Criteria for Viability Assessment of Discarded Human Donor Livers during Ex Vivo Normothermic Machine Perfusion

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37°C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial.     

Age-Related Longitudinal Changes in Metabolic Energy Expenditure during Walking in Boys with Duchenne Muscular Dystrophy

Objective

The aim of this study was to evaluate age-related changes in metabolic walking energy expenditure in ambulant boys affected by Duchenne muscular dystrophy over a follow-up period of 12 months.

Methods

At baseline (T1) and 12 months later (T2), metabolic walking energy expenditure was assessed during a 6-minute walk test at comfortable speed in 14 ambulant boys with Duchenne (age range: 6.0-12.5 years, mean 8.2). Outcome measures derived from the assessment included the 6-minute comfortable walking distance (m) and net-nondimensional energy cost relative to speed-matched control cost (SMC-EC, %). Statistical comparisons were made using a two-way repeated measures ANOVA (factors: time (T1 versus T2) and age (<8 years of age (yoa) versus ≥8 yoa)).

Results

Over the course of the study, a significant decrease of -28m (−8.2%, p = 0.043) was noted in the walked distance at comfortable speed. Besides, SMC-EC increased with 4.4%, although this change was not significant (p = 0.452). Regarding age groups, boys below 8 yoa showed a smaller annual decrease in the walked distance (−15 m) compared to boys above 8 yoa (−37 m). SMC-EC increased with 10% in the older boys, while in the younger boys it decreased (−2.1%). The main effect of age group on walking distance and SMC-EC however was not significant (p>0.158), and also there were no interaction effects (p>0.248).

Conclusions

The results of our small study suggest that the natural course of walking performance in ambulant boys with Duchenne is characterized by a decrease in comfortable walking distance and an increase in walking energy cost. The rate of energy cost seems to increase with age, while walking distance decreases, which is opposite from the trend in typically developing children.     

A Conserved Mitochondrial ATP-binding Cassette Transporter Exports Glutathione Polysulfide for Cytosolic Metal Cofactor Assembly

An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe²⁺ alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.     

Panmixia defines the genetic diversity of a unique arthropod‐dispersed fungus specific to Protea flowers

Knoxdaviesia proteae, a fungus specific to the floral structures of the iconic Cape Floral Kingdom plant, Protea repens, is dispersed by mites phoretic on beetles that pollinate these flowers. Although the vectors of K. proteae have been identified, little is known regarding its patterns of distribution. Seed bearing infructescences of P. repens were sampled from current and previous flowering seasons, from which K. proteae individuals were isolated and cultured. The genotypes of K. proteae isolates were determined using 12 microsatellite markers specific to this species. Genetic diversity indices showed a high level of similarity between K. proteae isolates from the two different infructescence age classes. The heterozygosity of the population was high (0.74 ± 0.04), and exceptional genotypic diversity was encountered (Ĝ = 97.87%). Population differentiation was negligible, owing to the numerous migrants between the infructescence age classes (N_m = 47.83) and between P. repens trees (N_m = 2.96). Parsimony analysis revealed interconnected genotypes, indicative of recombination and homoplasies, and the index of linkage disequilibrium confirmed that outcrossing is prevalent in K. proteae ( = 0.0067; P = 0.132). The high diversity and panmixia in this population is likely a result of regular gene flow and an outcrossing reproductive strategy. The lack of genetic cohesion between individuals from a single P. repens tree suggests that K. proteae dispersal does not primarily occur over short distances via mites as hypothesized, but rather that long-distance dispersal by beetles plays an important part in the biology of these intriguing fungi.     

Interphase in situ hybridization to disaggregated and intact tissue specimens of prostatic adenocarcinomas

A comparative study was performed of interphase in situ hybridization (ISH) to deparaffinized 4-m tissue sections and nuclear suspensions from eight prostatic adenocarcinomas, as well as one normal prostatic control. Whole nuclear suspensions were derived from the same tumor areas to evaluate differences of ISH to truncated versus whole nuclei. DNA probes specific for the centromeres of chromosome 1, 7, 8, 10, and Y were used for detection of numerical chromosomal changes and aneuploidy. In six adenocarcinomas chromosome aberrations (+7, +8, –8, –10, –Y) were seen. However, ISH to sections revealed focal aberrations (–10, –Y) in four cases that could not be distinguished in the suspensions. Chromosomal alterations occurring in larger tumor areas were also detected in the nuclear suspensions. Chromosome copy number changes, especially gains, were better discriminated in the nuclear suspensions. The rate of ISH aneuploidy seen in nuclear suspensions corresponded with that observed in the tissue sections (P<0.01). Ploidy patterns as assessed by ISH to sections and nuclear suspensions were in concordance with DNA flow cytometry (bothP<0.001). We conclude that both section and suspension ISH were able to accurately detect aneuploidy and numerical chromosomal aberrations occurring in larger histological areas. However, section ISH was also capable of revealing (small) focal cytogenetic abnormalities, due to a precise analysis of only target cells. Focal abnormalities were not detected by suspension ISH, probably due to an admixture of non-aberrant tumor cells and stromal elements.     

Postischemic injury in isolated rat hearts is not aggravated by prior depletion of myocardial glutathione

The aim of this study was to test the hypothesis that a decreased myocardial concentration of reduced glutathione (GSH) during ischemia renders the myocardium more susceptible to injury by reactive oxygen species generated during early reperfusion. To this end, rats were pretreated with L-buthionine-S,R-sulfoximine (2 mmol/kg), which depleted myocardial GSH by 55%. Isolated buffer-perfused hearts were subjected to 30 min of either hypothermic or normothermic no-flow ischemia followed by reperfusion. Prior depletion of myocardial GSH did not lead to oxidative stress during reperfusion, as myocardial concentration of glutathione disulfide (GSSG) was not increased after 5 and 30 min of reperfusion. In addition, prior depletion of GSH did not exacerbate myocardial enzyme release, nor did it impair the recoveries of tissue ATP, coronary flow rate and left ventricular developed pressure during reperfusion after either hypothermic or normothermic ischemia. Even administration of the prooxidant cumene hydroperoxide (20 M) to postischemic GSH-depleted hearts during the first 10 min of reperfusion did not aggravate postischemic injury, although this prooxidant load induced oxidative stress, as indicated by an increased myocardial concentration of GSSG. These results do not support the hypothesis that a reduced myocardial concentration of GSH during ischemia increases the susceptibility to injury mediated by reactive oxygen species generated during reperfusion. Apparently, myocardial tissue possesses a large excess of GSH compared to the quantity of reactive oxygen species generated upon reperfusion. (Mol Cell Biochem 156: 79-85, 1996)     

Prevalence of peripheral and extra-articular disease in ankylosing spondylitis versus non-radiographic axial spondyloarthritis: a meta-analysis

BackgroundPeripheral disease (arthritis, enthesitis and dactylitis) and extra-articular disease (uveitis, psoriasis and inflammatory bowel disease) is common in ankylosing spondylitis (AS) and non-radiographic axial spondyloarthritis (nr-axSpA). So far, however, summary data on the prevalence are lacking. The objective of this meta-analysis was to assess the prevalence of peripheral and extra-articular manifestations in ankylosing spondylitis (AS) and nr-axSpA.MethodsWe performed a systematic literature search to identify publications describing the prevalence of peripheral and extra-articular disease manifestations in patients with AS and nr-axSpA. We assessed the risk of bias and between-study heterogeneity, and extracted data. Pooled prevalence and prevalence differences were calculated.ResultsEight studies comprising 2236 patients with AS and 1242 with nr-axSpA were included: 7 of the studies were longitudinal cohort studies. There was male predominance in AS (70.4 %, 95 % CI 64.4, 76.0 %) but not in nr-axSpA (46.8 %, 95 % CI 41.7, 51.9), which was independent of the prevalence of human leukocyte antigen (HLA)-B27. The prevalence of HLA-B27 was similar in AS (78.0 % (95 % CI 73.9, 81.9 %) and nr-axSpA (77.4 %, 95 % CI 68.9, 84.9 %)). The pooled prevalence of arthritis (29.7 % (95 % CI 22.4, 37.4 %) versus 27.9 % (95 % CI 16.0, 41.6 %)), enthesitis (28.8 % (95 % CI 2.6, 64.8) versus 35.4 % (95 % CI 6.1, 71.2)). dactylitis (6.0 % (95 % CI 4.7, 7.5 %) versus 6.0 % (95 % CI 1.9, 12.0 %)), psoriasis (10.2 % (95 % CI 7.5, 13.2 %) versus 10.9 % (95 % CI 9.1, 13.0 %)) and inflammatory bowel disease (4.1 % (95 % CI 2.3, 6.5 %) versus 6.4 % (95 % CI 3.6, 9.7 %)) were similar in AS and nr-axSpA. The pooled prevalence of uveitis was higher in AS (23.0 % (95 % CI 19.2, 27.1 %)) than in nr-axSpA (15.9 % (95 % CI 11.8, 20.4 %)).ConclusionPeripheral and extra-articular manifestations are equally prevalent in AS and nr-axSpA, except for uveitis, which is slightly more prevalent in AS. These data provide evidence for the largely equal nature of disease manifestations in nr-axSpA and AS.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-016-1093-z) contains supplementary material, which is available to authorized users.