Immunosuppressive effects of radiation on human dendritic cells: reduced IL-12 production on activation and impairment of naïve T-cell priming

Dendritic cells (DC) are professional antigen-presenting cells (APC) of the immune system, uniquely able to prime naïve T-cell responses. They are the focus of a range of novel strategies for the immunotherapy of cancer, a proportion of which include treating DC with ionising radiation to high dose. The effects of radiation on DC have not, however, been fully characterised. We therefore cultured human myeloid DC from CD14+ precursors, and studied the effects of ionising radiation on their phenotype and function. Dendritic cells were remarkably resistant against radiation-induced apoptosis, showed limited changes in surface phenotype, and mostly maintained their endocytic, phagocytic and migratory capacity. However, irradiated DC were less effective in a mixed lymphocyte reaction, and on maturation produced significantly less IL-12 than unirradiated controls, while IL-10 secretion was maintained. Furthermore, peptide-pulsed irradiated mature DC were less effective at naïve T-cell priming, stimulating fewer effector cells with lower cytotoxicity against antigen-specific targets. Hence irradiation of DC in vitro, and potentially in vivo, has a significant impact on their function, and may shift the balance between T-cell activation and tolerisation in DC-mediated immune responses

The use of mesenchymal stem cells for cartilage repair and regeneration: a systematic review.

BACKGROUND: The management of articular cartilage defects presents many clinical challenges due to its avascular, aneural and alymphatic nature. Bone marrow stimulation techniques, such as microfracture, are the most frequently used method in clinical practice however the resulting mixed fibrocartilage tissue which is inferior to native hyaline cartilage. Other methods have shown promise but are far from perfect. There is an unmet need and growing interest in regenerative medicine and tissue engineering to improve the outcome for patients requiring cartilage repair. Many published reviews on cartilage repair only list human clinical trials, underestimating the wealth of basic sciences and animal studies that are precursors to future research. We therefore set out to perform a systematic review of the literature to assess the translation of stem cell therapy to explore what research had been carried out at each of the stages of translation from bench-top (in vitro), animal (pre-clinical) and human studies (clinical) and assemble an evidence-based cascade for the responsible introduction of stem cell therapy for cartilage defects. This review was conducted in accordance to PRISMA guidelines using CINHAL, MEDLINE, EMBASE, Scopus and Web of Knowledge databases from 1st January 1900 to 30th June 2015. In total, there were 2880 studies identified of which 252 studies were included for analysis (100 articles for in vitro studies, 111 studies for animal studies; and 31 studies for human studies). There was a huge variance in cell source in pre-clinical studies both of terms of animal used, location of harvest (fat, marrow, blood or synovium) and allogeneicity. The use of scaffolds, growth factors, number of cell passages and number of cells used was hugely heterogeneous. SHORT CONCLUSIONS: This review offers a comprehensive assessment of the evidence behind the translation of basic science to the clinical practice of cartilage repair. It has revealed a lack of connectivity between the in vitro, pre-clinical and human data and a patchwork quilt of synergistic evidence. Drivers for progress in this space are largely driven by patient demand, surgeon inquisition and a regulatory framework that is learning at the same pace as new developments take place

Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability

The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics

Effects of Aspirin on Endothelial Function and Hypertension

PURPOSE OF REVIEW: Endothelial dysfunction is intimately related to the development of various cardiovascular diseases, including hypertension, and is often used as a target for pharmacological treatment. The scope of this review is to assess effects of aspirin on endothelial function and their clinical implication in arterial hypertension. RECENT FINDINGS: Emerging data indicate the role of platelets in the development of vascular inflammation due to the release of proinflammatory mediators, for example, triggered largely by thromboxane. Vascular inflammation further promotes oxidative stress, diminished synthesis of vasodilators, proaggregatory and procoagulant state. These changes translate into vasoconstriction, impaired circulation and thrombotic complications. Aspirin inhibits thromboxane synthesis, abolishes platelets activation and acetylates enzymes switching them to the synthesis of anti-inflammatory substances. SUMMARY: Aspirin pleiotropic effects have not been fully elucidated yet. In secondary prevention studies, the decrease in cardiovascular events with aspirin outweighs bleeding risks, but this is not the case in primary prevention settings. Ongoing trials will provide more evidence on whether to expand the use of aspirin or stay within current recommendations

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field