Correction to: Assessment of cetacean–fishery interactions in the marine food web of the Gulf of Taranto (Northern Ionian Sea,Central Mediterranean Sea)

Reviews in Fish Biology and Fisheries - In the original publication of the article, the given name and surname of the authors are inverted in the author’s affiliation and in the citation of...     

Effective use of nitrocellulose-blotted antigens for phage display monoclonal antibody selection

The combinatorial phage display library approach to antibody repertoire cloning offers a powerful tool for the isolation of specific antibodies to defined target antigens. Panning strategy is often a very critical point for selecting antibody displayed on the surface of bacteriophages. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well defined antigen source. However, when the antigen is difficult to purify by means of laborious and time-consuming chromatography procedures, panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental complications. In the present work, we successfully generated a mouse monoclonal antibody fragment from a phage display library directed against protein E7 of HPV18 avoiding antigen purification as for immunizing mice as for antibody library selection. Our work demonstrates the feasibility of phage antibody selections on antigens transferred to a nitrocellulose membrane as solid support, using one-dimensional polyacrylamide gel electrophoresis system as the only practice to separate a given antigen present in bacterial crude cell lysate.     

The Cardioprotective Protein Apolipoprotein A1 Promotes Potent Anti-tumorigenic Effects

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b⁺ F4/80⁺ macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.     

Short-term effects of soil solarization in suppressing Calonectria microsclerotia

Background and aims

Calonectria species have been reported as devastating pathogens mostly on horticultural and forest crops worldwide. Since these pathogens represent a serious threat for the nursery production, the aim of this study was to investigate on the short-term potential of soil solarization for eradicating Calonectria microsclerotia.

Methods

Twenty Calonectria isolates collected in Italy from different hosts and locations were identified by using DNA sequencing of β-tubulin. The effect of thermal regimes and innovative solarizing films on the soil survival of Calonectria microsclerotia was evaluated through time at different sampling periods in growth chamber and greenhouse experiments.

Results

Eleven and nine isolates were identified as Calonectria pauciramosa and Calonectria polizzii, respectively. No viable Calonectria inoculum was recovered after 12 days from all solarized plots inside ethylene-tetrafluoroethylene (ETFE) greenhouse and at 15-cm depth from solarized plots inside ethylene-vinyl-acetate (EVA) greenhouse. Under EVA cover, solarization killed C. pauciramosa microsclerotia within 9 and 17 d at 15- and 30-cm depths in soil, respectively, whereas no viable inoculum was retrieved within 6 and 12 days from solarized plots inside ETFE greenhouse.

Conclusions

This paper demonstrates that short-term soil solarization is effective for Calonectria microsclerotia suppression in nurseries, and shows that ETFE film as well as other innovative materials could improve this technique.     

Efficient ex vivo induction of T cells with potent anti-tumor activity by protein antigen encapsulated in nanoparticles

Protein antigen (Ag)-based immunotherapies have the advantage to induce T cells with a potentially broad repertoire of specificities. However, soluble protein Ag is generally poorly cross-presented in MHC class I molecules and not efficient in inducing robust cytotoxic CD8⁺ T cell responses. In the present study, we have applied poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) which strongly improve protein Ag presentation by dendritic cells (DC) in the absence of additional Toll-like receptor ligands or targeting devices. Protein Ag-loaded DC were used as antigen presenting cells to stimulate T cells in vitro and subsequently analyzed in vivo for their anti-tumor effect via adoptive transfer, a treatment strategy widely studied in clinical trials as a therapy against various malignancies. In a direct comparison with soluble protein Ag, we show that DC presentation of protein encapsulated in plain PLGA-NP results in efficient activation of CD4⁺ and CD8⁺ T cells as reflected by high numbers of activated CD69⁺ and CD25⁺, interferon (IFN)-γ and interleukin (IL)-2-producing T cells. Adoptive transfer of PLGA-NP-activated CD8⁺ T cells in tumor-bearing mice displayed good in vivo expansion capacity, potent Ag-specific cytotoxicity and IFN-γ cytokine production, resulting in curing mice with established tumors. We conclude that delivery of protein Ag through encapsulation in plain PLGA-NP is a very efficient and simple procedure to stimulate potent anti-tumor T cells.     

Phenotypic and Genotypic Characterisation of Burkholderia cenocepacia J2315 Mutants Affected in Homoserine Lactone and Diffusible Signal Factor-Based Quorum Sensing Systems Suggests Interplay between Both Types of Systems

Many putative virulence factors of Burkholderia cenocepacia are controlled by various quorum sensing (QS) circuits. These QS systems either use N-acyl homoserine lactones (AHL) or cis-2-dodecenoic acid (“Burkholderia diffusible signal factor”, BDSF) as signalling molecules. Previous work suggested that there is little cross-talk between both types of systems. We constructed mutants in B. cenocepacia strain J2315, in which genes encoding CepI (BCAM1870), CciI (BCAM0239a) and the BDSF synthase (BCAM0581) were inactivated, and also constructed double (ΔcepIΔBCAM0581, ΔcciIΔBCAM0581 and ΔcepIΔcciI) mutants and a triple (ΔcepIΔcciIΔBCAM0581) mutant. Subsequently we investigated phenotypic properties (antibiotic susceptibility, biofilm formation, production of AHL and BDSF, protease activity and virulence in Caenorhabditis elegans) and measured gene expression in these mutants, and this in the presence and absence of added BDSF, AHL or both. The triple mutant was significantly more affected in biofilm formation, antimicrobial susceptibility, virulence in C. elegans, and protease production than either the single or double mutants. The ΔBCAM0581 mutant and the ΔcepIΔBCAM0581 and ΔcciIΔBCAM0581 double mutants produced significantly less AHL compared to the WT strain and the ΔcepI and ΔcciI single mutant, respectively. The expression of cepI and cciI in ΔBCAM0581, was approximately 3-fold and 7-fold (p<0.05) lower than in the WT, respectively. The observed differences in AHL production, expression of cepI and cciI and QS-controlled phenotypes in the ΔBCAM0581 mutant could (at least partially) be restored by addition of BDSF. Our data suggest that, in B. cenocepacia J2315, AHL and BDSF-based QS systems co-regulate the same set of genes, regulate different sets of genes that are involved in the same phenotypes and/or that the BDSF system controls the AHL-based QS system. As the expression of the gene encoding the C6-HSL synthase CciI (and to a lesser extent the C8-HSL synthase CepI) is partially controlled by BDSF, it seems likely that the BDSF QS systems controls AHL production through this system.     

Subcutaneous Interferon β-1a May Protect against Cognitive Impairment in Patients with Relapsing–Remitting Multiple Sclerosis: 5-Year Follow-up of the COGIMUS Study

Objective

To assess the effects of subcutaneous (sc) interferon (IFN) -1a on cognition over 5 years in mildly disabled patients with relapsing–remitting multiple sclerosis (RRMS).

Methods

Patients aged 18–50 years with RRMS (Expanded Disability Status Scale score ≤4.0) who had completed the 3-year COGIMUS study underwent standardized magnetic resonance imaging, neurological examination, and neuropsychological testing at years 4 and 5. Predictors of cognitive impairment at year 5 were identified using multivariate analysis.

Results

Of 331 patients who completed the 3-year COGIMUS study, 265 participated in the 2-year extension study, 201 of whom (75.8%; sc IFN β-1a three times weekly: 44 µg, n = 108; 22 µg, n = 93) completed 5 years'' follow-up. The proportion of patients with cognitive impairment in the study population overall remained stable between baseline (18.0%) and year 5 (22.6%). The proportion of patients with cognitive impairment also remained stable in both treatment groups between baseline and year 5, and between year 3 and year 5. However, a significantly higher proportion of men than women had cognitive impairment at year 5 (26.5% vs 14.4%, p = 0.046). Treatment with the 22 versus 44 µg dose was predictive of cognitive impairment at year 5 (hazard ratio 0.68; 95% confidence interval 0.48–0.97).

Conclusions

This study suggests that sc IFN β-1a dose-dependently stabilizes or delays cognitive impairment over a 5-year period in most patients with mild RRMS. Women seem to be more protected against developing cognitive impairment, which may indicate greater response to therapy or the inherently better prognosis associated with female sex in MS.     

Absence of TLR4 Reduces Neurovascular Unit and Secondary Inflammatory Process after Traumatic Brain Injury in Mice

Background

Traumatic brain injury (TBI) initiates a neuroinflammatory cascade that contributes to neuronal damage and behavioral impairment. Toll-like receptors (TLRs) are signaling receptors in the innate immune system, although emerging evidence indicates their role in brain injury. We have therefore investigated the role played by TLR4 signaling pathway in the development of mechanisms of secondary inflammatory process in traumatic brain injury (TBI) differ in mice that lack a functional TLR4 signaling pathway.

Methods/Principal Findings

Controlled cortical impact injury was performed on TLR4 knockout (KO) mice (C57BL/10ScNJ) and wild-type (WT) mice (C57BL/10ScNJ). TBI outcome was evaluated by determination of infarct volume and assessment of neurological scores. Brains were collected at 24 h after TBI. When compared to WT mice, TLR4 KO mice had lower infarct volumes and better outcomes in neurological and behavioral tests (evaluated by EBST and rotarod test). Mice that lacked TLR4 had minor expression of TBI-induced GFAP, Chymase, Tryptase, IL-1β, iNOS, PARP and Nitrotyrosine mediators implicated in brain damage. The translocation of expression of p-JNK, IκB-α and NF-κB pathway were also lower in brains from TLR4 KO mice. When compared to WT mice, resulted in significant augmentation of all the above described parameters. In addition, apoptosis levels in TLR4 KO mice had minor expression of Bax while on the contrary with Bcl-2.

Conclusions/Significance

Our results clearly demonstrated that absence of TLR4 reduces the development of neuroinflammation, tissues injury events associated with brain trauma and may play a neuroprotective role in TBI in mice.