Nuevo inhibidor de serina proteasa y su uso

La invención se refiere a una nueva proteína, identificada mediante rastreo de una genoteca de cDNA de Anisakis simplex, así como a su secuencia codificante y a sus vectores de expresión. Dicha proteína, miembro de la familia de las serpinas, es capaz de inhibir de manera dependiente de dosis a la trombina humana, sin afectar al factor Xa de la coagulación, y sin que la heparina altere su actividad inhibitoria. Así, la invención se refiere también al uso de esta proteína para la preparación de un medicamento, especialmente si se trata de un anticoagulante. Su administración tendría especial interés en los casos de hipercoagulopatías asociadas a la administración de heparina. Además, representa una alternativa a los anticoagulantes derivados de la cumarina, a los que podría sustituir en cualquiera de las situaciones en las que se administran.REIVINDICACIONES: 1. Un polipéptido cuya secuencia de aminoácidos comprende: a) la secuencia representada por SEQ ID NO:3, o b) una secuencia idéntica al menos en un 40% a SEQ ID NO:3 que presenta estructura de serpina con capacidad de inhibir a la trombina humana, o c) la secuencia de aminoácidos representada por SEQ ID NO:2, o d) una secuencia idéntica al menos en un 40% a SEQ ID NO:2, que presenta estructura de serpina con capacidad de inhibir a la trombina humana. 2. Polipéptido según la reivindicación 1, que comprende la secuencia de aminoácidos representada por SEQ ID NO:3. 3. Polipéptido según la reivindicación 1, que comprende una secuencia de aminoácidos que presenta estructura de serpina con capacidad de inhibir a la trombina humana, en el que dicha secuencia es idéntica a SEQ ID NO:3 en un porcentaje que se selecciona del grupo del 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99, 5% y 99, 9%. 4. Polipéptido según la reivindicación 1, que comprende la secuencia de aminoácidos representada por SEQ ID NO:2. 5. Polipéptido según la reivindicación 1, que comprende una secuencia de aminoácidos que presenta estructura de serpina con capacidad de inhibir a la trombina humana, en el que dicha secuencia es idéntica a SEQ ID NO:2 en un porcentaje que se selecciona del grupo del 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99, 5% y 99, 9%. 6. Polipéptido según una cualquiera de las reivindicaciones anteriores, que es una proteína de fusión que comprende la secuencia de una segunda proteína. 7. Polipéptido según la reivindicación 6, en el que la segunda proteína es la glutatión-S-transferasa. 8. Polipéptido según una cualquiera de las reivindicaciones anteriores, cuya capacidad de inhibición de la trombina humana no se ve afectada por la presencia de heparina. 9. Polipéptido según una cualquiera de las reivindicaciones anteriores, que presenta también capacidad de inhibir las catepsinas G y L. 10. Una molécula aislada de ácido nucleico que comprende una secuencia que codifica una serpina con capacidad de inhibir la trombina humana seleccionada del grupo que consiste en: a) una secuencia de ácido nucleico que codifica una secuencia polipeptídica idéntica al menos en un 40% a la secuencia representada por SEQ ID NO:3 b) una secuencia de ácido nucleico que codifica una secuencia polipeptídica idéntica al menos en un 40% a la secuencia representada por SEQ ID NO:2; c) una molécula de ácido nucleico complementaria a una de las anteriores. 11. Molécula de ácido nucleico según la reivindicación 10, que comprende una secuencia que codifica una secuencia polipeptídica idéntica a la representada por SEQ ID NO:3. 12. Molécula de ácido nucleico según la reivindicación 10, que comprende una secuencia que codifica una secuencia polipeptídica idéntica a la representada por SEQ ID NO:2. 13. Molécula de ácido nucleico según la reivindicación 12, que comprende la secuencia representada por SEQ ID NO: 1. 14. Molécula de ácido nucleico según la reivindicación 10, que comprende un fragmento de secuencia que codifica una secuencia polipeptídica que es idéntica a la representada por SEQ ID NO:2 al menos en un porcentaje que se selecciona del grupo de 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99, 5% y 99, 9%. 15. Un vector de expresión que comprende la secuencia de ADN correspondiente a la molécula de ácido nucleico de una cualquiera de las reivindicaciones 10 a 14 unida operativamente a una secuencia de control de la expresión. 16. Una célula hospedadora transformada con un vector de expresión de la reivindicación 15. 17. Célula hospedadora según la reivindicación 16, que es una bacteria, una levadura, o una célula eucariótica. 18. Un método para producir una proteína que comprende las etapas de: a) cultivar la célula hospedadora transformada con un vector de expresión de la reivindicación 15 que permite la expresión del polipéptido con capacidad de inhibir la trombina humana codificado en dicho vector de expresión; b) purificar el polipéptido sintetizado en la etapa a) de la célula o del medio celular. 19. Uso de un polipéptido de una cualquiera de las reivindicaciones 1 a 9 para la preparación de un medicamento. 20. Uso según la reivindicación 19, en el que el medicamento está destinado a ser utilizado como anticoagulante. 21. Uso según la reivindicación 20, en el que el medicamento está destinado a pacientes en los que se sospeche que puede haber alguna alteración en proteínas de la cascada de coagulación distintas de la trombina, a pacientes inmunodeprimidos, a pacientes con trastornos hepáticos, a pacientes con coagulopatías de consumo con coagulación intravascular diseminada, a pacientes que presenten estados de hipercoagulabilidad asociados al uso de heparina o a pacientes con déficit de antitrombina III. 22. Uso según la reivindicación 20, en el que el medicamento está destinado a ser administrado como anticoagulante durante períodos perioperatorios. 23. Uso según la reivindicación 22, en el que el medicamento está destinado a ser administrado durante el período perioperatorio de la cirugía cardiopulmonar, y de la cirugía traumatológica. 24. Uso según la reivindicación 23, en el que el medicamento está destinado a ser administrado durante el período perioperatorio de la cirugía traumatológica en tratamientos de sustitución completa de cadera y rodilla. 25. Uso según la reivindicación 19, en el que el medicamento está destinado a pacientes con síndrome coronario agudo, fibrilación auricular, enfermedad coronaria crónica, prótesis mecánicas o articulares y válvulas cardíacas o vasculares. 26. Uso según una cualquiera de las reivindicaciones 20 a 25, en el que el medicamento está destinado a ser administrado sin la administración simultánea de heparina.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idioma.Instituto de Salud Carlos III; Universidad Central de VenezuelaSolicitud de patent

A study of eosinophilia and helminths in migrant sub-Saharan patients in a primary care center (Madrid, Spain)

We determine the association between eosinophilia and certain parasites diagnosed by serology in patients of subsaharan origin of a Primary Care Center from Madrid region, Spain. It was implemented a complete protocol for migrant patient to study eosinophilia and realized serology tests for parasites detection. All variable and data were evaluated by statistical methods. A total of 184 patients with eosinophilia were included in the study, 115 patients (62.5%) were seronegative for helminths and 69 were seropositive. Strongyloides stercoralis (55.07%), Schistosoma spp (39.13%) and Toxocara canis (20.29%) were the most prevalent helminths immunodetected in the study. So, 49 patients (26.6%) had abdominal pain, 50 patients (27.17%) had problems related with skin conditions and 38 patients (20.65%) had respiratory disorder, symptoms not related with the helminth parasites detected. Regarding number of parasites by patient, one specie was identified in 49 patients (26.63%) and two or more was identified in 20 patients (10.86%). Eosinophilia was resolved in 91.4% of parasite serpositive patients after received one specific adequate antiparasitic treatment, but this was resolved in 98.3% after received two tratments, and 100% after the third. The results obtained allow us to make some reflections on the difficulty of managing these patients in the Primary Care Center and on whether to diagnose and treat individuals from endemic areas, with or without eosinophilia and being asymptomatic or not, given the benefit it has for the individual and public health, as possible to minimize any chance of transmission

Fasciola spp: Mapping of the MF6 epitope and antigenic analysis of the MF6p/HDM family of heme-binding proteins

MF6p/FhHDM-1 is a small cationic heme-binding protein which is recognized by the monoclonal antibody (mAb) MF6, and abundantly present in parenchymal cells and secreted antigens of Fasciola hepatica. Orthologs of this protein (MF6p/HDMs) also exist in other causal agents of important foodborne trematodiasis, such as Clonorchis sinensis, Opisthorchis viverrini and Paragonimus westermani. Considering that MF6p/FhHDM-1 is relevant for heme homeostasis in Fasciola and was reported to have immunomodulatory properties, this protein is expected to be a useful target for vaccination. Thus, in this study we mapped the epitope recognized by mAb MF6 and evaluated its antigenicity in sheep. The sequence of the MF6p/FhHDM-1 ortholog from F. gigantica (MF6p/FgHDM-1) was also reported. By means of ELISA inhibitions with overlapping synthetic peptides, we determined that the epitope recognized by mAb MF6 is located within the C-terminal moiety of MF6p/FhHDM-1, which is the most conserved region of MF6p/HDMs. By immunoblotting analysis of parasite extracts and ELISA inhibitions with synthetic peptides we also determined that mAb MF6 reacted with the same intensity with F. hepatica and F. gigantica, and in decreasing order of intensity with C. sinensis, O.viverrini and P. westermani orthologs. On the contrary, mAb MF6 showed no reactivity against Dicrocoelium dendriticum and Schistosoma mansoni. The study of the recognition of peptides covering different regions of MF6p/FhHDM-1 by sera from immunized sheep revealed that the C-terminal moiety is the most antigenic, thus being of potential interest for vaccination. We also demonstrated that the production of antibodies to MF6p/FhHDM-1 in sheep infected by F. hepatica occurs relatively early and follows the same pattern as those produced against L-cathepsins.This work was funded by the Ministerio de Ciencia e Innovación, Spain (Grants AGL2011-30563-C03-01, AGL2011-30563-C03-02 and AGL2011-30563-C03-03); Xunta de Galicia, Spain (Grants GPC 2014/058 and ED431B 2017/18); Network of Biomedical Research on Tropical Diseases (RICET), Spain (Grant RD12/0018/0013) and the European Fund for Regional Development (FEDER). VMS holds a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Evaluation of onchocerciasis seroprevalence in Bioko Island (Equatorial Guinea) after years of disease control programmes

BACKGROUND: Onchocerciasis or "river blindness" is a chronic parasitic disease caused by the filarial worm Onchocerca volvulus, transmitted through infected blackflies (Simulium spp.). Bioko Island (Equatorial Guinea) used to show a high endemicity for onchocerciasis. During the last years, the disease control programmes using different larvicides and ivermectin administration have considerably reduced the prevalence and intensity of infection. Based on this new epidemiological scenario, in the present work we aimed to assess the impact of the strategies applied against onchocerciasis in Bioko Island by an evaluation of IgG4 antibodies specific for recombinant Ov-16 in ELISA. METHODS: A cross-sectional study was conducted in Bioko Island from mid-January to mid-February, 2014. Twenty communities were randomly selected from rural and urban settings. A total of 140 households were chosen. In every selected household, all individuals aged 5 years and above were recruited; 544 study participants agreed to be part of this work. No previous data on onchocerciasis seroprevalence in the selected communities were available. Blood samples were collected and used in an "ELISA in-house" prepared with recombinant Ov-16, expressed and further purified. IgG4 antibodies specific for recombinant Ov-16 were evaluated by ELISA in all of the participants. RESULTS: Based on the Ov-16 ELISA, the onchocerciasis seroprevalence was 7.9 %, mainly concentrated in rural settings; samples from community Catedral Ela Nguema (# 16) were missed during the field work. Among the rural setups, communities Inasa Maule (# 7), Ruiché (# 20) and Barrios Adyacentes Riaba (# 14), had the highest seropositivity percentages (29.2, 26.9 and 23.8 %, respectively). With respect to the urban settings, we did not find any positive case in communities Manzana Casa Bola (# 3), Colas Sesgas (# 6), Getesa (# 8), Moka Bioko (# 9), Impecsa (# 10), Baney Zona Baja (# 12) and Santo Tomás de Aquino (# 1). No onchocerciasis seropositive samples were found in 10-year-old individuals or younger. The IgG4 positive titles increased in older participants. CONCLUSIONS: A significant decline in onchocerciasis prevalence was observed in Bioko Island after years of disease-vector control and CDTI strategy. The seroprevalence increased with age, mainly in rural settings that could be due to previous exposure of population to the filarial parasite, eliminated by the control programmes introduced against onchocerciasis. A new Ov-16 serological evaluation with a larger sample size of children below 10 years of age is required to demonstrate the interruption of transmission of O. volvulus in the human population of Bioko Island (Equatorial Guinea) according to the WHO criteria.We would like to thank the National Program for Control of Onchocerciasis and other Filariasis in Equatorial Guinea for supporting us to obtain the information on which this study is based. We are grateful to the study participants for volunteering to participate in the study and the data collectors for performing field work. Our gratitude to the Unit of Serological Diagnosis, Department of Parasitology, Centro Nacional de Microbiología, and the Spanish Red Cross for providing some control samples. Thanks are also due to Diana Gomez-Barroso, from the National Centre of Epidemiology (ISCIII) for her help with the mapping.S

ANISERP: a new serpin from the parasite Anisakis simplex

Background: Serine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP). Methods: The full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted. Results: The AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity. Conclusions: Our findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site.We thank Biomol-Informatics SL (http://www.biomol-informatics.com/) for bioinformatic consultation. We also thank Juan Francisco Alcaide and Julia Medrano for their invaluable help in the ANISERP patenting process. We thank Dr Raúl Iglesias (Laboratorio de Parasitología, Facultad de Biología, Universidad de Vigo) for his helpful comments on the interpretation of the IHQ studies

In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

Recombinant proteins expressed in E. coli are frequently purified by immobilized metal affinity chromatography (IMAC). By means of this technique, tagged proteins containing a polyhistidine sequence can be obtained up to 95% pure in a single step, but some host proteins also bind with great affinity to metal ions and contaminate the sample. A way to overcome this problem is to include a second tag that is recognized by a preexistent monoclonal antibody (mAb) in the gene encoding the target protein, allowing further purification. With this strategy, the recombinant protein can be directly used as target in capture ELISA using plates sensitized with the corresponding mAb. As a proof of concept, in this study we engineered a Trichinella-derived tag (MTFSVPIS, recognized by mAb US9) into a His-tagged recombinant Fasciola antigen (rFhLAP) to make a new chimeric recombinant protein (rUS9-FhLAP), and tested its specificity in capture and indirect ELISAs with sera from sheep and cattle. FhLAP was selected since it was previously reported to be immunogenic in ruminants and is expressed in soluble form in E. coli, which anticipates a higher contamination by host proteins than proteins expressed in inclusion bodies. Our results showed that a large number of sera from non-infected ruminants (mainly cattle) reacted in indirect ELISA with rUS9-FhLAP after single-step purification by IMAC, but that this reactivity disappeared testing the same antigen in capture ELISA with mAb US9. These results demonstrate that the 6XHis and US9 tags can be combined when double purification of recombinant proteins is required.This work was supported by: Ministerio de Economía y Competitividad (Spain) [grant number AGL2011-30563-C03 and AGL2014-57125R], Ministerio de Economía, Industria y Competitividad (INIA, Spain) [grants numbers RTA2017-00010-C02-01 and RTA2017-00010-C02-02] and the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia, Spain) [grant number ED431B 2017/18]. RAOM holds a predoctoral fellowship from the Spanish Ministerio de Economía y Competitividad (Programa de Formación de Personal Investigador). VMS is supported by a contract under the grant ED431B 2017/18. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

An enduring rapidly moving storm as a guide to Saturn's Equatorial jet's complex structure

This work is licensed under a Creative Commons Attribution 4.0Saturn has an intense and broad eastward equatorial jet with a complex three-dimensional structure mixed with time variability. The equatorial region experiences strong seasonal insolation variations enhanced by ring shadowing, and three of the six known giant planetary-scale storms have developed in it. These factors make Saturn’s equator a natural laboratory to test models of jets in giant planets. Here we report on a bright equatorial atmospheric feature imaged in 2015 that moved steadily at a high speed of 450 ms-1 not measured since 1980–1981 with other equatorial clouds moving within an ample range of velocities. Radiative transfer models show that these motions occur at three altitude levels within the upper haze and clouds. We find that the peak of the jet (latitudes 10ºN to 10º S) suffers intense vertical shears reaching þ2.5 ms-1 km-1, two orders of magnitude higher than meridional shears, and temporal variability above 1 bar altitude level.Peer ReviewedPostprint (published version

An enduring rapidly moving storm as a guide to Saturn’s Equatorial jet’s complex structure

Saturn has an intense and broad eastward equatorial jet with a complex three-dimensional structure mixed with time variability. The equatorial region experiences strong seasonal insolation variations enhanced by ring shadowing, and three of the six known giant planetary-scale storms have developed in it. These factors make Saturn's equator a natural laboratory to test models of jets in giant planets. Here we report on a bright equatorial atmospheric feature imaged in 2015 that moved steadily at a high speed of 450 ms(-1) not measured since 1980-1981 with other equatorial clouds moving within an ample range of velocities. Radiative transfer models show that these motions occur at three altitude levels within the upper haze and clouds. We find that the peak of the jet ( latitudes 10 degrees N to 10 degrees S) suffers intense vertical shears reaching + 2.5 ms(-1) km(1), two orders of magnitude higher than meridional shears, and temporal variability above 1 bar altitude level. Palabras claveThis work is based on observations and analysis from Hubble Space Telescope (GO/DD program 14064), Cassini ISS images (NASA pds), and Calar Alto Observatory (CAHA-MPIA). A.S.-L. and UPV/EHU team are supported by the Spanish projects AYA2012-36666 and AYA2015-65041-P with FEDER support, Grupos Gobierno Vasco IT-765-13, Universidad del Pais Vasco UPV/EHU program UFI11/55, and Diputacion Foral Bizkaia (BFA). We acknowledge the contribution of Saturn images by T. Olivetti, M. Kardasis, A. Germano, A. Wesley, P. Miles, M. Delcroix, C. Go, T. Horiuchi and P. Maxon. We also acknowledge the wind model data provided by J. Friedson

Tumor cells in light-chain amyloidosis and myeloma show distinct transcriptional rewiring of normal plasma cell development

Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation–related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies