Grupo MVeIT – Página 8 – Grupo de Investigación del Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS) de la Universidad de Las Palmas de Gran Canaria.

23
Sep 13
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Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies

PLoS ONE
Volume 8, Issue 9, 23 September 2013, Article number e75126
Salema, V., Marín, E., Martínez-Arteaga, R., Ruano-Gallego, D., Fraile, S., Margolles, Y., Teira, X., Gutierrez, C., Bodelón, G., Fernández, L.Á.

Abstract

Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or V_HH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM_EHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirM_EHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirM_EHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.

01
Sep 13
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Is Wolbachia participating in the bronchial reactivity of cats with heartworm associated respiratory disease?

Veterinary Parasitology
Volume 196, Issue 1-2, 1 September 2013, Pages 130-135
García-Guasch, L., Caro-Vadillo, A., Manubens-Grau, J., Carretón, E., Morchón, R., Simón, F., Kramer, L.H., Montoya-Alonso, J.A.

Abstract

Heartworm associated respiratory disease is a pulmonary syndrome in cats that results from the vascular and parenchymal inflammatory response associated with the arrival and death of Dirofilaria immitis worms into the distal pulmonary arteries. This parasite harbors intracellular Wolbachia, an endosymbiont bacteria. The association between the parasite and the bacteria is obligatory. Some studies suggest the involvement of Wolbachia in the development of the inflammatory reaction and in the polarization of the host immune response against the parasite. Barometric whole-body plethysmography is a non-invasive pulmonary function test that allows a dynamic study of breathing patterns and is useful to study airway disease and the response to different treatments. The aim of this prospective non-blinded study was to compare the influence of Wolbachia upon the respiratory function variables in a population of cats seropositive to D. immitis by use of Barometric whole-body plethysmography. Fourteen seropositive cats to Wolbachia and eight seronegative cats were put into the plethysmograph chamber and different respiratory variables were measured. The results were analyzed and compared between the two groups of animals. Significant differences were found for bronchoconstriction index variables PAU (pause) (P-value < 0.05) and Penh (enhanced pause) (P-value < 0.05). The results obtained in our study suggest that Wolbachia seems to produce a greater acute inflammatory response at bronchial, vascular and parenchymal level worsening the state of broncho-reactivity associated with the presence of seropositivity to D. immitis in cats.

01
Sep 13
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Surface associated antigens of Dirofilaria immitis adult worms activate the host fibrinolytic system

Veterinary Parasitology. Volume 196, Issue 1-2, 1 September 2013, Pages 235-240
González-Miguel, J. , Morchón, R., Carretón, E., Montoya-Alonso, J.A., Simón, F.

Abstract

Cardiopulmonary dirofilariosis (Dirofilaria immitis) is characterized by apparent contradictory events, like the long-term survival of adult worms in the circulatory system of the infected hosts and the development of life-threatening events like thromboembolisms and others. Thus parasite mechanisms, like the activation of fibrinolytic system, are key to the survival of both the worms and the host. The aim of this study was to investigate the interaction between D. immitis adult worms surface-associated antigens (DiSAA) and the fibrinolytic system of the host. We demonstrate that DiSAA extract is able to bind plasminogen and generate plasmin, with the latter occurring in a tissue plasminogen activator (t-PA) dependent manner. Additionally, 11 plasminogen-binding proteins from DiSAA extract were identified by proteomics and mass spectrometry (MS) (actin-5C, actin-1, enolase, fructose-bisphosphate aldolase, GAPDH, MSP domain protein, MSP 2, beta-galactosidase-binding-lectin, galectin, immunoglobulin I-set domain-containing protein and cyclophilin Ovcyp-2). Because in a previous work we have shown the positive interaction between the excretory/secretory antigens of D. immitis (DiES) and the host fibrinolytic system and many of the molecules identified here are shared by both antigens, we hypothesize that DiSAA cooperate in host fibrinolytic system activation promoting the fibrin clot lysis.

29
Ago 13
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SDS-PAGE analysis of urinary proteins in dogs with heartworm disease

Iranian Journal of Veterinary Research.Volume 14, Issue 3, 2013, Pages 245-249
Beristain-Ruiz, D.M., Carretón, E., Rodríguez-Alarcón, C.A. , Montoya-Alonso, J.A., Barrera, R.

Abstract

The aim of the study was to describe the urinary electrophoretic pattern of dogs with heartworm disease. Urine samples from 15 heartworm-infected and 15 healthy dogs were taken. Urinary specific gravity, urinary protein concentration and the urine protein/creatinine (U P/C) ratio were determined. Urine proteins were fractionated using SDS-PAGE. Results showed statistically significant differences for the U P/C ratio (P<0.05) but not for USG and urinary protein concentrations (P>0.05) between groups. Urinary protein SDSPAGE analysis showed eight distinct bands in the urine of heartworm-infected dogs. The presence of proteins exclusively found in the urine of infected dogs suggests renal damage, even in cases of light proteinuria, indicating that SDS-PAGE is a sensitive method for the identification and characterisation of renal proteinuria in dogs with heartworm disease.

01
Jul 13
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VHH (nanobody) directed against human glycophorin A: A tool for autologous red cell agglutination assays (Article)

Analytical Biochemistry
Volume 438, Issue 1, 1 July 2013, Pages 82-89
Habib, I., Smolarek, D., Hattab, C., Grodecka, M., Hassanzadeh-Ghassabeh, G., Muyldermans, S., Sagan, S., Gutiérrez, C., Laperche, S., Le-Van-Kim, C., Aronovicz, Y.C., Wasniowska, K., Gangnard, S., Bertrand, O.

Abstract

The preparation of a V_HH (nanobody) named IH4 that recognizes human glycophorin A (GPA) is described. IH4 was isolated by screening a library prepared from the lymphocytes of a dromedary immunized by human blood transfusion. Phage display and panning against GPA as the immobilized antigen allowed isolating this V_HH. IH4, representing 67% of the retrieved V_HH sequences, was expressed as a soluble correctly folded protein in SHuffle Escherichia coli cells, routinely yielding approximately 100 mg/L fermentation medium. Because IH4 recognizes GPA independently of the blood group antigens, it recognizes red cells of all humans with the possible exception of those with some extremely rare genetic background. The targeted linear epitope comprises the GPA Y₅₂PPE₅₅sequence. Based on surface plasmon resonance results, the dissociation constant of the IH4-GPA equilibrium is 33 nM. IH4 is a stable protein with a transition melting temperature of 75.8 C (measured by differential scanning calorimetry). As proof of concept, we fused HIV p24 to IH4 and used the purified construct expressed in E. coli to show that IH4 was amenable to the preparation of autologous erythrocyte agglutination reagents: reconstituted blood prepared with serum from an HIV-positive patient was readily agglutinated by the addition of the bifunctional reagent.

01
Jul 13
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Variation of d-dimer values as assessment of pulmonary thromboembolism during adulticide treatment of heartworm disease in dogs

Veterinary Parasitology
Volume 195, Issue 1-2, 1 July 2013, Pages 106-111
Carretón, E. , Morchón, R., González-Miguel, J., Simón, F., Juste, M.C., Montoya-Alonso, J.A.

Abstract

Dirofilaria immitis adult worms lodged in the pulmonary arteries are the causative agent of heartworm disease, characterized by endarteritis and obstruction of the blood flow. Dying worms form embolic fragments which trigger thrombosis. Thus, pulmonary thromboembolism is an inevitable consequence of successful adulticide therapy. Clinical signs of pulmonary thromboembolisms are highly variable and non-specific, and its diagnosis is often difficult to obtain. The d-dimer assay is considered the marker of choice for dogs with a suspicion of pulmonary thromboembolism. The aim of the present study was to evaluate the variation of d-dimer and platelets in 15 heartworm-infected dogs during the adulticide treatment with ivermectin, doxycycline and melarsomine. Nine dogs were considered to have a low parasite burden and 6 had a high burden. Measurements were carried out on days 0, 60, and afterwards weekly on days 67, 75, 82, 91, 98, 106, 113, 120, and finally on day 271 after treatment. 40% of dogs showed pathological concentrations of d-dimer and 40% showed slight or mild thrombocytopenia on day 0. The levels of d-dimer decreased by day 60, probably due to the joint action of the ivermectin, doxycycline and exercise restriction. All dogs with high parasite burden showed elevated values of d-dimer on several occasions during the treatment, reaching the highest values the first and second week after the injections of melarsomine. Only 33.3% of the dogs with low parasite burden showed elevated d-dimer levels at some point during the treatment, and all of them showed undetectable levels from the third week after the first injection of melarsomine. On day 271, all dogs showed undetectable levels of d-dimer. There was no correlation between thrombocytopenia and levels of d-dimer during the treatment. The evaluation of serum d-dimer appears to be a supportive test in the assessment and monitoring of pulmonary thromboembolism in dogs with heartworm disease during the adulticide treatment.

01
Jun 13
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Trypanosoma evansi assessment in equines: A study in one decade in an endemic area of the Canary Islands, Spain (Article)

Journal of Equine Veterinary Science
Volume 33, Issue 6, June 2013, Pages 406-409
Rodríguez, N.F., Tejedor-Junco, M.T., González-Martín, M., Doreste, F., Gutierrez, C.

Abstract

Trypanosoma evansi was diagnosed in a camel in the Canary Islands for the first time in 1997, and the parasite is still present in a little area of the Archipelago. Since then, the disease has exclusively affected camel herds, and clinical evidence of the infection has not been demonstrated in any other animal hosts. In the frame of a control plan of the disease in the Archipelago, 864 equines located in the infected area were examined during one decade (2001-2010), of which a total of 26 (3%) were serologically positive but showed negative results at parasitological and molecular examinations. FreeCalc, an epidemiological tool to detect presence/absence of disease, showed that the population would be diseased at a confidence level of 99.916%. These results must suppose to implement the control plan against the disease including the equine population present in the surrounding area of the infected camel farms.

12
Mar 13
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Prevalencia de Dirofilaria immitis en perros en Portugal

Montoya-Alonso JA y Carretón E

Editorial (año): Servicio de Medicina Veterinaria de la ULPGC. Las Palmas de Gran Canaria, 2013

ISBN: 978-84-616-7471-8

12
Mar 13
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Datos actuales de prevalencia de Dirofilaria immitis en perros en España y Portugal

Montoya-Alonso JA y Carretón E

Editorial (año): Servicio de Medicina Veterinaria de la ULPGC. Las Palmas de Gran Canaria, 2013

ISBN: 978-84-616-2778-3

09
Mar 13
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Arrhythmogenic right ventricular cardiomyopathy in boxer dogs: A retrospective study of survival.

Veterinary Record. Volume 172, Issue 10, 9 March 2013, Page 268
Caro-Vadillo, A. , Garcia-Guasch, L., Carreton, E., Montoya-Alonso, J.A., Manubens, J.

Abstract

The aim of the present study was to retrospectively evaluate survival in a population of 62 boxer dogs with arrhythmogenic right ventricular cardiomyopathy (ARVC), without left ventricular systolic failure, based on the following factors: age at diagnosis, presence of syncopal episodes, Holter arrhythmia classification and administered treatment. Medical records of boxer dogs with a diagnosis of ARVC between 2000 and 2010 were reviewed. Results showed that median survival time (MST) was longer in younger ARVC dogs than in the older ones P<0.001). MST was statistically different (P=0.012) between dogs with syncope (365 days) and dogs without syncope episodes (693 days), the probability of death within a year being 4.8 times greater in dogs with syncope (95% CI 1.48 to 15.99) than in dogs without syncope. Regarding Holter classification results, MST was 547.5 days in Holter class-2 dogs and 365 days in Holter class-4 dogs (P=0.030). There were no differences regarding treatment options; MST was 365 days (95% CI 193.615 to 536.4) in the sotalol group, 365 days (95% CI 92.86 to 637.14) in the mexiletine plus atenolol group, and 547.50 days (95% CI 170.45 to 924.55) in the procainamide group (P=0.383). According to this study, the best prognosis is for the younger boxer dog without syncope. There were no differences in survival times in relation to the different treatment options used.

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