OBJECTIVES: Availability of donor lungs suitable for transplant falls short of current demand and contributes to waiting list mortality. Ex vivo lung perfusion (EVLP) offers the opportunity to objectively assess and recondition organs unsuitable for immediate transplant. Identifying robust biomarkers that can stratify donor lungs during EVLP to use or non-use or for specific interventions could further improve its clinical impact. METHODS: In this pilot study, 16 consecutive donor lungs unsuitable for immediate transplant were assessed by EVLP. Key inflammatory mediators and tissue injury markers were measured in serial perfusate samples collected hourly and in bronchoalveolar lavage fluid (BALF) collected before and after EVLP. Levels were compared between donor lungs that met criteria for transplant and those that did not. RESULTS: Seven of the 16 donor lungs (44%) improved during EVLP and were transplanted with uniformly good outcomes. Tissue and vascular injury markers lactate dehydrogenase, HMGB-1 and Syndecan-1 were significantly lower in perfusate from transplanted lungs. A model combining IL-1β and IL-8 concentrations in perfusate could predict final EVLP outcome after 2 h assessment. In addition, perfusate IL-1β concentrations showed an inverse correlation to recipient oxygenation 24 h post-transplant. CONCLUSIONS: This study confirms the feasibility of using inflammation and tissue injury markers in perfusate and BALF to identify donor lungs most likely to improve for successful transplant during clinical EVLP. These results support examining this issue in a larger study.
The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, highmolecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergenspecific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-kB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.Keywords: dendritic cell; hyaluronan; tolerance; allergy; T cell Clinical RelevanceWe describe a novel tolerogen that can be used to induce allergen-specific tolerance in animals sensitized previously to those allergens. These effects were durable for 2 weeks, providing an advantage over current desensitization protocols.
Canine atopic dermatitis (CAD) is known to involve IgE‐mediated mechanisms, but the role of IgG in the pathogenesis is less well understood. Previous studies have shown that total serum IgG concentrations are increased in CAD and suggested that a subclass of IgG, IgGd, is involved in the pathogenesis. Furthermore, induction of IgG blocking antibodies has been suggested as a possible mode of action of allergen‐specific immunotherapy (ASIT). The aim of this study was to characterize the changes in Dermatophagoides farinae (DF)‐specific total IgG and IgG subclasses during treatment of atopic dogs with ASIT. Twenty‐one dogs with confirmed atopic dermatitis and showing positive reactions to DF on intradermal testing were treated with alum‐precipitated vaccines for 9 months. Blood samples were collected before and after 3, 6, and 9 months of ASIT. Lyophilized whole‐body DF allergens were reconstituted to 1 mg/mL in sterile phosphate‐buffered saline (PBS), and SDS–PAGE was used to separate mite proteins. Separated proteins were transferred onto blotting membranes, cut into strips, and incubated with canine sera. For DF‐specific total IgG responses, strips were probed with horseradish peroxidase‐conjugated goat anti‐dog IgG and subsequently incubated with the colorimetric substrate DAB (3,3’‐diaminobenzidine). For DF‐specific IgG subclass responses, strips were probed sequentially with one of a panel of four monoclonal antibodies with specificity for each subclass, bovine anti‐mouse IgG conjugated to horseradish peroxidase, and finally, the chemiluminescent substrate ECL that was used for its higher sensitivity. Images of the positive bands were captured using Kodak Image Station, and the number of bands and their intensity was analysed by Kodak 1D Image Analysis Software. Prior to ASIT, a number of proteins of different molecular weights were recognized by antisera specific for total IgG and the subclasses IgG1 and IgG4, the most common being the high‐molecular‐weight chitinase, Df 15. There was virtually no IgG2 or IgG3 response to DF allergens. During ASIT, the total IgG response to DF allergens varied widely between dogs and could increase, decrease, fluctuate or remain the same. For each dog, the changes in total DF‐specific IgG were paralleled by equivalent changes in IgG1 and IgG4, but there was no induction of IgG2 or IgG3 antibodies. There were no significant increases in total IgG or IgG subclass responses in dogs showing a good response to ASIT. These findings suggest that atopic dogs mount an IgG1 and IgG4 response against multiple antigens of Dermatophagoides farinae, but these responses are not consistently augmented by ASIT. This suggests that successful ASIT in dogs is not associated with the production of blocking antibodies. Funding: Self‐funded.
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