Profiling inflammation and tissue injury markers in perfusate and bronchoalveolar lavage fluid during human ex vivo lung perfusion.

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-1ß and IL-8 concentrations in perfusate could predict final EVLP outcome after 2 h assessment. In addition, perfusate IL-1ß 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 effect of ex vivo lung perfusion on microbial load in human donor lungs.

BACKGROUND: Ex vivo lung perfusion (EVLP) has emerged as a technique to potentially recondition unusable donor lungs for transplantation. Beneficial effects of EVLP on physiologic function have been reported, but little is known about the effect of normothermic perfusion on the infectious burden of the donor lung. In this study, we investigated the effect of EVLP on the microbial load of human donor lungs. METHODS: Lungs from 18 human donors considered unusable for transplantation underwent EVLP with a perfusate containing high-dose, empirical, broad-spectrum anti-microbial agents. Quantitative cultures of bacteria and fungi were performed on bronchoalveolar lavage fluid from the donor lung before and after 3 to 6 hours of perfusion. The identification of any organisms and changes in number of colony forming units before and after EVLP were assessed and anti-microbial susceptibilities identified. RESULTS: Thirteen out of 18 lungs had positive cultures, with bacterial loads significantly decreasing after EVLP. Yeast loads increased when no anti-fungal treatment was given, but were reduced when prophylactic anti-fungal treatment was added to the circuit. Six lungs were ultimately transplanted into patients, all of whom survived to hospital discharge. There was 1 death at 11 months. CONCLUSIONS: Our study shows that EVLP with high-dose, empirical anti-microbial agents in the perfusate is associated with an effective reduction in the microbial burden of the donor lung, a benefit that has not previously been demonstrated.

Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation.

The recruitment of T lymphocytes during diseases such as rheumatoid arthritis is regulated by stimulation of the chemokine receptors expressed by these cells. This study was designed to assess the potential of a CXCR3-specific small-molecule agonist to inhibit the migration of activated human T cells toward multiple chemokines. Further experiments defined the molecular mechanism for this anti-inflammatory activity. Analysis in vitro demonstrated agonist induced internalization of both CXCR3 and other chemokine receptors coexpressed by CXCR3(+) T cells. Unlike chemokine receptor-specific antagonists, the CXCR3 agonist inhibited migration of activated T cells toward the chemokine mixture in synovial fluid from patients with active rheumatoid arthritis. A humanized mouse air-pouch model showed that intravenous treatment with the CXCR3 agonist prevented inflammatory migration of activated human T cells toward this synovial fluid. A potential mechanism for this action was defined by demonstration that the CXCR3 agonist induces receptor cross-phosphorylation within CXCR3-CCR5 heterodimers on the surface of activated T cells. This study shows that generalized chemokine receptor desensitization can be induced by specific stimulation of a single chemokine receptor on the surface of activated human T cells. A humanized mouse model was used to demonstrate that this receptor desensitization inhibits the inflammatory response that is normally produced by the chemokines present in synovial fluid from patients with active rheumatoid arthritis.

Xenobiotic incorporation into pyruvate dehydrogenase complex can occur via the exogenous lipoylation pathway.

UNLABELLED: Lipoylated enzymes such as the E2 component of pyruvate dehydrogenase complex (PDC-E2) are targets for autoreactive immune responses in primary biliary cirrhosis, with lipoic acid itself forming a component of the dominant auto-epitopes. A candidate mechanism for the initiation of tolerance breakdown in this disease is immune recognition of neo-antigens formed by xenobiotic substitution of normal proteins. Importantly, sensitization with proteins artificially substituted with the lipoic acid analogue xenobiotic 6-bromohexanoic acid (6BH) can induce an immune response that cross-reacts with PDC-E2. This study investigated the potential of recombinant lipoylation enzymes lipoate activating enzyme and lipoyl-AMP(GMP):N-lysine lipoyl transferase to aberrantly incorporate xenobiotics into PDC-E2. It was found that these enzymes could incorporate lipoic acid analogues including octanoic and hexanoic acids and the xenobiotic 6BH into PDC-E2. The efficiency of incorporation of these analogues showed a variable dependence on activation by adenosine triphosphate (ATP) or guanosine triphosphate (GTP), with ATP favoring the incorporation of hexanoic acid and 6BH whereas GTP enhanced substitution by octanoic acid. Importantly, competition studies showed that the relative incorporation of both 6BH and lipoic acid could be regulated by the balance between ATP and GTP, with the formation of 6BH-substituted PDC-E2 predominating in an ATP-rich environment. CONCLUSION: Using a well-defined system in vitro we have shown that an important xenobiotic can be incorporated into PDC in place of lipoic acid by the exogenous lipoylation system; the relative levels of lipoic acid and xenobiotic incorporation may be determined by the balance between ATP and GTP. These observations suggest a clear mechanism for the generation of an auto-immunogenic neo-antigen of relevance for the pathogenesis of primary biliary cirrhosis.