Osteoprotegerin and receptor activator of nuclear factor-kappaB ligand system in the early post-operative period of liver transplantation

Abstract: Background: The precise mechanism that leads to accelerated bone resorption in the early post‐transplant period remains unclear. Recent data suggest that osteoprotegerin (OPG) and its ligand receptor activator of nuclear factor‐κB ligand (RANKL) constitute a novel cytokine system that can influence the function of both bone and immune cells. The aim of our study was to assess OPG and RANKL concentrations in the early post‐operative period of liver transplantation. Methods: Serum OPG and RANKL levels were measured in 30 patients who underwent liver transplantation at 1, 7 and 14 d post‐operatively. These values were compared with 22 age‐ and sex‐matched healthy controls. Plasma sodium, creatinine, aspartate‐aminotransferase, alanine‐amino transferase, γ‐glutamyl transferase, alkaline phosphatase, bilirubin, albumin, prothrombin time, tacrolimus and cyclosporine levels were measured in each patient. Results: We found a significant increase in OPG levels in the early post‐operative period compared with the control group: day 1 (10.42 pmol/L, range 3.80–17.50 vs. 3.91 pmol/L, range 1.20–6.60; p = 0.0001), day 7 (6.90 pmol/L, range 3.00–15.30 vs. 3.91 pmol/L, range 1.20–6.60; p = 0.0001) and day 14 (5.76 pmol/L, range 2.60–10.70 vs. 3.91 pmol/L, range 1.20–6.60; p = 0.001). Similarly, serum RANKL levels were significantly higher than in the control group in this period, day 1 (0.123 pmol/L, range 0.010–0.420 vs. 0.054 pmol/L, range 0.010–0.300; p = 0.02), day 7 (0.236 pmol/L, range 0.010–0.720 vs. 0.054 pmol/L, range 0.010–0.300; p = 0.0004) and day 14 (0.137 pmol/L, range 0.010–0.520 vs. 0.054 pmol/L, range 0.010–0.300; p = 0.007). No correlation was found between OPG levels and RANKL, ischemic times, liver function tests, albumin, sodium or creatinine concentrations and tacrolimus or cyclosporine levels. Conclusions: A significant amount of OPG and RANKL is released in the early post‐transplant period of liver transplantation. This might be explained by an activation of the immune system caused by the allograft. Therefore, the RANKL/OPG system may be involved in the pathophysiological evolution of transplantation osteoporosis.     

Enhancing circulation to lower limbs during head-down tilt by warming upper body and thighs

BACKGROUND: Long-duration spaceflight results in deconditioning of the cardiovascular system, loss of fluid volume, bone demineralization, and atrophy of skeletal muscles, particularly affecting the lower limbs. We hypothesized that it is possible to improve blood circulation to the lower extremities in simulated microgravity by forcing the blood to deliver heat to the feet through heating parts of the upper body and thighs. METHODS: In Study 1, seven men and four women were assessed in an environmental chamber with head-down tilt (HDT) at 14 degrees, wearing a newly developed shortened multi-compartment liquid cooling/warming garment (SLCWG) with local tubing networks covering parts of the head, torso, thigh, arms, and hands, with fingers, lower leg, and feet exposed. Study 2 was the same as Study 1 with a new cohort of four men and two women, and the assessment of toe blood perfusion on all subjects. Heat was applied as follows: Stage 1--SLCWG inlet water temperature 33 degrees C to stabilize comfort; Stage 2--inlet water temperature 8-10 degrees C (in combination with HDT) to reach a criterion of 25 degrees C finger temperature (Tfing); and Stage 3--inlet water temperature 45 degrees C to restore Tfing to 33 degrees C. RESULTS: Improvement of foot circulation by delivering more heat to the upper body and thighs was noted; increases in toe temperature (Ttoe) suggest enhanced perfusion. From Stage 2 to 3, there were significant increases in Ttoe (p < 0.05), a significant decrease in diastolic BP (DBP) (p < 0.05), and a significant change across stages in subjective perception of foot comfort (p < 0.001) and foot heat (p < 0.06). Further, toe blood perfusion increased significantly from Stage 2 to 3 (p < 0.05). CONCLUSIONS: Moderate partial heating of the upper body/thighs improved blood circulation in the feet in simulated microgravity by delivering heat to the lower extremities through restriction of heat exchange with the environment in the heated body parts. This technique could serve as a supplemental countermeasure for increasing blood circulation to the lower extremities.     

Copy number variation underlies complex phenotypes in domestic dog breeds and other canids

Extreme phenotypic diversity, a history of artificial selection, and socioeconomic value make domestic dog breeds a compelling subject for genomic research. Copy number variation (CNV) is known to account for a significant part of inter-individual genomic diversity in other systems. However, a comprehensive genome-wide study of structural variation as it relates to breed-specific phenotypes is lacking. We have generated whole genome CNV maps for more than 300 canids. Our data set extends the canine structural variation landscape to more than 100 dog breeds, including novel variants that cannot be assessed using microarray technologies. We have taken advantage of this data set to perform the first CNV-based genome-wide association study (GWAS) in canids. We identify 96 loci that display copy number differences across breeds, which are statistically associated with a previously compiled set of breed-specific morphometrics and disease susceptibilities. Among these, we highlight the discovery of a long-range interaction involving a CNV near MED13L and TBX3, which could influence breed standard height. Integration of the CNVs with chromatin interactions, long noncoding RNA expression, and single nucleotide variation highlights a subset of specific loci and genes with potential functional relevance and the prospect to explain trait variation between dog breeds.

Dogs have been the subject of intense study over many decades (Vilà et al. 1999; Ostrander and Wayne 2005; Freedman et al. 2014; Ostrander et al. 2019), providing valuable insight into human history, disease, and evolution (Coelho et al. 2018; Ní Leathlobhair et al. 2018; Wang et al. 2019). Much has been learned about canines through traditional approaches, including genotype studies with microsatellites (Irion 2003), single nucleotide polymorphisms (SNPs) (Gundry et al. 2007; Boyko et al. 2010; Vaysse et al. 2011), and, finally, whole genome sequencing (WGS) (Lindblad-Toh et al. 2005; Freedman et al. 2014; Plassais et al. 2019).As a result of the extensive history of genetic studies in dogs, remarkable advances have been made toward the resolution of the canine phylogeny (vonHoldt et al. 2010; Parker et al. 2017) and the temporal, geographic, and demographic history of dog domestication (Freedman et al. 2014; Shannon et al. 2015; Skoglund et al. 2015). Studies suggest that dogs were initially domesticated from gray wolves 15,000 to 40,000 yr ago (Freedman et al. 2014; Skoglund et al. 2015; Freedman and Wayne 2017; Ostrander et al. 2019), with a rapid diversification of breeds occurring within the past few hundred years. Currently, about 400 dog breeds exist worldwide, 193 recognized by the American Kennel Club and 360 by the Fédération Cynologique Internationale. Breed classification schemes have been proposed based on occupation, morphology, and geographic origin (American Kennel Club 2007; Wucher et al. 2017). The most recent genetic analysis, encompassing nearly 200 breeds and populations, suggests a monophyletic origin for most modern breeds and provides data regarding their origins and timing (Parker et al. 2017). Clusters of genetically similar breeds were identified and assigned to clades, which often reflected occupational and geographical origins.Targeted and genome-wide genotyping approaches have led to the discovery of nearly 400 variants associated with more than 270 traits, over 220 of which correspond to possible models for human diseases (Online Mendelian Inheritance in Animals [OMIA], Sydney School of Veterinary Science, https://omia.org/). Particularly, genome-wide association studies (GWASs) involving modest size cohorts of dogs have led to the identification of variants controlling a variety of morphological, behavioral, and disease traits (Akey et al. 2010; Vaysse et al. 2011; Rimbault et al. 2013; Hayward et al. 2016; MacLean et al. 2019; Plassais et al. 2019).The recent and intense artificial selective pressure exerted on dogs has induced pronounced inter-breed phenotypic differences while preserving intra-breed homogeneity. This process makes dogs of the same breed more likely to share not only morphometric traits but also disease susceptibilities (Karlsson and Lindblad-Toh 2008; Chase et al. 2009; Akey et al. 2010; Boyko et al. 2010; Marchant et al. 2017; Mansour et al. 2018; Ostrander et al. 2019). The level of anatomic similarity among dogs of any one breed is sufficiently strong that genetic studies have been successfully executed using breed standards as phenotypes, thus unraveling the genetic bases of some complex traits such as body size or behavior (Akey et al. 2010; Boyko et al. 2010; Vaysse et al. 2011; Hayward et al. 2016; MacLean et al. 2019; Plassais et al. 2019), which remain elusive, even in humans.However, all these analyses have been performed using a subset of indicative SNPs and, more recently, SNPs from WGSs (Jagannathan et al. 2019; Plassais et al. 2019), but other forms of genomic variation have rarely been studied systematically. In fact, there is still a lack of fine-scale, genome-wide analyses of any variants other than SNPs across dog breeds, a notable exception when compared to humans and other model organisms (Yalcin et al. 2011; Brown et al. 2012; Sudmant et al. 2015). Copy number variation (CNV) has been previously studied in canines to elucidate specific phenotypes (Karyadi et al. 2013; Arendt et al. 2014; Waldo and Diaz 2015; Deane-Coe et al. 2018). However, most studies have focused on the comparison of dogs and wolves using array-based technologies, rather than undertaking a comprehensive and unbiased examination of all CNVs across the genome of distinct breeds (Berglund et al. 2012; Schoenebeck et al. 2012). Most CNV-related studies published to date only aimed to identify segmentally duplicated regions and did not aim to produce quantitative copy-number (CN) genotypes (Quilez et al. 2012; Molin et al. 2014). Knowing the exact number of copies at a locus is crucial for an accurate comparison of closely related organisms, such as distinct dog breeds and wild canids.Here, we present a fine-scale CNV map of over 300 canid samples using WGS to produce the most extensive, high-resolution CNV panel in dogs to date. We examine more than 145 individual breeds, as well as nonbreed dogs, including village dogs, dingoes, captive New Guinea singing dogs, and wild canids such as wolves. We employ this data set to determine the ability of CNVs to recreate a current dog phylogeny. Moreover, we test for breed-phenotype associations using an extensive data set of breed standards as individual phenotypes in the first CNV-based GWAS performed in dogs to date.     

The Effect of Obesity Class on the Energetics and Mechanics of Walking

Higher mass-normalized net energy cost of walking (NetC_w/kg) and mechanical pendular recovery are observed in obese compared to lean adults. This study aimed to investigate the effect of different classes of obesity on the energetics and mechanics of walking and to explore the relationships between body mass, NetC_w/kg and gait mechanics by using principal component analysis (PCA). NetC_w/kg and gait mechanics were computed in severely obese (SOG; n = 18, BMI = 40.1 ± 4.4 kg·m⁻²), moderately obese (MOG; n = 17, BMI = 32.2 ± 1.5 kg·m⁻²) and normal-weight (NWG; n = 13, BMI = 22.0 ± 1.5 kg·m⁻²) adults during five walking trials (0.56, 0.83, 1.11, 1.39, 1.67 m·s⁻¹) on an instrumented treadmill. NetC_w/kg was significantly higher in SOG compared to NWG (p = 0.019), with no significant difference between SOG and MOG (p = 0.14), nor between MOG and NWG (p = 0.27). Recovery was significantly higher in SOG than in NWG (p = 0.028), with no significant difference between SOG and MOG (p = 0.13), nor between MOG and NWG (p = 0.35). PCA models explained between 17.0% and 44.2% of the data variance. This study showed that: (1) obesity class influences the gait energetics and mechanics; (2) PCA was able to identify two components, showing that the obesity class is associated with lower walking efficiency and better pendulum-like characteristics.     

Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis

Myofibroblasts infiltrate malignant liver tumors, although their pathogenic implications are unclear. Immunohistochemical detection of alpha-smooth muscle actin, glial fibrillary acidic protein (GFAP), and CD31 and CD34 expression was used to analyze the contribution of myofibroblasts to angiogenesis in hepatic metastasis produced by intrasplenically-injected B16 melanoma (B16M). Because activated hepatic stellate cells (HSCs) are oxygen-sensing myofibroblasts producing vascular endothelial growth factor (VEGF), the effect of B16M and human A375 melanoma supernatants on VEGF production by immortalized rat HSC line T6 and primary cultured human HSCs also was studied under an hypoxic atmosphere mimicking a tumor microenvironment. Myofibroblast infiltration preceded endothelium recruitment in avascular micrometastasis and generated specific stroma for sinusoidal-type and portal-type angiogeneses. Thereafter, myofibroblasts and endothelial cells colocalized within both angiogenic patterns and their numerical densities correlated with metastasis development. Myofibroblasts often were GFAP-positive, suggesting an HSC origin. Melanoma supernatants stimulated VEGF messenger RNA and protein synthesis by HSCs. These effects were potentiated by hypoxia. VEGF up-regulation was accompanied by increased expression of cyclooxygenase type 2 (COX-2) and PGE2 synthesis. HSC production of VEGF decreased under COX-2 inhibition, whereas it was increased by exogenous PGE2. The high VEGF expression in HSCs induced by melanoma factors and hypoxia resulted in mitogenic, antiapoptotic, and motogenic stimulation of both murine hepatic sinusoidal endothelium and human umbilical vein endothelium. In conclusion, temporal and positional relationships evolve between myofibroblast and endothelium recruitment during metastasis development. Mechanistically, hypoxic induction of VEGF in tumor-activated HSCs may create a proangiogenic microenvironment, facilitating endothelial cell recruitment and survival during hepatic metastasis transition from an avascular to a vascular stage.