Increased IL-4 and decreased regulatory cytokine production following relocation of Icelandic horses from a high to low endoparasite environment

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation into continental Europe where Culicoides are present, >or=50% of Icelandic horses (1st generation) develop IBH but

Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.

Harry Potter and the Theory of Things

This thesis investigates the boundaries between body and object in J.K. Rowling’s Harry Potter series, seven children’s literature novels published between 1997 and 2007. Lord Voldemort, Rowling’s villain, creates Horcruxes—objects that contain fragments of his soul—in order to ensure his immortality. As vessels for human soul, these objects rupture the boundaries between body and object and become “things.” Using contemporary thing theorists including John Plotz and materialists Jean Baudrillard and Walter Benjamin, I look at Voldemort’s Horcruxes as transgressive, liminal, unclassifiable entities in the first chapter. If objects can occupy the juncture between body and object, then bodies can as well. Dementors and Inferi, dark creatures that Rowling introduces throughout the series, live devoid of soul. Voldemort, too, becomes a thing as he splits his soul and creates Horcruxes. These soulless bodies are uncanny entities, provoking fear, revulsion, nausea, and the loss of language. In the second chapter, I use Sigmund Freud’s theorization of the uncanny as well as literary critic Kelly Hurley to investigate how Dementors, Inferi, and Voldemort exist as body-turned-object things at the juncture between life and death. As Voldemort increasingly invests his immaterial soul into material objects, he physically and spiritually degenerates, transforming from the young, handsome Tom Marvolo Riddle into the snake-like villain that murdered Harry’s parents and countless others. During his quest to find and destroy Voldemort’s Horcruxes, Harry encounters a different type of object, the Deathly Hallows. Although similarly accessing boundaries between body/object, life/death, and materiality/immateriality, the three Deathly Hallows do not transgress these boundaries. Through the Deathly Hallows, Rowling provides an alternative to thingification: objects that enable boundaries to fluctuate, but not breakdown. In the third chapter, I return to thing theorists, Baudrillard, and Benjamin to study how the Deathly Hallows resist thingification by not transgressing the boundaries between body and object.

Equine insect bite hypersensitivity: immunoblot analysis of IgE and IgG subclass responses to Culicoides nubeculosus salivary gland extract

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of Culicoides and sometimes Simulium spp. The allergens causing IBH are probably salivary gland proteins from these insects, but they have not yet been identified. The aim of our study was to identify the number and molecular weight of salivary gland extract (SGE) proteins derived from Culicoides nubeculosus which are able to bind IgE antibodies (ab) from the sera of IBH-affected horses. Additionally, we sought to investigate the IgG subclass (IgGa, IgGb and IgGT) reactivity to these proteins. Individual IgE and IgG subclass responses to proteins of C. nubeculosus SGE were evaluated by immunoblot in 42 IBH-affected and 26 healthy horses belonging to different groups (Icelandic horses born in Iceland, Icelandic horses and horses from different breeds born in mainland Europe). Additionally, the specific antibody response was studied before exposure to bites of Culicoides spp. and over a period of 3 years in a cohort of 10 Icelandic horses born in Iceland and imported to Switzerland. Ten IgE-binding protein bands with approximate molecular weights of 75, 66, 52, 48, 47, 32, 22/21, 19, 15, 13/12 kDa were found in the SGE. Five of these bands bound IgE from 50% or more of the horse sera. Thirty-nine of the 42 IBH-affected horses but only 2 of the 26 healthy horses showed IgE-binding to the SGE (p<0.000001). Similarly, more IBH-affected than healthy horses had IgGa ab binding to the Culicoides SGE (19/22 and 9/22, respectively, p<0.01). Sera of IBH-affected horses contained IgE, IgGa and IgGT but not IgGb ab against significantly more protein bands than the sera of the healthy horses. The cohort of 10 Icelandic horses confirmed these results and showed that Culicoides SGE specific IgE correlates with onset of IBH. IBH-affected horses that were born in Iceland had IgGa and IgGT ab (p< or =0.01) as well as IgE ab (p=0.06) against a significantly higher number of SGE proteins than IBH-affected horses born in mainland Europe. The present study shows that Culicoides SGE contains at least 10 potential allergens for IBH and that IBH-affected horses show a large variety of IgE-binding patterns in immunoblots. These findings are important for the future development of a specific immunotherapy with recombinant salivary gland allergens.

Evaluation of an in vitro sulphidoleukotriene release test for diagnosis of insect bite hypersensitivity in horses

REASONS FOR PERFORMING STUDY: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. OBJECTIVES: The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. METHODS: A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluated, and 32 horses were tested repeatedly over one year. The kappa reliability test was used to assess agreement of the test results with different insect extracts. RESULTS: Horses with IBH had significantly higher sLT release than controls with C. nubeculosus and S. vittatum. The highest diagnostic sensitivity and specificity levels were attained when using adult C. nubeculosus extracts with the CAST (78% and 97%, respectively), suggesting that most horses with IBH are sensitised against Culicoides allergens. A proportion of IBH-affected horses was found to be sensitised to allergens of Simulium spp. in addition to those of C. nubeculosus. The CAST with C. nubeculosus had positive and negative predictive values > or = 80% for a true prevalence of IBH of 12-52%. In the follow-up study, the proportion of IBH-affected horses with a positive test result ranged from 90% in November to 68% in March. Severity of clinical signs or age did not influence test results significantly. However, IBH-affected males achieved significantly more positive test results than IBH-affected females. CONCLUSIONS: The CAST with adult C. nubeculosus has high specificity and good sensitivity for diagnosis of IBH. Horses with IBH are mainly sensitised to Culicoides allergens, and some horses are additionally also sensitised to allergens in Simulium spp. POTENTIAL RELEVANCE: The CAST is likely to be a useful test for diagnosis of IBH, even allowing the identification of IBH-affected but asymptomatic horses. This test may also help in further characterisation of allergens involved in this condition.

Production of monoclonal antibodies specific for native equine IgE and their application to monitor total serum IgE responses in Icelandic and non-Icelandic horses with insect bite dermal hypersensitivity

Immunoglobulin E forms a minor component of serum antibody in mammals. In tissues IgE is bound by FcvarepsilonRI receptors on the surface of mast cells and mediates their release of inflammatory substances in response to antigen. IgE and mast cells have a central role in immunity to parasites and the pathogenesis of allergic diseases in horses and other mammals. This paper describes the production of several novel monoclonal antibodies that detect native equine IgE in immunohistology, ELISA and Western blotting. An antigen capture ELISA to quantify equine IgE in serum has been developed using two of these antibodies. The mean serum IgE concentration of a group of 122 adult horses was 23,523ng/ml with a range of 425-82,610ng/ml. Total serum IgE of healthy horses was compared with that of horses with insect bite dermal hypersensitivity (IBDH) an allergic reaction to the bites of blood feeding insects of Culicoides or Simulium spp. IBDH does not occur in Iceland where Culicoides spp. are absent, but following importation into mainland Europe native Icelandic horses have an exceptionally high incidence of this condition. In the present study Icelandic horses with IBDH had significantly higher total IgE than healthy Icelandic horse controls (P<0.05). By contrast in horses of other breeds the difference in total serum IgE between those affected with IBDH and healthy controls was not statistically significant. Total serum IgE was also monitored in a cohort of Icelandic horses prior to import into Switzerland and for a period of 3 years thereafter. High levels of serum IgE were present in all horses at the start of the study but dropped in the first year after import. Thereafter the total serum IgE remained low in Icelandic horses that remained healthy but rose significantly (P<0.05) in those that developed IBDH. These results support the conclusion that IBDH is a type I hypersensitivity response to insect allergens but indicate that IBDH in Icelandic horses may have a different pathogenesis from the same condition in other breeds.

Glycoprotein Ib cross-linking/ligation on echicetin-coated surfaces or echicetin-IgMkappa in stirred suspension activates platelets by cytoskeleton modulated calcium release

Cross-linking platelet GPIb with the snake C-type lectin echicetin provides a specific technique for activation via this receptor. This allows GPIb-dependent mechanisms to be studied without the necessity for shear stress-induced binding of von Willebrand factor or primary alpha(IIb)beta(3) involvement. We already showed that platelets are activated, including tyrosine phosphorylation, by echicetin-IgMkappa-induced GPIb cross-linking. We now investigate the mechanism further and demonstrate that platelets, without modulator reagents, spread directly on an echicetin-coated surface, by a GPIb-specific mechanism, causing exocytosis of alpha-granule markers (P-selectin) and activation of alpha(IIb)beta(3). This spreading requires actin polymerization and release of internal calcium stores but is not dependent on external calcium nor on src family tyrosine kinases. Cross-linking of GPIb complex molecules on platelets, either in suspension or via specific surface attachment, is sufficient to induce platelet activation.

Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper)

Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein.

Translocation of GPIb and Fc receptor gamma-chain to cytoskeleton in mucetin-activated platelets

Recent studies have implied that GPIb-IX-V as well as functioning as an adhesion receptor may also induce signaling to mediate binding of platelets to damaged vessel wall to prevent bleeding. Reorganization of the cytoskeleton and redistribution of platelet structural proteins and signaling molecules are thought to be important in this early activation process, though the molecular mechanisms remain to be fully defined. In this study, we have used mucetin, a snake venom lectin protein that activates platelets via GPIb, to study the redistribution of GPIb in platelets. In unstimulated platelets, a minor portion of GPIb localized to Triton-insoluble cytoskeleton fractions (TIC). This portion increased considerably after platelet activation by mucetin. We also find increased contents of the FcRgamma chain in TIC. Anti-GPIb antibodies, mocarhagin or cytochalasin D completely inhibited the cytoskeletal translocation. In addition, BAPTA-AM, a cytoplasmic calcium chelator, strongly inhibited this process. On the other hand, inhibitors of alphaIIbbeta3, PLCgamma, PKC, tyrosine kinases, ADP receptor, PI3-kinase or EDTA are effective in preventing GPIb relocation in convulxin- but not in mucetin-activated platelets. We propose that cytoskeletal translocation of GPIb is upstream of alphaIIbbeta3 activation and cross-linking of GPIb is sufficient to induce this event in mucetin-activated platelets.

Ophioluxin, a convulxin-like C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI

Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca(2+)](i) elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 microg/ml, whereas ophioluxin does not, even at 80 microg/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin- or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fcgamma complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (malayan pit viper), stimulates platelets by binding to alpha 2beta 1 integrin and glycoprotein Ib, activating Syk and phospholipase Cgamma 2, but does not involve the glycoprotein VI/Fc receptor gamma chain collagen receptor

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.

Alboaggregin A activates platelets by a mechanism involving glycoprotein VI as well as glycoprotein Ib

The snake venom C-type lectin alboaggregin A (or 50-kd alboaggregin) from Trimeresurus albolabris was previously shown to be a platelet glycoprotein (GP) Ib agonist. However, investigations of the signal transduction induced in platelets showed patterns of tyrosine phosphorylation that were different from those of other GPIb agonists and suggested the presence of an additional receptor. In this study, the binding of biotinylated alboaggregin A to platelet lysates, as well as affinity chromatography evaluations of platelet lysates on an alboaggregin A-coated column, indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this finding. These experiments also showed that both GPIb and GPVI have a role in the combined signaling and that the overall direction this takes can be influenced by inhibitors of one or the other receptor pathway.

The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

Platelet collagen receptors: a new target for inhibition?

Collagen is a major component of extracellular matrix and a wide variety of types exist. Cells recognise collagen in different ways depending on sequence and structure. They can recognise predominantly primary sequence, they may require triple-helical structure or they can require fibrillar structures. Since collagens are major constituents of the subendothelium that determine the thrombogenicity of the injured or pathological vessel wall, a major role is induction of platelet activation and aggregation as the start of repair processes. Platelets have at least two direct and one indirect (via von Willebrand factor) receptors for collagen, and collagen has specific recognition motifs for these receptors. These receptors and recognition motifs are under intensive investigation in the search for possible methods to control platelet activation in vivo. A wide range of proteins has been identified and, in part, characterised from both haematophageous insects and invertebrates but also from snake venoms that inhibit platelet activation by collagen or induce platelet activation via collagen receptors on platelets. These will provide model systems to test the effect of inhibition of specific collagen-platelet receptor interactions for both effectiveness as well as for side effects and should provide assay systems for the development of small molecule inhibitors. Since platelet inhibitors for long-term prophylaxis of cardiovascular diseases are still in clinical trials there are many unanswered questions about long-term effects both positive and negative. The major problem which still has to be definitively solved about these alternative approaches to inhibition of platelet activation is whether they will show advantages in terms of dose-response curves while offering decreased risks of bleeding problems. Preliminary studies would seem to suggest that this is indeed the case.