Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells

Vaccination of pigs against Classical swine fever virus (CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3¿NS4A were used to identify viral genes encoding CTL epitopes. Monocyte-derived dendritic cells (DCs) and fibrocytes served as the APCs. In vitro translation of the mRNA and microscopic analysis of transfected cells demonstrated that E2 and NS3¿NS4A could be identified. APCs transfected with either of the mRNA molecules restimulated CSFV-specific T cells to produce gamma interferon and specific cytotoxic activity against CSFV-infected target cells. The presence of CTL epitopes on E2 was confirmed by using d/d-haplotype MAX cells expressing E2 constitutively as target cells in d/d-haplotype CTL assays. A potent CTL activity against E2 was detected early (1¿3 weeks) after CSFV challenge. This work corroborates the existence of CTL epitopes within the non-structural protein domain NS3¿NS4A of CSFV. Furthermore, epitopes on the E2 protein can also now be classified as targets for CTLs, having important implications for vaccine design, especially subunit vaccines. As for the use of mRNA-transfected APCs, this represents a simple and efficient method to identify viral genes encoding CTL epitopes in outbred population

MODELO DE ATENCIÓN DEL CÁNCER EN LA INFANCIA Y ADOLESCENCIA

La atención integral de niños y adolescentes con cáncer es uno de los grandes desafíos para el sistema de salud pública de nuestros países donde el cáncer infantil representa un problema de salud pública y un problema social. El cáncer pediátrico en Paraguay, un país de escasos recursos, es un problema social y de salud pública por las consecuencias que se infringen a los pacientes, sus familias, las comunidades y los sistemas de salud. Un modelo descentralizado con clínicas más cercanas y dedicadas a cuidados primarios y referencias de niños con diagnóstico potencial de cáncer mejoraron el acceso a cuidados especializados y seguimiento del cáncer. Estas clínicas, implementadas dentro de los hospitales regionales de los sistemas nacionales de salud, ofrecen soluciones sostenibles y efectivas para un mejor acceso y seguimiento del cuidado de los niños con cáncer. El análisis de los desafíos, el éxito y la rentabilidad de estas clínicas regionales de cáncer pediátrico para referencias y seguimiento, permite sugerir un modelo óptimo para tales clínicas en entornos de bajos ingresos. Este modelo podría ser replicado para el cuidado de otras enfermedades y en otros grupos de edad. Presentamos aquí el resultado de la evaluación de los resultados de los pacientes de las cuatro clínicas regionales desde su implementación inicial

Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins

The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where ‘n’ can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5′ of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel α-helical ‘tweezer’-like structure

Development of a diagnostic protocol for dizziness in elderly patients in general practice: a Delphi procedure

<p>Abstract</p> <p>Background</p> <p>Dizziness in general practice is very common, especially in elderly patients. The empirical evidence for diagnostic tests in the evaluation of dizziness is scarce. Aim of our study was to determine which set of diagnostic tests should be part of a diagnostic protocol for evaluating dizziness in elderly patients in general practice.</p> <p>Methods</p> <p>We conducted a Delphi procedure with a panel of 16 national and international experts of all relevant medical specialities in the field of dizziness. A selection of 36 diagnostic tests, based on a systematic review and practice guidelines, was presented to the panel. Each test was described extensively, and data on test characteristics and methodological quality (assessed with the Quality Assessment of Diagnostic Accuracy Studies, QUADAS) were presented. The threshold for in- or exclusion of a diagnostic test was set at an agreement of 70%.</p> <p>Results</p> <p>During three rounds 21 diagnostic tests were selected, concerning patient history (4 items), physical examination (11 items), and additional tests (6 items). Five tests were excluded, although they are recommended by existing practice guidelines on dizziness. Two tests were included, although several practice guidelines question their diagnostic value. Two more tests were included that have never been recommended by practice guidelines on dizziness.</p> <p>Conclusion</p> <p>In this study we successfully combined empirical evidence with expert opinion for the development of a set of diagnostic tests for evaluating dizziness in elderly patients. This comprehensive set of tests will be evaluated in a cross-sectional diagnostic study.</p

Activation and modulation of antiviral and apoptotic genes in pigs infected with classical swine fever viruses of high, moderate or low virulence

The immune response to CSFV and the strategies of this virus to evade and suppress the pigs’ immune system are still poorly understood. Therefore, we investigated the transcriptional response in the tonsils, median retropharyngeal lymph node (MRLN), and spleen of pigs infected with CSFV strains of similar origin with high, moderate, and low virulence. Using a porcine spleen/intestinal cDNA microarray, expression levels in RNA pools prepared from infected tissue at 3 dpi (three pigs per virus strain) were compared to levels in pools prepared from uninfected homologue tissues (nine pigs). A total of 44 genes were found to be differentially expressed. The genes were functionally clustered in six groups: innate and adaptive immune response, interferon-regulated genes, apoptosis, ubiquitin-mediated proteolysis, oxidative phosphorylation and cytoskeleton. Significant up-regulation of three IFN-γ-induced genes in the MRLNs of pigs infected with the low virulence strain was the only clear qualitative difference in gene expression observed between the strains with high, moderate and low virulence. Real-time PCR analysis of four response genes in all individual samples largely confirmed the microarray data at 3 dpi. Additional PCR analysis of infected tonsil, MRLN, and spleen samples collected at 7 and 10 dpi indicated that the strong induction of expression of the antiviral response genes chemokine CXCL10 and 2′–5′ oligoadenylate synthetase 2, and of the TNF-related apoptosis-inducing ligand (TRAIL) gene at 3 dpi, decreased to lower levels at 7 and 10 dpi. For the highly and moderately virulent strains, this decrease in antiviral and apoptotic gene expression coincided with higher levels of virus in these immune tissues

Effective-one-body Hamiltonian with next-to-leading order spin-spin coupling for two nonprecessing black holes with aligned spins

The canonical Arnowitt-Deser-Misner (ADM) Hamiltonian with next-to-leading order spin-spin coupling [J. Steinhoff, S. Hergt, and G. Schäfer, Phys. Rev. D 77, 081501 (2008); 78, 101503 (2008)] is converted into the effective-one-body (EOB) formalism of [T. Damour, P. Jaranowski, and G. Schäfer, Phys. Rev. D 78, 024009 (2008)] for the special case of spinning black hole binaries whose spins are aligned with the angular momentum. In particular, we propose to include the new terms by adding a dynamical term of next-to-leading order to the Kerr parameter squared entering the effective metric. The modified EOB Hamiltonian consistently reduces to the Kerr Hamiltonian as the mass ratio tends to zero; moreover, it predicts the existence of an innermost stable circular orbit. We also derive, for the general case of arbitrarily oriented spins but in the vanishing mass-ratio limit, a coordinate transformation that maps the next-to-leading order spin-spin contribution of the ADM Hamiltonian to the EOB Hamiltonian