Preparation of silk fibroin–poly(ethylene glycol) conjugate films through click chemistry

Azide silk fibroin (azido SF) and alkyne terminal poly(ethylene glycol) (PEG) 2000 (acetylene-terminal PEG 2000) were synthesized. Azido SF was reacted with acetylene-terminal PEG 2000 to produce films via a copper-mediated 1,3-cycloaddition (‘click’ chemistry) generating a triazole linkage as the networking forming reaction. Through click chemistry, novel silk-based films with various weight ratios were prepared and investigated. Fourier transform infrared, X-ray diffraction and differential scanning calorimetry analyses showed that the ordered association of the PEG molecules is strongly constrained by the presence of the SF molecules and crosslinking and that the presence of acetylene-terminal PEG 2000 in the films induced crystallization to a β-sheet of SF chains.Water content and contact angle measurements indicated that the hydrophilicity of the films increased compared with SF. SF–PEG films exhibited smooth and rough structures, depending on degree of crosslinking and on the weight ratio of SF and PEG, as shown by scanning electron microscopy

Heterogeneity assessment of functional T cell avidity.

The potency of cellular immune responses strongly depends on T cell avidity to antigen. Yet, functional avidity measurements are rarely performed in patients, mainly due to the technical challenges of characterizing heterogeneous T cells. The mean functional T cell avidity can be determined by the IFN-γ Elispot assay, with titrated amounts of peptide. Using this assay, we developed a method revealing the heterogeneity of functional avidity, represented by the steepness/hillslope of the peptide titration curve, documented by proof of principle experiments and mathematical modeling. Our data show that not only natural polyclonal CD8 T cell populations from cancer patients, but also monoclonal T cells differ strongly in their heterogeneity of functional avidity. Interestingly, clones and polyclonal cells displayed comparable ranges of heterogeneity. We conclude that besides the mean functional avidity, it is feasible and useful to determine its heterogeneity (hillslope) for characterizing T cell responses in basic research and patient investigation

Relevance of the Diversity among Members of the Trypanosoma Cruzi Trans-Sialidase Family Analyzed with Camelids Single-Domain Antibodies

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8+ T-Cell Response: Reversal by Adenoviral Vaccine

MHC class Ia-restricted CD8+ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8+ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8+ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8+ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8+ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8+ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8+ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination

Wound dressings for a proteolytic-rich environment

Wound dressings have experienced continuous and significant changes over the years based on the knowledge of the biochemical events associated with chronic wounds. The development goes from natural materials used to just cover and conceal the wound to interactive materials that can facilitate the healing process, addressing specific issues in non-healing wounds. These new types of dressings often relate with the proteolytic wound environment and the bacteria load to enhance the healing. Recently, the wound dressing research is focusing on the replacement of synthetic polymers by natural protein materials to delivery bioactive agents to the wounds. This article provides an overview on the novel protein-based wound dressings such as silk fibroin keratin and elastin. The improved properties of these dressings, like the release of antibiotics and growth factors, are discussed. The different types of wounds and the effective parameters of healing process will be reviewed

2017 HRS/EHRA/ECAS/APHRS/SOLAECE expert consensus statement on catheter and surgical ablation of atrial fibrillation: executive summary.

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2017 HRS/EHRA/ECAS/APHRS/SOLAECE expert consensus statement on catheter and surgical ablation of atrial fibrillation: executive summary.

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