets demonstrated that the platelets attached to the fibrinogen coated materials did not promote significant thrombin generation. Such low prothrombinase activity of adherent platelets suggests that adsorbed fibrinogen, while capable of supporting platelet adhesion and spreading on biomaterials, does not necessarily enhance the procoagulant activity of adherent platelets.

Retropharyngeal infection in a pregnant diabetic. A case report.

A retropharyngeal infection occurred in a pregnant class C diabetic at 31 weeks' gestation. Early diagnosis, prompt therapy with antibiotics and possibly surgery for drainage if an abscess develops constitute optimal management.

Isolation and Partial Nucleic Acid Characterization of a New Isolate of Potato virus V with Distinct Biological and Serological Properties.

In vitro cultures of potato (Solanum tuberosum cv. Papa Amarillo) were imported into the United States and were subjected to routine pathogen screening for newly introduced varieties. These cultures indexed positive for several of the six viruses tested and thus were treated using standard methods for eliminating viruses in in vitro cultures. Although the therapy apparently freed the potato of the tested viruses, some of the cultures continued to exhibit mosaic symptoms when planted in the greenhouse. When examined by electron microscopy, these plants contained flexuous rods. Tested samples did not index positive with any potato virus-specific polyclonal antisera but reacted with the "universal" potyvirus monoclonal antibody. These results indicated that these samples were likely infected with an as yet undescribed potyvirus. Further investigation indicated that this virus is a new strain of Potato virus V (PVV) that is serologically distinct from the common strain. This is the first description of a distinct strain of PVV. This particular strain has diverged sufficiently from other isolates of PVV to no longer be detectable by some commonly used antisera, and therefore is a concern for the future restriction of PVV spread into regions of the world where it is not currently present.

A New Geminivirus Associated with Tomato in the State of São Paulo, Brazil.

The apical growth of about 20% of young tomato plants in observed fields near Campinas, State of São Paulo, Brazil, had yellow streaking of veins. Leaf symptoms developed into patches of yellow mosaic and the leaves became wavy. The whitefly Bemisia tabaci Genn. transmitted a pathogen from the infected tomato plants to healthy tomato and potato plants, reproducing the original symptoms in tomato. The apical leaves of infected potatoes showed yellow or green mottle that developed into leaf distortion with yellow blotches, symptoms indistinguishable from potato-deforming mosaic disease (2). DNA was extracted from these tomato and potato plants (1). Using DNA from the infected tomato plant, polymerase chain reaction (PCR) with the degenerate primer pair PAC1v1978/ PAV1c715 (1), which amplifies part of the rep gene (AC1 ORF), the common region (CR), and part of the cp gene (AV1 ORF), and with the primer pair PBC1v2039/PBV1c800, which amplifies part of BC1 ORF, CR, and part of BV1 ORF, gave virus-specific DNA fragments of the sizes expected from a whitefly-transmitted geminivirus. These were cloned and the complete nucleotide (sequences for DNA-A (pToYA, GenBank accession no. U79998) and DNA-B (pToYB, GenBank accession no. U80042) fragments obtained. Nucleotide identity between the CRs (184 nucleotides) was 90%, strongly indicating that those fragments correspond to a bipartite subgroup III geminivirus. PCR with the DNA from infected potato gave the expected size fragment for DNA-A. The partial sequence of the rep gene was 100% identical to the homologous sequence from the PCR fragment from the infected tomato. A search in the GenBank, EMBL, DDBJ, and PDB databases, using the BLAST program, found no identical geminivirus. The highest identity for the CR was 75% to tomato mottle geminivirus-Florida (ToMoV) and 74% to bean golden mosaic virus-Brazil. For the rep gene, the highest identity was 73% to tomato yellow leaf curl virus-Israel, an Old World geminivi-rus, followed by 71% to tomato golden mosaic virus-Brazil (TGMV) and ToMoV. For the cp gene, the highest identity was 86% to TGMV, followed by 83% to squash leaf curl geminivirus. Therefore, we propose the name tomato yellow vein streak geminivirus (ToYVSV) for this distinct virus (2). References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) J. A. C. Souza-Dias et al. Summa Phytopathol. 22:57, 1996.

The hand in Charcot-Marie-Tooth disease.

The upper extremity was evaluated in 68 symptomatic individuals previously diagnosed as having Charcot-Marie-Tooth disease. Consistent patterns of motor and sensory involvement were found noted as well as a predictable progression of neuromuscular deficits. Improved understanding of the clinical behaviour of this disorder is necessary to develop better treatment strategies.

Clearing the cervical spine of paediatric trauma patients.

OBJECTIVES: To review the evidence available for clearance of the cervical spine in children under 16 years of age after trauma, and to provide guidance to enable this to be practised safely. METHODS: A comprehensive literature review was carried out, and combined with a review of standard texts and liaison with experts. RESULTS: 241 papers were identified, of which 71 papers were thought possibly relevant. These were obtained and appraised. Children in whom there is concern about possible cervical spine injury may be divided into three groups. Alert, asymptomatic children with a normal examination may be clinically cleared without need for radiology. Children with cervical spine symptoms or signs require plain radiology in the first instance. Those areas that are poorly visualised or suspicious should be discussed with a paediatric radiologist and are likely to undergo computed tomography. Children with impaired conscious level require careful evaluation. Plain radiology, if normal, can be usefully complemented by early magnetic resonance imaging to exclude ligamentous and spinal cord damage. CONCLUSIONS: There is limited evidence to guide clinicians on how to clear the paediatric cervical spine. The approach suggested is similar to adult recommendations made elsewhere, and the differences are highlighted.

The effect of fluid shear and co-adsorbed proteins on the stability of immobilized fibrinogen and subsequent platelet interactions.

The conformation adopted by the plasma protein fibrinogen upon its adsorption onto synthetic surfaces has been implicated to play an important role in determining the blood compatibility of biomaterials. It has recently been shown that adsorbed fibrinogen undergoes biologically significant conformational changes with increasing residence time on the surface of selected biomaterials. The purpose of this study was to examine the effects of co-adsorbed proteins and shear forces on such time-dependent functional changes in fibrinogen adsorbed onto polyethylene (PE), polytetrafluoroethylene (PTFE), and silicone rubber (SR). Fibrinogen was adsorbed onto these materials for 1 min and then allowed to 'reside' on these surfaces for up to 2 h prior to assessing its biological activity. Changes in fibrinogen reactivity were determined by measuring the adhesion of 51Cr-labeled platelets and the ability of blood plasma to displace previously adsorbed fibrinogen. The magnitude of platelet adhesion to substrates adsorbed with pure fibrinogen increased in the presence of shear, compared with static conditions; at the lowest shear rate of 200 s(-1), samples exhibited a 20-fold increase in adhered platelet levels. In contrast, at a higher shear rate of 1000 s(-1), the three polymers supported minimal levels of platelet attachment. Surfaces pre-adsorbed with 10% plasma did not promote a significant increase in the number of adherent platelets with increasing shear when compared with the pure fibrinogen-coated substrates. The presence of shear also significantly altered the materials' ability to retain fibrinogen. Under static conditions, the amount of fibrinogen retained following incubation in blood plasma increased on all materials with increasing fibrinogen residence time. However, the materials varied distinctly in their ability to retain adsorbed fibrinogen with increasing fibrinogen residence time, shear rate, and nature of the co-adsorbed proteins. Thus, the results from this study indicate that fluid shear, residence time of the adsorbed protein, nature of the co-adsorbed proteins, and surface chemistry of the material all play important roles in influencing platelet-surface interactions and that they act in a complex manner to influence the biocompatibility of a material.

Changes in fibrinogen adsorbed to segmented polyurethanes and hydroxyethylmethacrylate-ethylmethacrylate copolymers.

Fibrinogen adsorption from blood to biomaterials may regulate platelet adhesion and thrombus formation because of fibrinogen's central role in the coagulation cascade and its ability to bind specifically to the platelet membrane glycoprotein (GP) IIb-IIIa. Adsorption of fibrinogen from blood plasma to many materials exhibits a maximum with respect to plasma dilution and exposure time (the Vroman effect). In this study fibrinogen adsorption to several polymers was examined to ascertain the influence of controlled changes in surface chemistry on the Vroman effect. The materials included hydroxyethylmethacrylate-ethylmethacrylate (HEMA/EMA) copolymers, Biomer, and a series of segmented polyurethanes (PEUs), two of which contained fluorinated chain extenders. Each material exhibited maximal adsorption of fibrinogen at intermediate plasma concentrations. Little effect of soft-segment type or molecular weight was observed and no significant differences in fibrinogen adsorption to the fluorinated PEUs were seen. Changes in the strength of fibrinogen attachment to these materials with time after adsorption were also assessed. Fibrinogen adsorbed for 1 min was displaced more readily by blood plasma than that adsorbed for 1 h, regardless of the material. The more hydrophobic polymers exhibited greater retention of adsorbed fibrinogen. In addition, the fraction of fibrinogen retained by polyethylene depended on the amount of fibrinogen adsorbed to the surface, being greatest when the surface loading was the least. These studies indicate that spreading or transition of adsorbed fibrinogen molecules from a weakly to tightly bound state is a general consequence of protein adsorption to solid surfaces.

The effects of temperature and buffer on fibrinogen adsorption from blood plasma to glass.

[125I]-Fibrinogen was used to measure the adsorption of fibrinogen from baboon plasma to two types of glass (Pyrex and a borosilicate glass) at 25 and 37 degrees C using two different buffers to dilute the plasma, the first being citrate-phosphate buffered saline (CPBSz) and the second isotonic Tris-saline (TRIS), both pH 7.4. In addition, the effects of hydration conditions, rinsing techniques, and glass-cleaning treatments on fibrinogen adsorption were evaluated. The data reveal that lower temperatures and the use of TRIS to dilute the plasma significantly enhance fibrinogen adsorption to both types of glass. As has been observed in the past, fibrinogen adsorption peaked at intermediate plasma concentrations on both Pyrex and borosilicate glass (the so-called Vroman effect), but almost twice as much fibrinogen adsorbed to glass when TRIS was used to dilute the plasma instead of CPBSz. Moreover, up to five times as much fibrinogen adsorbed to both types of glass at 25 degrees C compared with 37 degrees C. No effects of the rinsing technique or glass-cleaning treatment were observed.

Tissue factor expression by rat osteosarcoma cells adherent to tissue culture polystyrene and selected orthopedic biomaterials.

Tissue factor (TF), a transmembrane glycoprotein expressed by numerous cell types, plays a critical role in the initiation of blood coagulation at sites of vascular injury. Activated products of the coagulation cascade may then enhance the inflammatory responses associated with wound healing. In the present investigation the ability of rat osteosarcoma (ROS) cells to express TF activity was examined following their growth on tissue-culture polystyrene (TCPS) and selected orthopedic biomaterials (titanium and zirconium alloys, and stainless steel). ROS cells exhibited significant TF activity as evidenced by the conversion of Factor X to Factor Xa in the presence of TF, Factor VIIa, and Ca2+. Factor Xa concentrations ranged from 1.0 fM per cell at 10 min to 6.0 fM per cell after 60 min. Additionally, ROS cells stimulated with calcium ionophore (A23187) exhibited approximately twice the activity of non-stimulated cells when grown on TCPS but not on the metallic substrates. ROS cells (stimulated or unstimulated) adherent to the zirconium alloy generated lower amounts of Factor Xa compared to those bound to the other alloys and unstimulated cells grown on TCPS. These results indicate that ROS cells cultured on these synthetic surfaces differentially express procoagulant activity and that cells grown on TCPS, but not the metallic alloys, exhibit increased TF activity in response to stimulation by calcium ionophore. This procoagulant activity may potentiate subsequent inflammatory responses associated with the use of orthopedic biomaterials and thereby influence the tissue compatibility of the implant.