Estudio de los sueros ricos en factores de crecimiento y de su efecto sobre células de epitelio corneal

Objetivos: Estudiar cómo afecta a la concentración de determinados factores de crecimiento presentes en los sueros la filtración (utilizado como método de esterilización) y el tratamiento por calor (utilizado para la inactivación del complemento). Además de estudiar el efecto de un bioadhesivo (ácido hialurónico, HaNa), aplicado solo o conjuntamente con el suero rico en factores de crecimiento (s-PRGF), sobre la capacidad de las células de epitelio corneal (HCE) para proliferar y migrar. Materiales y métodos: Se midió mediante kits ELISA comerciales la concentración en las diferentes condiciones de filtración y calentamiento de las siguientes biomoléculas EGF (Epidermal Growth Factor), VEGF (Vascular Endothelial Growth Factor), HGF (Hepatocyte Growth Factor), PDGF (Platelet-derived Growth Factor) y la Fibronectina. Teniendo en cuenta el papel de la proliferación y migración celular en los procesos de cicatrización se han realizado dos ensayos diferentes in vitro: un ensayo MTT para estudiar la viabilidad y la proliferación celular y el método de la herida (Scratch wound-healing assay) para determinar la capacidad migratoria de células bajo ciertos tratamientos: BSA (Bovine Serum Albumin) al 1% como control, FBS (Fetal Bovine Serum) al 10%, s-PRGF al 45%, s-PRGF al 45% con HaNa 0,1% y HaNa al 0,1% Resultados: En el caso de la filtración, se observa una mayor pérdida de factores utilizando un filtro con una membrana de PVDF (Durapore®) para todos los factores estudiados. El calentamiento produce una reducción de la concentración superior al 50% en el caso del HGF y EGF, manteniéndose constante en el caso del VEGF.La mezcla de diferentes muestras con el complemento inactivado para formar un pool no presenta cambios en la concentración al compararlo con la media de las muestras utilizadas. Por tanto, la utilización de un pool del hemoderivado no supone perdida de factores de crecimiento, haciendo de ello un procedimiento perfectamente aceptable para los ensayos celulares. El tratamiento con s-PRGF y el combinado con el bioadhesivo promueven la proliferación y migración de las células de epitelio corneal humano(HCE) in vitro de manera similar, no encontrándose diferencias estadísticamente significativas entre ambos. Conclusiones: La adicción del bioadhesivo no produce efecto tóxico en las células, sin embargo, no se han encontrado efectos beneficiosos en cuanto a proliferación y migración se refiere. A este respecto, creemos que hay que dar un paso más haciendo comprobaciones in vivo, ya que, a diferencia de la experimentación in vitro los componentes de los hemoderivados no están indefinidamente en contacto con las células sino por un espacio de tiempo muy reducido. Por ello, la concentración de factores de crecimiento en la aplicación in vivo es especialmente importante, y no sería conveniente reducirla mediante procedimientos físicos como la filtración o el calentamiento.

HIV-1 Capture and Transmission by Dendritic Cells: The Role of Viral Glycolipids and the Cellular Receptor Siglec-1

Dendritic cells (DCs) are essential in order to combat invading viruses and trigger antiviral responses. Paradoxically, in the case of HIV-1, DCs might contribute to viral pathogenesis through trans-infection, a mechanism that promotes viral capture and transmission to target cells, especially after DC maturation. In this review, we highlight recent evidence identifying sialyllactose-containing gangliosides in the viral membrane and the cellular lectin Siglec-1 as critical determinants for HIV-1 capture and storage by mature DCs and for DC-mediated trans-infection of T cells. In contrast, DC-SIGN, long considered to be the main receptor for DC capture of HIV-1, plays a minor role in mature DC-mediated HIV-1 capture and trans-infection.

Association analyses of DNA polymorphisms in bovine SREBP-1, LXRα,FADS1 genes with fatty acid composition in Canadian commercial crossbred beef steers

Two previously reported DNA polymorphisms of sterol regulatory element binding transcription factor 1 (SREBP1) and liver X receptor alpha (LXRα) and two DNA polymorphisms of fatty acid desaturase 1 (FADS1) were evaluated for associations with fatty acids in brisket adipose tissue of Canadian cross-bred beef steers. The polymorphism of 84 bp insert/deletion in intron 5 of SREBP1 was significantly associated with the concentration of 9c C17:1 (P=0.013). The G>A single nucleotide polymorphism (SNP) in the exon 4 of LXRα gene was associated with the concentration of 9c, 11t C18:2 (P=0.04), sum of conjugated linoleic acids (CLA) (P=0.025) and 11c C20:1(P=0.042). Two DNA polymorphisms in the promoter region of FADS1, deletion/insertion of ->GTG in rs133053720 and SNP A>G in rs42187276, were significantly associated with concentrations of C17:0 iso, C17:0 ai, total branched chain fatty acids (BFA), 12t C18:1, 13t/14t C18:1, 15t C18:1, and 13c C18:1 (P<0.05). Further studies are needed to validate the associations and to delineate the roles of the gene polymorphisms in determining the fatty acid composition in beef tissues.

Recognition of Membrane-Bound Fusion-Peptide/MPER Complexes by the HIV-1 Neutralizing 2F5 Antibody : Implications for Anti-2F5 Immunogenicity

13 p.

Inoculation of Triatoma Virus (Dicistroviridae: Cripavirus) elicits a non-infective immune response in mice

Background: Dicistroviridae is a new family of small, non-enveloped, +ssRNA viruses pathogenic to both beneficial arthropods and insect pests. Little is known about the dicistrovirus replication mechanism or gene function, and any knowledge on these subjects comes mainly from comparisons with mammalian viruses from the Picornaviridae family. Due to its peculiar genome organization and characteristics of the per os viral transmission route, dicistroviruses make good candidates for use as biopesticides. Triatoma virus (TrV) is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of the human trypanosomiasis disease called Chagas disease. TrV was postulated as a potential control agent against Chagas' vectors. Although there is no evidence that TrV nor other dicistroviruses replicate in species outside the Insecta class, the innocuousness of these viruses in humans and animals needs to be ascertained. Methods: In this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice. Results: In this study we have observed that there is no significant difference in the ratio IgG2a/IgG1 in sera from animals inoculated with TrV when compared with non-inoculated animals or mice inoculated only with non-infective TrV protein capsids. Conclusions: We conclude that, under our experimental conditions, TrV is unable to replicate inmice. This study constitutes the first test to evaluate the infectivity of a dicistrovirus in a vertebrate animal model.

Chiral gold(I) vs chiral silver complexes as catalysts for the enantioselective synthesis of the second generation GSK-hepatitis C virus inhibitor

The synthesis of a GSK 2(nd) generation inhibitor of the hepatitis C virus, by enantioselective 1,3-dipolar cycloaddition between a leucine derived iminoester and tert-butyl acrylate, was studied. The comparison between silver(I) and gold(I) catalysts in this reaction was established by working with chiral phosphoramidites or with chiral BINAP. The best reaction conditions were used for the total synthesis of the hepatitis C virus inhibitor by a four step procedure affording this product in 99% ee and in 63% overall yield. The origin of the enantioselectivity of the chiral gold(I) catalyst was justified according to DFT calculations, the stabilizing coulombic interaction between the nitrogen atom of the thiazole moiety and one of the gold atoms being crucial.

Novel Papillomaviruses in Free-Ranging Iberian Bats: No Virus-Host Co-evolution, No Strict Host Specificity, and Hints for Recombination

Papillomaviruses (PVs) are widespread pathogens. However, the extent of PV infections in bats remains largely unknown. This work represents the first comprehensive study of PVs in Iberian bats. We identified four novel PVs in the mucosa of free-ranging Eptesicus serotinus (EserPV1, EserPV2, and EserPV3) and Rhinolophus ferrumequinum (RferPV1) individuals and analyzed their phylogenetic relationships within the viral family. We further assessed their prevalence in different populations of E. serotinus and its close relative E. isabellinus. Although it is frequent to read that PVs co-evolve with their host, that PVs are highly species-specific, and that PVs do not usually recombine, our results suggest otherwise. First, strict virus-host co-evolution is rejected by the existence of five, distantly related bat PV lineages and by the lack of congruence between bats and bat PVs phylogenies. Second, the ability of EserPV2 and EserPV3 to infect two different bat species (E. serotinus and E. isabellinus) argues against strict host specificity. Finally, the description of a second noncoding region in the RferPV1 genome reinforces the view of an increased susceptibility to recombination in the E2-L2 genomic region. These findings prompt the question of whether the prevailing paradigms regarding PVs evolution should be reconsidered.

Viral metagenomic analysis of feces of wild small carnivores

Background: Recent studies have clearly demonstrated the enormous virus diversity that exists among wild animals. This exemplifies the required expansion of our knowledge of the virus diversity present in wildlife, as well as the potential transmission of these viruses to domestic animals or humans. Methods: In the present study we evaluated the viral diversity of fecal samples (n = 42) collected from 10 different species of wild small carnivores inhabiting the northern part of Spain using random PCR in combination with next-generation sequencing. Samples were collected from American mink (Neovison vison), European mink (Mustela lutreola), European polecat (Mustela putorius), European pine marten (Martes martes), stone marten (Martes foina), Eurasian otter (Lutra lutra) and Eurasian badger (Meles meles) of the family of Mustelidae; common genet (Genetta genetta) of the family of Viverridae; red fox (Vulpes vulpes) of the family of Canidae and European wild cat (Felis silvestris) of the family of Felidae. Results: A number of sequences of possible novel viruses or virus variants were detected, including a theilovirus, phleboviruses, an amdovirus, a kobuvirus and picobirnaviruses. Conclusions: Using random PCR in combination with next generation sequencing, sequences of various novel viruses or virus variants were detected in fecal samples collected from Spanish carnivores. Detected novel viruses highlight the viral diversity that is present in fecal material of wild carnivores.

Novel Papillomaviruses in Free-Ranging Iberian Bats: No Virus-Host Co-evolution, No Strict Host Specificity, and Hints for Recombination

Papillomaviruses (PVs) are widespread pathogens. However, the extent of PV infections in bats remains largely unknown. This work represents the first comprehensive study of PVs in Iberian bats. We identified four novel PVs in the mucosa of free-ranging Eptesicus serotinus (EserPV1, EserPV2, and EserPV3) and Rhinolophus ferrumequinum (RferPV1) individuals and analyzed their phylogenetic relationships within the viral family. We further assessed their prevalence in different populations of E. serotinus and its close relative E. isabellinus. Although it is frequent to read that PVs co-evolve with their host, that PVs are highly species-specific, and that PVs do not usually recombine, our results suggest otherwise. First, strict virus-host co-evolution is rejected by the existence of five, distantly related bat PV lineages and by the lack of congruence between bats and bat PVs phylogenies. Second, the ability of EserPV2 and EserPV3 to infect two different bat species (E. serotinus and E. isabellinus) argues against strict host specificity. Finally, the description of a second noncoding region in the RferPV1 genome reinforces the view of an increased susceptibility to recombination in the E2-L2 genomic region. These findings prompt the question of whether the prevailing paradigms regarding PVs evolution should be reconsidered.

Seroprevalence of Triatoma virus (Dicistroviridae: Cripaviridae) antibodies in Chagas disease patients

Background: Chagas disease is caused by Trypanosoma cruzi, and humans acquire the parasite by exposure to contaminated feces from hematophagous insect vectors known as triatomines. Triatoma virus (TrV) is the sole viral pathogen of triatomines, and is transmitted among insects through the fecal-oral route and, as it happens with T. cruzi, the infected insects release the virus when defecating during or after blood uptake. Methods: In this work, we analysed the occurrence of anti-TrV antibodies in human sera from Chagas disease endemic and non-endemic countries, and developed a mathematical model to estimate the transmission probability of TrV from insects to man, which ranged between 0.00053 and 0.0015. Results: Our results confirm that people with Chagas disease living in Bolivia, Argentina and Mexico have been exposed to TrV, and that TrV is unable to replicate in human hosts. Conclusions: We presented the first experimental evidence of antibodies against TrV structural proteins in human sera.