Bactericidal synergism between daptomycin and the phage lysin Cpl-1 in a mouse model of pneumococcal bacteraemia.

Combination therapy may improve the outcome of Streptococcus pneumoniae-induced bacteraemia. Here we tested the combination of two antipneumococcal agents, daptomycin and Cpl-1 (the pneumococcal Cp-1 bacteriophage lysin), in a mouse model of pneumococcal bacteraemia. Mice were challenged intraperitoneally (i.p.) with 10(6)CFU of the extremely virulent serotype 2 S. pneumoniae D39 isolate. Subtherapeutic doses of daptomycin (0.4mg/kg) and Cpl-1 (0.4mg/kg and 1mg/kg) were administrated i.p. either alone or in combination by a single bolus injection 1h after bacterial challenge. Survival rates of animals were followed over a period of 7 days. Daptomycin (0.4mg/kg) in combination with Cpl-1 (0.4mg/kg) significantly increased the percentage of surviving mice at Day 7 (80%) compared with the untreated control (0%) and daptomycin or Cpl-1 monotherapy (35% and 0%, respectively). Whilst increasing the concentration of Cpl-1 to 1.0mg/kg did not improve survival when injected alone, its combination with 0.4mg/kg daptomycin further increased the survival rate to 95%. Thus, it was found that the combination of daptomycin with Cpl-1 was synergistic and bactericidal against S. pneumoniae in a mouse model of pneumococcal bacteraemia. To our knowledge, this is the first report of synergism between daptomycin and a phage lysin demonstrated in vivo. Such a combination could represent an interesting alternative therapy for the treatment of pneumococcal bacteraemia/sepsis and possibly other severe pneumococcal infections.

Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin.

Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires. However, the affinity of the selected antibodies is usually low and current methods of affinity maturation are complex and time-consuming. In this paper, we describe an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their site-specific biotinylation by the enzyme BirA. Expression vectors have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on selected scFvs. Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-immune and murine immune phage display libraries. The scFvs were first synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni-NTA affinity chromatography. Purified biotinylated scFvs were tetramerized on the streptavidin molecule to create a streptabody (StAb). The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the scFv monomer counterparts. Furthermore, the percentage of direct binding of 125I-labeled StAb or monomeric scFv on CEA-Sepharose beads and on CEA-expressing cells showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%). Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv.

In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of Streptococcus agalactiae bacteremia.

Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.

Análisis arqueomorfológico y dinámica territorial en el Vallés Oriental (Barcelona) de la Protohistoria s. VI-V a.C.) a la alta Edad Media (s. IX-X)

La investigación sobre el paisaje en zonas urbanas o periurbanas implica importantes limitaciones metodológicas. En el presente estudio, el trabajo se centra en un llano pre-litoral próximo a la ciudad de Barcelona, el Vallès Oriental, profundamente urbanizado en las últimas dos décadas, hecho que condiciona la recuperación de nuevos datos arqueológicos y la implementación de programas de prospección arqueológica. Asimismo, la particular topografía que presenta el llano, caracterizado por unos relieves suaves, ha obligado a adaptar la metodología del análisis arqueomorfológico a este contexto geográfico. El artículo presenta los resultados del análisis arqueomorfológico realizado, que han sido cruzados con la documentación histórica y arqueológica para caracterizar —desde una perspectiva diacrónica— la red viaria, la estructuración territorial y la evolución del poblamiento de esta área y, finalmente, determinar las dinámicas del paisaje en época romana. La investigación sobre la morfología del territorio se ha llevado a cabo a partir de un intenso trabajo de fotointerpretación y análisis de la cartografía histórica en entorno SIG, especialmente útil en un paisaje marcado por las importantes trasformaciones del medio rural. Igualmente, los datos generados en los últimos años por la investigación arqueológica han sido revisados de forma detallada, a fin de contribuir a la planificación de las prospecciones arqueológicas y arqueomorfológicas desarrolladas.

Del Mujal a Xanten: noves visions del comerç romà de vi de la Tarraconense

Ja fa uns anys des que J. Miró va publicar el seu llibre sobre les àmfores de la Tarraconense; des d’aleshores noves proves han canviat el panorama d’aquestes àmfores romanes catalanes. El present article tracta d’actualitzar el nostre coneixement actual sobre la pauta de consum i de distribució d’aquests envasos. Nous mètodes i dades quantificades i ben datades (per exemple, segells i fragments d’àmfora) ens permeten reconsiderar la distribució d’aquesta àmfora per l’occident de l’Imperi romà.

El mercat del vi espanyol als Estats Units

L’objecte d’estudi d’aquest projecte final, és el mercat del vi espanyol als Estats Units. Es tracta d’un mercat relativament jove degut a que la cultura del vi per un nord-americà amb una renda mitja es nova. Encara així, els Estats Units es el tercer país amb major consum per volum del món. Per tal de poder entendre millor aquest mercat, s’estudiaran les diferents característiques del país, les regions on es produeix el vi i la seva distribució i els diferents agents que s’impliquen en aquest procés en el país. Un cop analitzat els aspectes econòmics i les característiques més importants d’Estats Units, es realitzarà una anàlisi de l’oferta i de la demanda del sector del vi en aquest país per poder realitzar una comparativa més endavant amb el mercat del vi espanyol i de la perspectiva que es té a Estats Units sobre aquest tipus de vi. Un aspecte molt important que s’ha observat es la dificultat d’accés al mercat del vi a Estats Units. Per això, s’ha dedicat un capítol sencer a analitzar els diferents controls i regulacions que s’han de tenir en compte alhora d’importar vi a aquest país americà. Per últim, es realitza un anàlisi del mercat del vi espanyol i la perspectiva que es té dels vins espanyols a Estats Units. En aquest capítol s’analitzarà sobre tot la producció, el consum i les exportacions espanyoles i s’explicarà amb detall el concepte de denominacions d’origen.

Targeting Cytokines: Production and Characterization of Anti-TNF-α scFvs by Phage Display Technology.

The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF- α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF- α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μ g), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α , 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α . The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications.

Dictionnaire historique et critique. T. VI, Drabicius-Furius / de Pierre Bayle... ; éd. augm. de notes extraites de Chaufepié, Joly, La Monnoie, Leduchat, L.-J. Leclerc, Prosper Marchand, etc...

Collection : Archives de la linguistique française ; 30