Hepatic fibrosis and cirrhosis cause strong human suffering and necessitate a monetary burden worldwide. Therefore, there is an urgent need for the development of therapies. Pre-clinical animal models are indispensable in the drug discovery and development of new anti-fibrotic compounds and are immensely valuable for understanding and proofing the mode of their proposed action. In fibrosis research, inbreed mice and rats are by far the most used species for testing drug efficacy. During the last decades, several hundred or even a thousand different drugs that reproducibly evolve beneficial effects on liver health in respective disease models were identified. However, there are only a few compounds  that were translated from bench to bedside. In contrast, the large number of drugs successfully tested in animal studies is repeatedly tested over and over engender findings with similar or identical outcome. This circumstance undermines the 3R  principle of Russell and Burch that was introduced to minimize the suffering of laboratory animals. This ethical framework, however, represents the basis of the new animal welfare regulations in the member states of the European Union. Consequently, the legal authorities in the different countries are halted to foreclose testing of drugs in animals that were successfully tested before. This review provides a synopsis on anti-fibrotic compounds that were tested in classical rodent models. Their mode of action, potential sources and the observed beneficial effects on liver health are discussed. This review attempts to provide a reference compilation for all those involved in the testing of drugs or in the design of new clinical trials targeting hepatic fibrosis. HoweverIn the present review, a comprehensive synopsis on experimentally tested anti-fibrotic compounds is given. The chemical structure, potential sources, mode of action, molecular target and their experimental pharmacological activity in hepatic fibrogenesis of each drug is discussed.4) to induce liver fibrosis, they showed reduced hepatic inflammation than wild type mice. Moreover, culture-activated HSC derived from these mice had overall reduced expression of pro-fibrogenic genes and the dual NOX1/4 inhibitor GKT137831 suppressed ROS production and expression of inflammation-associated genes . In cultured HSC, TGF-β increases the production of H2O2  formation is one key driver of hepatic inflammation and fibrosis. Under normal physiological conditions, oxygen-containing reactive molecules control key physiological activities such as cell growth, proliferation, migration, differentiation, and apoptosis  contains two sulfur atoms that are connected by a redox-sensitive disulfide bond. It functions as a cofactor for some enzyme systems involved in acyl group transfer. Independent studies have shown that α-lipoic acid and its reduced form dihydrolipoic acid inhibit liver fibrosis in rats chronically treated with thioacetamide (TAA), most likely by preventing ROS generation and ROS-mediated signaling in HSC  and its water-soluble analog Trolox are effective peroxyl radical scavengers and absorber of oxygen radicals Figure . Moreove2+ homeostasis , avocado, celery, olive oil, chamomile, peppermint and many aromatic plants. It was supposed that this compound and some of its derivatives act as dopamine transporter activators , celery (Apium graveolens), and different chamomiles. The efficacy of this drug was proven in different toxic liver injury models  and phosphorylated (NADP+ and NADPH) forms. These compounds are indispensable for a multitude of dehydrogenase enzymes, act as mitochondrial electron carriers and electron donors for GSH, thioredoxin, and NADPH oxidases . They further act as ADP donors in ribosylation reactions, substrates for NAD+-dependent enzymes , and redox regulators that modify ion channel function  and its amide (vitamin B3) serve as the most fundamental nicotinamide adenine dinucleotide precursors  reductase that NADH- or NADPH-dependently forms mevalonate from HMG-CoA. There are a large number of drugs, i.e., the statins, which inhibit the activity of this enzyme. A growing number of statins such as lovastatin, atorvastatin, simvastatin, pravastatin, fluvastatin, pitavastatin, rosuvastatin, and others -BB and IL-6  and its intracellular signaling mediators, the Smad proteins, is the key cytokine axis driving collagen gene transcription. Most obvious the important role of TGF-β in collagen synthesis was shown in mice that carried a fusion gene consisting of modified porcine TGF-β1 cDNA directed under regulatory control of the mouse albumin gene promoter. Respective mice developed hepatic fibrosis and apoptotic death of hepatocytes  that can form pure or mixed dimers. In general, PDGFs are potent mitogens that drive cell proliferation and differentiation. Their involvement in the pathogenesis of hepatic fibrosis was proven in transgenic mice that expressed PDGF-B under control of the hepatocyte specific albumin promoter  is the prototype of the TNF family that consists of 19 different proteins sharing a homotrimeric or heterotrimeric structure. TNF-α is mainly secreted by macrophages and binds with different affinities to two receptors , that initiate several intracellular cascades leading to activation of NF-κB. This pathway triggers cellular activation, differentiation, cytokine production and cellular apoptosis. The activation of TNF-α-associated pathways is causatively linked to liver injury and hepatic inflammation. This connection is particularly demonstrable after Concanavalin A injection that induces acute hepatitis  binds to different receptors  and leads to activation of several pathways . During fibrotic liver injury, hepatocytes express NGF that dose-dependently leads to increase in HSC apoptosis  already suggest, the five members of this cytokine family  are important for vascular development and angiogenesis. They bind to tyrosine kinase receptors (VEGFRs) that trigger multiple downstream signals. It is generally assumed that VEGF-A is implicated in hepatic fibrosis. This growth factor is induced in cells upon oxygen tension. It modulates the density of microvessels and is involved in the production of portal hypertension  have hepatoprotective and anti-fibrotic effects. Therefore, several clinical studies actually examine their effects in liver disease. The high number of ILs and their receptors as well as the availability of IL agonists or antagonists offers infinite therapeutic modalities. Since ILs may evolve pro- or anti-inflammatory activities (as well as both activities), it is most likely that strategies targeting one particular IL or a specific IL receptor will be beneficial only in selected disease subsets. A comprehensive summary of experimental studies in mice targeting different ILs is given elsewhere  family bind to five distinct receptors . Signaling of these cytokines is mediated through several downstream pathways including the Ras-Raf-mitogen-associated protein kinase and phosphoinositol-3 kinase-AKT cascades. The FGF family is involved in the initiation of fibrosis and progression of hepatic fibrosis to cirrhosis  or scatter factor binds to the c-Met receptor that possesses tyrosine kinase activity. This cytokine has pleiotropic effects on liver cells and influences cell proliferation, apoptosis, differentiation, motility, invasion and angiogenesis. In experimental liver disease, HGF attenuated liver fibrosis  induces on its receptor (EGFR) tyrosine kinase activity. This cytokine has recently attracted much interest because EGFR inhibition by erlotinib was shown to attenuate liver fibrosis and development of HCC in DEN-treated mice and BDL rats  or in combination with other antiviral drugs is already clinical exploited in treatment of hepatitis virus associated liver disease since many years  and type II (IFN-γ) interferons (IFN), and their receptors (IFNR) transduce signals rs EASL, .The two insulin-like growth factors , their six binding proteins (IGFBP-1 to IGFBP-6) and their two receptors  form a complex network, i.e., the “IGF axis.” This axis is critically associated with the development of liver disease. In accordance with this assumption, the treatment with recombinant human IGF-1 attenuated and reversed the fibrotic degeneration of hepatic tissue  and autophagy (autophagocytosis) are physiological programs having fundamental function during development and differentiation. However, enhanced rates of apoptosis are also physiologically important when aberrant cellular proliferation occurs or unwanted cells should be removed. Similarly, exposure to tremendous factors can induce a cellular senescence response in which the growth of premalignant cells is haltered and overshooting cell proliferation in tissue repair prevented. Since the pathogenesis of hepatic fibrosis and cirrhosis is associated with a significant increase in ECM producing cells, it is obvious that the targeted induction of apoptosis, autophagy, or senescence in ECM-producing cells might be therapeutically beneficial. The development of respective strategies targeting profibrogenic cells in hepatic fibrosis is therefore a very important therapeutic option.Aspergillus fumigatus and many other fungi  that is also known as osteonectin is a 40-kDa calcium binding protein that binds to collagen, increases the production and activity of MMPs, modulates cell shape and disrupts cell-matrix interactions. The major source of SPARC in fibrotic livers are HSC, while other hepatic cell types produce only trace amounts of this matricellular protein  or secreted phosphoprotein 1 (SPP1), In HSC it is involved in the control of migration, activation of MMP2, and production of collagen and TGF-β type II receptor  protein family consists of six conserved small secreted cysteine-rich proteins. These matricellular proteins have a modular structure with up to four individual modules including an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, a thrombospondin type I module, and a carboxyl-terminal cystine knot. The individual domains of the CCN proteins confer different binding activities, intrinsic activities, modulator activities, and antagonistic activities  sharing multiple EGF-like repeats and several fibronectin-III domains. It is assumed that tenascins have affinity for fibronectin thereby blocking its interaction with syndecans that are heparin sulfate and chondroitin sulfate coupled single transmembrane proteins. TNC is upregulated during chronic hepatic disease and mice that lacked TNC were more prone to hepatic inflammation and fibrosis  or placebo (n = 275) for 2–6 years  and their physiological inhibitors, the tissue inhibitors of MMPs (TIMPs). The family of MMPs comprise 23 different members in human and mouse (MMP-1 to MMP-28) that are not numbered consecutively. These enzymes act as zinc-dependent endopeptidases that have a role in a diverse range of physiological and pathological processes  induce expression of TIMP-1 thereby attenuating MMP-2 and MMP-9 activities in HSC and portal MFB vβ3.This integrin and other integrins play essential role in angiogenesis and in fibroblasts where they are central in differentiation, migration, and proliferation. They have many ligands  that are important in initiation or progression of hepatic fibrosis. Although integrins have no intrinsic enzymatic activity, they are capable to initiate diverse signaling cascades after binding to its ECM partner. They not only signal on their own but can also cooperate with growth factor receptors or modify the activity of enzymes or cytokines that are involved in ECM homeostasis. As good ligands, the short RGD peptides are already able to induce a conformational change from a closed to an open integrin conformation  that has high affinity to the collagen type VI receptor binds preferentially to activated HSC and when coupled to sterically stable liposomes these particles accumulated in vivo 10-fold higher to HSC than uncoupled liposomes in BDL rats  was specifically targeted by RGD peptide-labeled liposomes to HSC in the CCl4 model in rats , Glu-Ile-Leu-Asp-Val (EILDV), or Arg-Glu-Asp-Val  consists of an amine and nitrile group that are linked 4 model in rats  are a family of enzymes that remove acetyl groups from histones thereby suppressing general gene transcription. Their activities are counterbalanced by histone acetyl transferases (HAT) that acetylate lysine amino acids on histones resulting in relaxed chromatin structures. The ratio of acetylation and de-acylation adjudicate gene regulation. Substances that prevent deacetylase or transferase activity are suitable to modulate gene expression, replication, mitosis, meiosis, and apoptosis. In addition, there are some non-histone protein substrates of histone deacylases that are involved in control of cell proliferation, cell migration, and cell death.The 18 mammalian HDACs are grouped into four major classes that are distinguished by their inhibitability by different substances. They counteract oxidative stress and inflammation and further influence MAP kinase driven pathways  and to a lesser content from ginger  has a catechin structure that is one of the major effective ingredients of green tea. It has antioxidant activity and inhibits HATs, DNA methyltransferases, and tyrosinases. EGCG is further effective in preventing NF-κB and TNF-α activation  have attracted the interest of hepatic fibrosis research  potent vasoconstrictive peptides that can bind with different affinity to two G-protein coupled receptors, i.e., ETLeptin is an adipokine that acts as a hormone and is majorly involved in the regulation of energy balance. It binds to a cerebral receptor that regulates appetite and body weight. Although predominantly synthesized in adipose tissue, leptin is also secreted from activated HSC  that belong to the seven transmembrane G-protein-coupled receptor family. These stimulate upon proteolytic activation cell proliferation and transdifferentiation in HSC, while their expression increases in parallel with the severity and duration of liver disease  to tyrosine residues in a protein substrate. This phosphorylation causes structural changes that induce altered functions or enzymatic activities. Tyrosine kinases can be classified into two groups, i.e., the transmembrane receptors and the cytoplasmic non-receptor tyrosine kinases. Both groups are linked to fundamental functions such as cell differentiation, proliferation, growth factor signaling, apoptosis, and malignant transformation. During the last decades, the understanding in the biochemical activity of receptor tyrosine kinases (RTKs) increased dramatically. Simultaneously, the number of RTK inhibitor substances  partly functions as an antioxidant and zinc supplementation is associated with decreased ROS formation  that use ingredients form plants, animals, humans, and mineral products for healing. Although most the knowledge of TCM drugs was developed over generations, it is thought that they have no real rational mechanism of action Nature, . HoweverIn the following, some herbs or mixtures including their most important compounds are discussed.Bupleurum falcatum , 5.0% root/stolon of Glycyrrhiza uralensis , 7.5% root w/o rootlets of Panax ginseng , 12.5% tuber w/o cork layer of Pinelliae ternatae , 7.5% rot w/o periderm of Scutellaria baicalensis , 2.5% root/rhizome of Zingiber officinale , 7.5% fruit of Ziziphus jujube , and 40% additives  in rats in which hepatic fibrosis was induced by TAA  as its main components. There are several studies that show that this herbal mixture evolves anti-fibrotic effects in vitro, most likely by counteracting activities of individual TGF-β signal components , Semen Persicae (Tan Ren), Gynostemma Pentaphyllum (Jiaogulan), Pollen Pini (Song Hua Fen), Fructus Schisandrae Chinensis (Wu Wei Zi)  and 10 herbs . Serum collected from rats that were perfused with CBJRGC decreased the proliferation and expression of desmin, synapsin, and PDGF in a HSC line , Fructus Lycii (Gou Qi Zi), Radix Glehnaia (Sha Shen), Radix Ophopogonis (Mai Men Dong), Radix Angelicae Sinensis (Dang Gui), and Fructus Meliae Toosendan (Chuan Lian Zi). In DMN-treated rats, the oral administration of this traditional TCM prevented body weight loss, collagen accumulation, and expression of TIMP-1 and α-SMA  were added to YGJ was shown to effectively induce HSC apoptosis through ROS accumulation and induction of intrinsic apoptotic pathways  that is composed out of Capparis spinosa (Capers), Cichorium intybus (Wild chicory), Mandur bhasma , Solanum nigrum (Black Nightshade), Terminalia arjuna (Arjuna), Cassia occidentalis (Negro coffee), Achillea millefolium (Yarrow), and Tamarix gallica (Tamarisk). Livol (IHF-100) is a purely herbal product containing different plant ingredients. Livomyn contains 18 herbals in different quantities. Both, Livol and Livomyn showed hepatoprotective effects in rats treated with CCl4 . Its active ingredient is silymarin that has been tested already in several experimental models of hepatic fibrosis and recently turned out to be particular suitable to block progression of fibrosis in the early stages of liver injury  that is native throughout the world. It contains four main components . Curative, intragastric delivery of high doses of silymarin in rats is sufficient to increase the resolution of CCl4-induced fibrosis . It is able to scavenge ROS and modulate the activity of a broad range of enzymes and pathways that are involved in pro-inflammatory signaling , transmembrane G-protein-coupled receptors, pregnane X receptor, vitamin D receptor, and constitutive androstane receptor  was applied in TCM already long before introduction of modern medicine. There it is used as an anti-inflammatory formulation reducing the effects of alcohol over-consumption and restoring liver homeostasis. Bile acids act as physiological emulgators and signal 4-induced hepatic fibrosis  polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have an inflammation dampering effect on hepatic inflammation by interfering with the arachinodonic acid pathway and inhibition of pro-inflammatory cytokines  and the Interdisciplinary Centre for Clinical Research within the Faculty of Medicine at the RWTH Aachen University .The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although TADs can be stable in genomic positions across cell lines, structural comparisons show that a considerable number of stable TADs undergo significantly structural rearrangements during cell changes. Moreover, the structural change of TAD is tightly associated with its transcription remodeling. Altogether, the theoretical parameter defined in this work provides a quantitative method to link structural characteristics and biological functions of TADs, and this linkage implies that chromatin interaction pattern has the potential to mark transcription activity in TADs.Recent chromosome conformation capture (3C) derived techniques have revealed that topologically associating domain (TAD) is a pervasive element in chromatin three-dimensional (3D) organization. However, there is currently no parameter to quantitatively measure the structural characteristics of TADs, thus obscuring our understanding on the structural and functional differences among TADs. Based on our finding that there exist intrinsic chromatin interaction patterns in TADs, we define a theoretical parameter, called aggregation preference (AP), to characterize TAD structures by capturing the interaction aggregation degree. Applying this defined parameter to 11 Hi-C data sets generated by both traditional and  Caulobacter crescentus  derived techniques ,2, especC derivedescentus , Plasmodlciparum , Arabidolciparum ,7, yeastlciparum , drosophlciparum , mouse , to represent the structural characteristics of TAD and then build its association with biological functions. First, the locally high-frequency chromatin interactions violating distance-dependence decay are selected from TAD. Then a density-based algorithm DBSCAN  is used recently . The Hi-in situ Hi-C data sets of human cell lines GM12878, IMR90, K562, HMEC, HUVEC and NHEK were downloaded from NCBI with accession number GSE63525  were downloaded from NCBI with accession number GSE35156 , and theThe pipeline of Hi-C data processing follows a previous procedure  by usingN is the number of beads in the chromatin and i and j. The gap regions arising from DNA repeat elements or the absence of enzyme restriction sites are eliminated from the observed interaction matrix to facilitate next calculations (Chromatin is partitioned into bins (or beads) at given resolution, and the kept reads are assigned to these beads to generate the observed interaction matrix ulations . To remoulations  with defel (HMM) .k in a n-beads TAD is calculated by k to obtain the smoothed interaction frequency  where P-value for observing interaction frequency p and w are set to 4 and 7 respectively at 5 kb resolution, 2 and 5 at 10 kb resolution, and 1 and 3 at 20 kb resolution.The statistically significant chromatin interactions are selected by considering both distance-dependent decay and local interaction background . Specifious work , the winThe selected chromatin interactions are clustered into spatially neighbored groups m DBSCAN . This alm DBSCAN  has discThe convex hull lgorithm . The so-http://code.google.com/p/align2rawsignal/). When calculating Pearson correlation between AP and given annotation signal, the processed signal in the domain are summed and then averaged by domain length to represent signal strength of TAD. To reduce the negative impact of confounding factors, such as GC-composition of the genome, in computing P-value for correlation coefficient, the Pearson correlation between AP and randomly shuffled annotation signal (less than 1 Mb independently for different chromosomes) is calculated. This shuffled correlation is repeated 500 times independently, and the final P-value is computed by testing the difference between original correlation coefficient and shuffled correlation average under Fisher's z transformation , short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs), were downloaded from ENCODE . LaminB1ormation .P-value for each investigated signal.TAD stability is measured by using the boundary overlap in genomic positions between two cell lines or between two experimental pipelines on the same cell line. The number of stable TADs decreases as stricter criterion is used . To compromise between TAD number and stability, TADs sharing similar boundaries (less than 5 bins in both left and right ones) in genomic positions are considered to be stable. The overlapped block regions of two stable TADs are divided by total blocks in each cell line, and the averaged overlap percentage is used to calculate Pearson correlation between AP change and block overlap. By using the biological replicates sharing similar intra-chromosomal interaction reads in different cell lines, statistical analysis shows that the stable TADs consistently exhibit higher structural variations arising from cell lines than those arising from biological replicates . To simplify the TAD selection when performing structural comparisons across cell lines in diverse cases, quantile regression is used to select stable TADs with top AP change by accounting for the difference in structural variations ,26. Finain situ Hi-C maps are denoted as GM12878-I, IMR90-I, K562-I, HMEC-I, HUVEC-I and NHEK-I to represent corresponding human cell lines. To compromise between higher resolution and limited sequencing depth, TADs were identified at 20 kb resolution for traditional Hi-C data sets and at 10 kb resolution for in situ Hi-C data sets by using DI based HMM proposed by a previously work  observations that statistically significant interactions are often physically preferred contacts . The cell lines hESC-T, HUVEC-I and NHEK-I are eliminated from reproducibility evaluation, either because significant difference exists in sequencing depth between two replicates or because there are no biological replicates. AP values calculated from merged replicates are used for next analysis in all cell lines. We next performed TAD identification and AP calculation for GM12878-I and IMR90-I at 20 kb resolution to further evaluate the robustness of parameter AP between two experimental pipelines in the same human cell lines. To reduce the impact of differences in sequencing depth and data quality, only the stable TADs shared by both Hi-C pipelines were used to calculate the reproducibility of parameter AP. Our result shows that the parameter AP is quite reproducible between two experimental pipelines . Finally, these two kinds of reproducibility analyses together indicate the robustness of our defined parameter.Pearson correlations between biological replicates show that the defined parameter AP is highly reproducible in both traditional and We explored the structural characteristics of TADs by using interaction blocks and corresponding parameter AP. Meanwhile, domain structure was functionally annotated by using DNA sequences, epigenomic signals and transcription activity. Among these signals, SINEs are often found in gene-rich regions  and LINEThe interaction blocks generated from selected chromatin interactions are the structural and functional bases to distinguish TADs. Though previous works ,33 have in situ Hi-C data sets . Some domains have high AP values, indicating that the selected chromatin interactions are aggregated to form high-density and small-size interaction blocks. By contrast, the domains exhibiting low AP values own the dispersedly distributed chromatin interactions. Nevertheless, the widely distributed values suggest the mixed situations in most domains. Consistent with Figure s Figure . These rWe next investigated the structural rearrangements of TADs across cell lines by using the parameter AP. Our analysis showed that TADs could be stable in genomic positions across cell lines, consistent with previous study . We thenThere exist different kinds of structural changes across cell lines, which can be captured by interaction blocks and corresponding AP values. Figure in situ Hi-C cell lines, indicating that most TADs becoming interaction-aggregated are activated and those becoming interaction-dispersed are repressed in regulatory activity. The observed exceptions are partly caused by the complicated nature of structural variations and biological functions of TADs. These results suggest that chromatin interaction pattern captured by parameter AP has the potential to mark regulatory functions at genome-wide scale in mammalian genomes.Genome-wide comparisons on the parameter AP revealed that a considerable number of TADs undergo structural changes in both human and mouse cell lines . Some domains become more aggregated in chromatin interactions, and some domains transit to disperse chromatin interaction patterns. To statistically analyze the association between structural change and transcription remodeling, the selected TADs were paired by AP values, and Wilcoxon signed rank test was performed on epigenomic and transcription signals . Figure Previous methods only focused on identifying functional chromatin interactions based on different purposes ,33, but Heterogeneous structures exist among TADs and this structural heterogeneity is probably encoded by DNA sequences and corresponding regulatory activities. Previous studies have shown that TAD is a pervasive element in chromatin 3D organization –11, but Chromatin interaction pattern has the potential to mark gene regulation and transcription activity, at least in TADs. Several recent works explored the relationship between chromatin interactions and biological functions. McCord et al. showed that the progress of Hutchinson–Gilford progeria syndrome was related to the reorganization of chromatin compartments . RousseaIn summary, we define a parameter, the aggregation preference of statistically significant chromatin interactions, to represent the structural characteristics of TADs in this work, allowing us to systematically investigate domain structures and their association with biological functions. Statistical analysis shows that TADs are different in chromatin 3D structure, and this structural difference may be encoded in DNA sequences and corresponding regulatory activities. TADs can also undergo significant structural rearrangements across cell lines though they may preserve the domain positions in the genome. Moreover, this structural change is tightly associated with transcription remodeling. With the rapid expansion of Hi-C data, the chromatin interaction parameter defined in this work can provide a useful tool to explore the biological functions underlying the structural characteristics of TADs, especially when the epigenomic and TF binding signals are poor.https://pypi.python.org/pypi/TADLib.A TAD library (TADLib) to automatically calculate the parameter is coded by Python and freely available at


Over the last few centuries, many cetacean species have witnessed dramatic global declines due to industrial overharvesting and other anthropogenic influences, and thus are key targets for conservation. Whale bones recovered from archaeological and palaeontological contexts can provide essential baseline information on the past geographical distribution and abundance of species required for developing informed conservation policies. Here we review the challenges with identifying whale bones through traditional anatomical methods, as well as the opportunities provided by new molecular analyses. Through a case study focused on the North Sea, we demonstrate how the utility of this (pre)historic data is currently limited by a lack of accurate taxonomic information for the majority of ancient cetacean remains. We then discuss current opportunities presented by molecular identification methods such as DNA barcoding and collagen peptide mass fingerprinting (zooarchaeology by mass spectrometry), and highlight the importance of molecular identifications in assessing ancient species’ distributions through a case study focused on the Mediterranean. We conclude by considering high-throughput molecular approaches such as hybridization capture followed by next-generation sequencing as cost-effective approaches for enhancing the ecological informativeness of these ancient sample sets.This article is part of the themed issue ‘From DNA barcodes to biomes’. Theiracialis)  and the obustus) . The pasobustus) ,13, cetaobustus) , but theIn this article, we review the challenges with identifying ancient whale bones, as well as the opportunities provided by new molecular identification methods. We begin by summarizing the limitations inherent to taxonomic identification based on traditional anatomical methods, illustrated with a case study from the North Sea on the proportion of unidentified archaeological cetacean remains housed in museums and repositories. We then discuss the opportunities for more accurate identifications made possible by molecular analyses, and demonstrate the need for molecular validation through a case study comparing anatomical and molecular identifications of whale bones from Mediterranean archaeological contexts. Finally, we conclude by presenting future perspectives for molecular methods, including high-throughput approaches for the study of ancient cetacean assemblages.2.Despite the millennia of human–cetacean interactions, the research potentials of palaeontological and archaeozoological cetaceans have received very little attention, in large part because of the difficulty in identifying (often fragmentary) ancient whale bones to the genus or species level using comparative anatomy methods. Compared with other large mammals, whale bone is extremely friable; composed primarily of oil-filled cancellous bone, with only a thin external cortical layer, whale bone easily breaks up into non-diagnostic fragments. When a whale is exploited through active hunting or scavenging of drift carcasses, its sheer size limits the viability for humans to transport complete anatomical elements far from the beach . Thus, iThese identification problems are compounded by a lack of comprehensive or easily accessible skeletal reference collections, which are usually restricted to a few large national natural history museums  . Unlike These difficulties create substantial gaps and biases in the archaeological record, with ramifications for understanding past human interactions with these marine mammals. For example, in a study of whale remains in the Western Isles (northwest of Scotland), Mulville  noted an(a)n = 306) could be morphologically identified to the species level through traditional comparative anatomy methods (electronic supplementary table S1). Furthermore, among these taxonomically identified bones 119 originated from a single site, the Early Medieval site of Flixborough  [A case study from the southern North Sea  illustraus orca) .Figure Overall, most of the identified specimens across all 102 sites represented dolphins or porpoises. The taxonomic identification of these small species is significantly easier, as a greater proportion of entire bones are preserved and more complete reference collections are available. Only 12 specimens (less than 4% of the identified assemblage) were identified as baleen whale species. The fact that half of these baleen whales were identified as North Atlantic right whale demonstrates the significance of these ancient sample sets. Indeed, this species has all but disappeared from the North Sea, but its prevalence in these archaeological remains  hints at its potential historic abundance within the region. It is highly likely that a significant proportion of the unidentified cetacean specimens are also from large  whales, as they are less likely to be taxonomically identified if they are in a fragmented state compared with smaller species (see discussion in ). Human 3.(a)b (cytb) gene to identify taxa and to estimate geographical provenience [a). By comparing the resulting sequences to a databank of known species and populations, it is possible to evaluate the relationships between known and unknown samples by parsimony or maximum-likelihood criteria, with the reliability of phylogenetic relationships analysed by bootstrapping procedures [Over the last two decades, molecular methods have been increasingly applied to the problem of cetacean identification, but their primary focus was the study of contemporary populations. Indeed, given the 1984 moratorium on commercial whaling by the IWC, their protected status under CITES and the many national laws protecting particular species and populations, accurate taxonomic identification of whale products has become essential to differentiate products obtained from legal versus illegal exploitation or trade. For example, identification to the species or even population level may be key to assessing whether whale products  sold in domestic markets have a legal origin  or not. However, such products are often processed in ways that render morphological identification impossible. Considering that such processing  may significantly degrade DNA, early molecular studies targeted mitochondrial DNA (mtDNA), amplifying relatively short diagnostic fragments (150–500 bp) of the control region or cytochrome venience ,29 m/z) ratios [b). In mammals, collagen is composed of two alpha 1 chains, and a third, more rapidly evolving alpha 2 chain. In collagen PMF approaches, collagen is extracted from archaeological bone, followed by enzymatic digestion, which cleaves proteins at specific amino acid sites producing a characteristic mixture of peptides. The peptides are analysed through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), producing a ‘peptide mass fingerprint’ based on their respective m/z ratios. Species identification of archaeological bones can thus be accomplished by comparing collagen peptide fingerprints with the fingerprints from known samples—i.e. zooarchaeology by mass spectrometry (ZooMS) [Peptide mass fingerprinting (PMF) has been widely used as a rapid and cost-effective protein identification method based upon the pattern of mass-to-charge  ratios . Most re (ZooMS) ,51. Coll (ZooMS) ,53. The  (ZooMS) ,54.The advantages of a collagen versus DNA-based approach for identifying ancient samples are numerous. First, collagen is a remarkably robust protein, and recent evidence suggests that collagen survives at least 10 times longer than DNA, preserving even in tropical climates where DNA preservation is poor ,55. UnliBiomolecular identification approaches such as DNA barcoding and ZooMS can offer robust taxonomic identifications of ancient cetaceans, however, they can be limited by taphonomic histories and biomolecular preservation. Some studies, for example, have noted a high presence of inhibitory substances in ancient whale bones, compromising the success of PCR amplifications –62. Also(c)E. robustus) [In order to illustrate the necessity of validating osteological identifications of cetaceans, we present a case study of 17 pre-industrial cetacean specimens from six sites around the Mediterranean Sea (obustus) . The graobustus)  as this obustus)  (figure obustus) . Owing tb mtDNA analysis) and ZooMS , identifying 11 fin whale , one sperm whale (Physeter catodon), one right whale  and one Cuvier's beaked whale (Ziphius cavirostris). The relative merits of both techniques in terms of their precision and robusticity are exemplified here. For example, although ZooMS identified two samples only as ‘beaked whale’ and ‘bowhead/right whale’, respectively, DNA provided the resolution to confirm these as Cuvier's beaked whale and right whale. Although ZooMS may be less precise, it may be more successful with poorly preserved samples: three samples that failed DNA analysis were identified through ZooMS as two fin whales and one baleen whale (Mysticeti), respectively. For the latter sample, a higher taxonomic resolution was not possible due to a lack of high molecular–weight diagnostic peptide markers in the mass spectra . Despite the advantages of applying both techniques to the same assemblage, two samples failed to produce identification using either molecular method, illustrating the limitations of working with degraded archaeological materials.We analysed the 17 ancient bones using DNA barcoding  for mod(a)The advent of high-throughput or NGS methods has revolutionized the application of ancient genetics, massively enhancing the ability to recover ancient DNA templates from degraded remains. Although whole-genome ‘shotgun’ approaches have been attempted for species identification , thiEnrichment approaches are particularly useful for increasingly old samples, or those from tropical climates where preserved DNA templates may be degraded beyond the fragment length feasible for traditional PCR amplicons ,84. More(b)E. robustus) [Balaena mysticetus) [E. glacialis) [Cephalorhynchus hectori) [NGS approaches are beginning to be applied more routinely to modern cetacean populations, recovering full mitogenomes –95, genoobustus) , bowheadticetus) ,104, Noracialis)  and Hecthectori) , which hhectori) ,70.5.Molecular methods are already proving crucial to our understanding of the past distribution and abundance of whale species and much scope remains to expand their application to existing zooarchaeological collections. With further refinement of these methods and the augmentation of cetacean genomic reference datasets, we will be able to obtain increasingly fine-grained identifications to the subspecies, ecotype and population levels. The systematic integration of well-dated archaeological and palaeontological remains with high-throughput molecular analysis methods will reveal changes in habitat, genetic diversity and population abundance associated with climatic and anthropogenic factors through millennial timescales ,106.


Platelet-rich plasma (PRP) is a whole blood fraction which contains several growth factors. Controlled clinical studies using different autologous PRP formulations have provided controversial results. However, favourable structural healing rates have been observed for surgical repair of small and medium rotator cuff tears. Cell-based approaches have also been suggested to enhance tendon healing. Bone marrow is a well known source of mesenchymal stem cells (MSCs). Recently, ex vivo human studies have isolated and cultured distinct populations of MSCs from rotator cuff tendons, long head of the biceps tendon, subacromial bursa, and glenohumeral synovia. Stem cells therapies represent a novel frontier in the management of rotator cuff disease that required further basic and clinical research.Rotator cuff injuries are a common source of shoulder pathology and result in an important decrease in quality of patient life. Given the frequency of these injuries, as well as the relatively poor result of surgical intervention, it is not surprising that new and innovative strategies like tissue engineering have become more appealing. Tissue-engineering strategies involve the use of cells and/or bioactive factors to promote tendon regeneration via natural processes. The ability of numerous growth factors to affect tendon healing has been extensively analyzed  Rotator cuff lesions represent the vast majority of shoulder injuries in adult patients and are a common contributing factor to shoulder pain and occupational disability.The incidence of this condition is increasing along with an aging population . The manImprovements in arthroscopic instrumentation and suture anchor technology have allowed the development of stronger constructs with multiple suture configurations, allowing repair of large and massive tears through minimally invasive means. However, although repair instrumentation and techniques have improved, healing rates have not. A high failure rate remains for large and massive rotator cuff tears , 3.A recent meta-analysis has shown that the development and introduction of novel surgical techniques are not related to an improvement of clinical and anatomical results over the investigated period (1980–2012) .To enhance tendon tissue regeneration, new biological solutions including growth factors, platelet-rich plasma (PRP), and stem cells are being investigated.This review will outline the current evidence for the novel frontier in the management of rotator cuff disease including growth factor and stem cell therapy.Growth factors are signal molecules involved in the control of cell growth and differentiation and are active in different phases of inflammation. They are produced by inflammatory cells, platelets, and fibroblasts.β), transforming growth factor-beta (TGF-β), and insulin-like growth factor-1 (IGF-1) [Rotator cuff healing occurs via a sequence of inflammation, repair, and remodeling . Several (IGF-1) , 23.Because rotator cuff healing results in reactive scar formation rather than a histologically normal insertion site, addition of these factors may enhance the repair-site biology.β1 expression [PDGF is a basic protein composed of two subunits, an A and a B chain, that exists in three main isoforms . These isoforms function as chemotactic agents for inflammatory cells and help to increase type I collagen synthesis and induce TGF-pression . The hompression , 25. Kobpression  studied Several studies have examined the role of PDGF as a mitogenic and chemotactic cytokine that can enhance tendon and ligament healing.Uggen et al.  showed rA similar study used an interpositional graft composed of a type I collagen matrix, enriched with recombinant human PDGF-BB (rhPDGF-BB), implanted in an ovine model for rotator cuff repair . At 12 wβ is a family of cytokines that includes three isoforms . Although this superfamily is responsible for numerous physiologic effects, it is of particular interest in biological augmentation, as it is thought to play an important role in tendon and ligament formation through cellular proliferation, differentiation, and matrix synthesis [TGF-ynthesis .β3 holds the greatest promise to enhance the local microenvironment of the rotator cuff repair site because its high expression during fetal wound healing correlates with no formation of scar tissue [β1 and TGF-β2 expression and low level of TGF-β3. Because TGF-β3 is not present within the adult healing environment, its exogenous application could promote a scarless regenerative healing process. Based on this assumption, three recent studies have specifically examined the use of TGF-β3 in a rat rotator cuff model [Of the three isoforms, TGF-r tissue . By contff model –31.β1 and TGF-β3 at the tendon-to-bone insertion of repaired rat supraspinatus tendons. The TGF-β1 group showed increased type III collagen production compared with control shoulders, which is consistent with a scar-mediated healing response. There was also a trend toward reduced mechanical properties within this group. By contrast, the TGF-β3 group showed no histological or biomechanical differences compared with the paired controls. While these results support the TGF- β1 mediated reparative healing response, the lack of improvement in the TGF-β3 group could be attributed to the delivery system used.Kim et al. . used anβ3 delivery system to the tendon-to-bone insertion demonstrated accelerated healing with increased inflammation, cellularity, vascularity, and cell proliferation at early time points [β3 can enhance tendon-to-bone healing in vivo in a rat model. In a further study the authors examined the delivery to the tendon-bone interface of repaired rat supraspinatus tendons of TGF-β3 in an injectable calcium-phosphate matrix (Ca-P) [β3 to the Ca-P matrix, there was a significant improvement in strength at the repair site 4 weeks postoperatively and a more favorable collagen type I/III ratio, which reflects more mature healing. These results further support the role of TGF-β3 in improving tendon healing after surgical repair; however, future studies are needed to optimize delivery techniques and dosing as well as to examine the effects of multiple growth factors administration on the healing process.A subsequent study by the same group using a heparin-/fibrin-based TGF-e points . In addix (Ca-P) . The autThe use of PRP as a biological solution to improve rotator cuff tendon healing has gained popularity over the last several years.PRP is a whole blood fraction containing high platelet concentrations that, once activated, provides a release of various growth factors which participate in tissue repair processes .In vitro studies on the effect of PRP on human tenocytes from rotator cuff with degenerative lesions showed that growth factors released by platelets may enhance cell proliferation of tenocytes and promote the synthesis of extracellular matrix [r matrix , 34.β (IL-1β) but also enhances TGF-β production. Increased concentration of IL-1β is significantly correlated with rotator cuff tendon degeneration; conversely, TGF-β enhances rotator cuff tendon repair strength [ in vivo animal study showed that different types of application did not influence the effect of PRP on rotator cuff healing [PRP not only inhibits the inflammatory effects of interleukin 1strength . Further healing .There are several different PRP formulations currently available. PRP can be classified into four main categories: pure PRP (P-PRP), leucocyte-rich PRP (L-PRP), pure platelet-rich fibrin (P-PRF), and leucocyte-rich platelet-rich fibrin (L-PRF). In each category, platelet concentration can be obtained by different processes, either in a fully automatized setup or by manual protocols . ex vivo with thrombin and/or calcium and those unactivated, which rely on in vivo activation via endogenous collagen [Among PRP formulations, a further division can be made between those which are activatedcollagen .The role of leucocytes in PRP is a controversial issue in the literature.Basic science studies showed that growth factors and cytokine concentrations are influenced by the cellular composition of PRP, with leukocytes increasing catabolic signaling molecules .Furthermore L-PRP has been found more proinflammatory when injected in rabbits  and incrMost clinical studies have used numerous different PRP formulations. The obtained results have never been analyzed using the leucocyte content of the final concentrate as a key parameter. Thus, differences between P-PRP and L-PRP preparations are still unknown.However the leukocyte content does not seem to induce negative effects or to impair the potentially beneficial effects of PRP and no uncontrolled immune reactions of L-PRPs have been also reported; on the contrary, the use of L-PRP could diminish pain and inflammation of the treated sites , 47. FurLiterature showed that PRP can be applied either by direct injection or by application of a PRP matrix scaffold on repaired tissues. The main characteristics of controlled clinical studies using PRP in arthroscopic rotator cuff repair are reported in Conflicting results on the effectiveness of PRP use in rotator cuff tendon repair were produced, making it now difficult to draw definitive conclusions.ex vivo activation with thrombin and/or calcium) or unactivated form (in vivo activation via endogenous collagen) [The clinical studies published to date have different experimental designs with a level of evidence that varies from 1 to 4. Moreover, there are differences in PRP formulations in terms of growth factor concentration and catabolic enzyme content . A PRP collagen) .Experimental protocols present differences among the trials, concerning volume of autologous blood collected, speed and time of centrifugation, method of administration, activating agent, presence of leukocytes, final volume of PRP, and final concentration of platelets and growth factors. The surgical technique  and the rehabilitation protocol (standard or rapid) were not the same among different studies.In spite of the differences in surgical techniques, PRP formulation, size of the lesions, retear rate have been recalculated by combining the available data from studies in order to determine the role of PRP in improving the rotator cuff healing after surgical repair.P value > 0.05).Differences in term of retear rate between PRP and control group were assessed by a chi-square test. The analysis of all studies examined showed that there was no significant difference in the retear rate between PRP and control group. The retear rate was 31% (101 out of 323) and 37% (115 out of 312), respectively  are necessary to define the role of PRP in healing of rotator cuff repair. in vitro studies [Injections of PRP have gained popularity in the treatment of tendinopathy because of their promoting effects on tendon cell proliferation, collagen synthesis, and vascularization, which have been shown in animal and studies , 50.In spite of this popularity and increasing use in clinical settings we have found only two controlled randomized trials evaluating the use of PRP injections in rotator cuff tendinopathy , 20.These studies have reported controversial results on the effectiveness of the use of PRP injection in chronic rotator cuff tendon diseases.The systems of PRP preparation were not the same among trials and different treatment protocols were used (single or double PRP injections). Furthermore, the presence of some bias including the concomitant standard exercise program and the needle stimulus effect can have influenced the results of studies. The nature of rotator cuff disease was also not the same throughout the studies and patients refractory to physical therapy and corticosteroid injection seem to have a benefit from the PRP use.Extrinsic and intrinsic factors including anatomical problems, joint kinematics alterations, and age- and vascularity-related degenerative changes may play a role in developing such disease.More studies with a high level of evidence are required to validate the role of PRP injections in the subacromial space for treatment of rotator cuff diseases.Stem cells are defined as unspecialized cells with a self-renewal potential, which are able to differentiate into various adult cell types. The most common stem cell sources are embryonic and adult stem cells.Embryonic stem cells are truly pluripotent; that is, they are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm.In contrast to embryonic stem cells, multipotent adult stem cells are characterized by a differentiation potential restricted to tissues of 1 germ layer. Those which can differentiate into various forms of mesenchymal tissue  are termed mesenchymal stem cells (MSCs).Most of the clinical related stem cells research to date has focused on adult stem cells rather than embryonic stem cells, as the latter are associated with numerous regulatory and ethical constraints.MSCs are progenitor cells that have the capacity to self-renew and differentiate into several different mesenchymal tissues including muscle, fat, bone, ligament, tendon, and cartilage . For thiBone marrow is the main source of MSCs for rotator cuff healing and can be easily accessed by surgeons to harvest cells, the extraction and culture techniques of which, as well as the conditions for propagation, have been extensively defined. For these reasons, bone marrow-derived MSCs (BMSCs) may have valid clinical use, and there is evidence showing that BMSCs can be manipulated to differentiate into a tenogenic lineage and produce tendon tissue when exposed to the appropriate stimuli.The iliac crest is the most common site for MSC harvesting, although a number of other sources have been recently identified. Recent research performed by Mazzocca et al.  demonstrBeitzel et al. , 55 showMSCs can also be collected from other sources, such as adipose tissue; this can be easily accessible although its cells have an apparently reduced ability to differentiate compared to BMSCs . ex vivo studies confirmed TDSCs isolation from animal and human rotator cuff tissues. Tsai et al. [Tendon derived stem cells (TDSCs) are considered of extreme interest for rotator cuff repair enhancement. Existence of TDSCs has been first shown in murine patellar and human hamstring tendons by Bi et al. . More rei et al.  showed oi et al.  confirmei et al.  isolated in vitro and in vivo. These results showed in animal models that the cells isolated from bursa tissue exhibited MSCs characteristics and high proliferative capacity, and differentiated toward cells of mesenchymal lineages  with high efficiency suggesting that bursa, a tissue usually discarded during rotator cuff tear repairs, is a new abundant source of MSCs with a high potential for application.Recently, Song et al.  isolatedThe use of MSCs to enhance tendon regeneration has been examined in multiple animal models of tendon healing. MSC can be applied directly to the site of injury or can be delivered on a suitable carrier matrix, which functions as a scaffold while tissue repair takes place. In an attempt to augment tendon-bone healing in a rat rotator cuff repair model, Gulotta et al.  in 2009 Yokoya et al.  studied Kim et al.  harvesteShen et al.  used a kUp to now, only one cohort study has evaluated the safety of clinical application of MSCs in shoulder surgery. In this study Gomes et al.  investigAutologous BMMCs were harvested from the iliac crest prior to the surgical repair and subsequently injected into tendon borders after being fixed down by transosseous stitches. The BMMC fractions were obtained by cell sorting and resuspended in saline enriched with 10% autologous serum. Each patient was monitored for a minimum of 12 months, and University of California, Los Angeles (UCLA), scores improved on average from 12 to 31, and magnetic resonance imaging showed tendon integrity in all 14 patients. No control group was included in this study, but for this procedure, overall rates of rerupture during the first postoperative year range from 25% to 65%, depending on lesion diameter. Only 1 patient in the following year relapsed with loss of strength and pain. Unfortunately, only 14 patients were enrolled in this study, making it difficult to determine the efficacy of BMMCs as an adjunct to cuff repair at this time. However, these results suggest that BMMC therapy is a safe treatment that has potential to enhance tendon repair. Further research will be critical to better investigate the use of this biologic approach.Several regenerative approaches have been investigated to augment tendon healing after arthroscopic cuff repair. in vitro and in animal models, showing promising results. However there is still no study on the use of growth factors in the treatment of rotator cuff on human.The ability of numerous growth factors to affect tendon healing has been extensively analyzedDifferent delivery systems for these factors, including simple injection, coated sutures, fibrin sealants, heparin-fibrin delivery systems, collagen, and hyaluronic acid sponges, are being tested. PRP is a whole blood fraction which contains several growth factors. Different PRP formulations exist: leucocyte-poor and leucocyte-rich, activated and unactivated. Moreover, PRP can be administrated either with a simple injection or in a fibrin-matrix clot. Clinical trials using different autologous PRP formulations after rotator cuff tear repairs have provided controversial results.However, favourable structural healing rates have been observed for surgical repair of small and medium rotator cuff tears. ex vivo human studies have isolated and cultured distinct populations of MSCs from rotator cuff tendons, long head of the biceps tendon, subacromial bursa, and glenohumeral synovia. A single clinical study has been conducted on stem cell-based therapies for rotator cuff healing, proving the injection of bone marrow mononuclear cells to be safe. Clinical research regarding the use of MSCs in shoulder surgery is very limited. Further basic and clinical investigations are required until a procedure can be defined for the routine use of these cells in shoulder surgery.Cell-based approaches have also been suggested to enhance tendon healing. Bone marrow is a well known source of MSCs; recently,


Immunomodulatory antibodies that enhance the immune system to fight cancer are revolutionizing the treatment of patients with an expanding variety of malignancies. There is a unique spectrum of side effects associated with immunomodulatory antibodies, termed immune-related adverse events (irAEs), which include colitis and hepatitis among others. The treatment of refractory or severe irAEs can occasionally require significant immunosuppression, involving steroids or tumor necrosis factor-alpha antagonists, placing these patients at risk for infections. We present the first reported case to our knowledge of an opportunistic infection in a patient treated with an immunomodulatory antibody. As the use of immunomodulatory antibodies expands and more patients develop irAEs that require treatment with immunosuppression, recognition of the potential for opportunistic infections in this emerging patient population will be critical. Prospective trials are needed to define the optimal immunosuppressive management of irAEs and determine whether prophylactic antiviral, antibacterial, or antifungal therapies are beneficial in this unique population. T-cell checkpoint blockade is one of the most active areas of cancer research and has demonstrated dramatic results in patients with a variety of cancers-5. IpiliT-cell checkpoint blockade can result in side effects called immune-related adverse events (irAEs), most commonly involving the skin (rash) and, particularly with ipilimumab, gastrointestinal tract . Refractory or severe irAEs often require treatment with prolonged immunosuppression including high-dose steroids and sometimes the addition of tumor necrosis factor-alpha blockade or other immunosuppressants. We repoA 48-year-old Caucasian man was diagnosed with primary cutaneous melanoma in 1991 when he was noted to have a pigmented lesion on his posterior right neck. Subsequent biopsy showed Breslow depth 2 mm melanoma. No information about the presence or absence of ulceration was available. He underwent wide local excision with pathology demonstrating no residual disease. A sentinel lymph node biopsy was not pursued.BRAF V600E mutation).The patient remained free of disease for 20 years until the age of 68 when he developed new hypertension, prompting a renal ultrasound that showed normal kidneys. Two hepatic lesions were incidentally noted. A subsequent positron emission tomography (PET) scan demonstrated bilobar hepatic metastases and multiple pulmonary metastases concerning for metastatic disease. Core biopsy of a lesion in the right hepatic lobe confirmed metastatic melanoma. Mass-spectrometry genotyping (Sequenom) revealed no known mutations that affect the gene encoding serine-threonine protein kinase BRAF . After three doses of ipilimumab (approximately two months of therapy), he developed significant diarrhea. Colonoscopy with biopsy showed active colitis. He received two doses of infliximab  and high-dose systemic corticosteroids  with ultimate resolution of his diarrhea.Aspergillus fumigatus pneumonia with a lavage fluid also positive for galactomannan. Voriconazole and liposomal amphotericin B treatment for a 14-day course resulted in ultimate radiographic improvement  scan three months after starting ipilimumab demonstrated response of pulmonary and hepatic metastases. However, new bilateral cavitary pulmonary consolidations were noted concerning for fungal pneumonia Figure a-b. At tAs the use of immunomodulatory antibodies that block T-cell checkpoints expands, so too may the complications associated with this treatment. The unique spectrum of immune-mediated toxicities from these agents has been well characterized and algorithms for suggested immunosuppression regimens have been developed. However, the potential for opportunistic infections to arise as a result of the immunosuppression necessary to treat an irAE has not previously been highlighted. Though we have chosen to describe this one illustrative case, we have observed additional cases at our institution, including patients with Fournier’s gangrene and cytomegalovirus viremia. Clinicians across the spectrum of internal medicine must have a high degree of suspicion for the development of these rare infections as early recognition, diagnosis, and treatment are essential to achieve favorable clinical outcomes.Consensus guidelines instruct clinicians on the prophylaxis and treatment of opportunistic infections arising in patients following hematopoietic stem cell transplantation. As we lWritten informed consent was obtained from the patient’s next of kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CTLA-4: Cytotoxic T-lymphocyte-associated antigen 4; PD-1: Programmed cell death-1; irAE: Immune-related adverse event.JDW and MP receive research support from Bristol-Myers Squibb and have served on advisory councils. CK, MDH, and PBC have no competing interests to disclose.CK and MAP conceived of this study report, collected the data, wrote and revised the manuscript. MH conceived this study report concept and reviewed the manuscript. JDW and PBC reviewed the manuscript. All authors read and approved the final manuscript.


All these approaches represent potentially promising therapeutic strategies for the treatment of patients with chronic kidney disease. Although many obstacles to kidney regeneration remain, we hope that innovative strategies and reliable research will ultimately allow the restoration of renal function in patients with end-stage kidney disease.Kidney regeneration is likely to provide an inexhaustible source of tissues and organs for immunosuppression-free transplantation. It is currently garnering considerable attention and might replace kidney dialysis as the ultimate therapeutic strategy for renal failure. However, anatomical complications make kidney regeneration difficult. Here, we review recent advances in the field of kidney regeneration, including (i) the directed differentiation of induced pluripotent stem cells/embryonic stem cells into kidney cells; (ii) blastocyst decomplementation; (iii) use of a decellularized cadaveric scaffold; (iv) embryonic organ transplantation; and (v) use of a nephrogenic niche for growing xenoembryos for  Transplantation represents the ideal method of restoring organ function in patients with organ failure. However, the lack of suitable available donor organs is a serious problem worldwide . RegenerThere are currently no published clinical reports regarding the regeneration of functional organs for the treatment for terminal organ failure. Kidney regeneration is particularly difficult because of the anatomical complexity of the organ , and recThe regeneration of an entire kidney may be difficult, but functional recovery of as little as 10% of kidney filtration function might allow for the withdrawal of dialysis in patients with end-stage kidney disease (ESKD) , thus gr in vitro. PSCs therefore represent an important cell type for bioengineering strategies aimed at kidney regeneration. PSCs include both embryonic stem cells (ESCs) and iPSCs. ESCs are derived from embryos and grown in primary culture [Previous studies have suggested that pluripotent stem cells (PSCs) have the potential to differentiate into any cell type in the body and self-assemble into heterogeneous tissues or organs –6. PSCs  culture , while i culture , 6. PSCs culture , hepatic culture , 9, neur culture , 11, hem culture , pancrea culture , 14, and culture . This apRecent progress in stem cell research has generated human nephron progenitor cells, including intermediate mesoderm (IM) and metanephric mesenchyme (MM) cells –21. An uHigh-throughput chemical screening and low-molecular-weight chemical compounds have recently been used in directed differentiation for kidney formation. An efficient method has been developed for inducing the differentiation of IM cells from hiPSCs/hESCs using a combination of activin A and CHIR99021 to generate mesoderm, followed by combined treatment with bone morphogenetic protein 7 (BMP7) and CHIR99021 , 18. Thi in vitro differentiation of PSCs generated cells with a UB-committed IM fate with the potential to assemble spontaneously into complex, chimeric three-dimensional (3D) structures upon coculture with murine embryonic kidney cells [Many studies have attempted to generate MM progenitor cells by direct differentiation from PSCs. Xia et al. tried to differentiate PSCs into ureteric bud (UB) lineage cells by stepwise treatment of iPSCs/hESCs with a combination of fibroblast growth factor 2 (FGF2) and BMP4 for 2 days, followed by combined treatment with activin A and BMP2 for another 2 days. After the 4 days, IM-like cells expressing PAX2, OSR1, WT1, and LHX1 were produced. The UB markers HOXB7, RET, and GFRA1 were upregulated in these cells after an additional 2 days of differentiation, implying that PSCs could be driven towards UB progenitor-like cells. Additionally,ey cells . HoweverμM) and BMP4 for posteriorization, followed by combined treatment with mid-CHIR99021 (3 μM) and BMP4 with the addition of retinoic acid and activin A. A final step involved the addition of low-CHIR99021 (1 μM) and FGF9 for 1 day each. They finally verified the temporal kinetics of gene expression at each step of the induction process. Wnt signals are important for posteriorization [The strategy of direct differentiation from PSCs has made significant advances in the past few years , 21. Takrization , 27. Ind de novo. The differentiation of induced IM cells into precise renal progenitor cells would allow a complete 3D kidney structure to be constructed from PSCs. However, the means of successfully regenerating a functional vascular system between the regenerated kidney and the recipient remain unknown. Additionally, the in vivo functioning of a regenerated kidney remains unclear. However, further advances in developmental biology and bioengineering may resolve these issues and allow whole kidney regeneration.Generating precise renal progenitor cells is essential for the development of a whole kidney/−− (pancreatogenesis-disabled) mouse blastocysts produced newborn rat/mouse chimeras with a pancreas derived almost entirely from rat iPSCs [ in vivo using a xenogeneic environment [Injection of PSCs into blastocysts, the initial embryonic stage after fertilization, synchronizes the development of two line cells, and the combined blastocyst generates a chimeric body. In the first report of this method, normal ESCs were injected into blastocysts of recombination-activating gene 2-deficient mice, which have no mature B or T lymphocytes, to generate somatic chimeras with ESC-derived mature B and T cells . This “bat iPSCs . The mouat iPSCs . This stironment , 37. Thiironment . Nondefiironment . Howeverironment . Preciseironment .The most important problem associated with blastocyst complementation is the ethical issue. It is impossible to exclude the possibility of generating interspecific chimeras containing brain derived from injected PSCs. Although it is difficult to establish a xenogeneic blastocyst complementation system that overcomes the xenogeneic barrier, this strategy appears to be one of the most promising methods for kidney regeneration. in vitro [Stem/progenitor cells isolated from many adult organs demonstrate self-renewing ability and can give rise to terminally differentiated cells. This kidney stem/progenitor population disappears in the adult kidney, possibly because of the loss of its niche , 38. Howin vitro . Kidney-in vitro , while 2in vitro . They asin vitro . In thisin vitro –47. ThesNative kidney extracellular matrix (ECM) has been reported to provide a scaffold for cell seeding and a niche for stem cells to differentiate into whole organs . The ECM in vitro and ex vivo [ in vivo. However, these transport functions were less efficient than those in native proximal tubules [The developing field of tissue engineering is an extension of cell therapy, in which biological and engineering science techniques are combined to create structures and devices to replace lost tissue or organ functions , 68. The ex vivo . Briefly ex vivo . The tub tubules . Further tubules –74. The We attempted to regenerate a functional, transplantable whole kidney able to produce urine and renal hormones, such as Epo, using a xenoembryo and human mesenchymal stem cells. The embryonic metanephros, which is the mammalian renal anlagen, is thought to represent a potential source for the regeneration of functional whole kidneys –83. An e per se could thus acquire some renal functions in the host omentum, as well as supplying a niche for host stem cells to regenerate renal tissues that can be rebuilt using host-cell components. These techniques may help to reduce the adverse effects of long-term immunosuppressant administration and to address the ethical issues surrounding xenotransplantation [We recently demonstrated that xenotransplanted metanephros could supply endogenous MSCs with a niche for differentiation into Epo-producing tissues . Polymerantation , 86.We exploited the developing xenoembryo as a niche for organogenesis using stem cells of renal lineage. Using this strategy, we previously showed that the xenobiotic developmental process for growing xenoembryos allows exogenous human MSCs to undergo epithelial conversion and form a nephron that produces urine and Epo –89 Figu. During Based on our previous promising results, we are currently investigating the possibility of large-scale harvesting of metanephroi from pigs, given that the volume of the kidney in pigs is almost identical to that in humans . The ult de novo. Despite significant recent advances, the reconstruction of a complete functional kidney remains difficult, and many problems are still unsolved. Direct differentiation of ESCs/iPSCs into nephron progenitors has not yet succeeded in generating mature functioning tissues in vivo. Before regenerated kidneys can be used in clinical practice, a method of generating fully functioning renal tissues that produce urine and Epo needs to be developed. Additionally, the regenerated tissue must be able to survive and function in the long term. Future research in stem cell biology and bioengineering will hopefully resolve these issues and open the door to new therapeutic strategies for kidney regeneration aimed at repairing kidney damage and restoring function.This review has summarized recent research involving the use of renal stem cells and renal bioengineering to regenerate functional whole kidneys


The role of serological tests such as IgA anti-transglutaminase autoantibodies has become increasingly important in celiac disease (CD) diagnosis. However, the efficiency of these tests for patient follow-up is controversial. We investigated the correlation of 12 different serological tests, including recent deamidated gliadin and actin IgA tests, with villous atrophy (VA) in a retrospective cohort of treated celiac patients.Serum samples were collected from 100 treated CD patients who had intestinal biopsy in the course of their follow-up. Antibodies against transglutaminase, deamidated gliadin peptides, and native gliadin were measured, along with IgA anti-actin. The biopsy slides were all blind-reviewed and scored according to Marsh classification.For all deamidated gliadin and transglutaminase tests, we found that a positive result was significantly associated with persistence of intestinal VA, with a diagnostic efficacy up to 80%. Furthermore, antibodies titers directly correlated with the degree of VA, indicating a strong link between disease activity and presence of antibodies in the serum. Interestingly, the tests with the highest association with persistent VA were those for deamidated gliadin IgG. Using a test positivity pattern analysis, we were also able to identify several groups of patients with distinct antibody profiles that showed significant differences in intestinal damage and diet compliance.Altogether, these results show that deamidated gliadin antibodies are strongly correlated with VA and should be considered valuable tools in CD follow-up and that multiplex serologic analysis for treated CD represents a promising tool for personalized patient management. Celiac disease (CD) is an intestinal auto-immune disease, the particularity of which is to be triggered by an exogenous antigen composed of peptides from gluten in genetically susceptible individuals. ClinicaHowever, in the course of adult disease follow-up, small bowel biopsies are common, either to confirm diagnosis, to assess diet efficiency or to detect refractory celiac disease. Therefore, a serological test correlating with villous atrophy in treated patients could reduce the number of endoscopic procedures needed. Despite the good performance of IgA anti-tTG antibodies in CD diagnosis, their efficiency for patient follow-up is less well documented. Under GFD, IgA anti-tTG titers decrease rather quickly in most patients, whereas it can take up to 2 years to observe normalization of the intestinal mucosa in adults, particularly in patients with high autoantibody titers. Of noteWe thus aimed to assess the efficiency and usefulness of these different serological tests in treated patient follow-up.We performed 12 different serological tests on a retrospective cohort of treated celiac adult patients at a center specializing in celiac disease and correlated the results with the analysis of intestinal biopsies performed concurrently in the course of disease follow-up.Our results show a strong association of some tests, particularly tests detecting IgG anti-deamidated gliadin, with biopsy results. Indeed, for most tests, antibodies levels correlated clearly with VA degree and GFD compliance. We then discuss the usefulness of these serological tools, alone or in combination, in celiac disease follow up.A retrospective study was performed on serum samples paired with a concurrent small bowel biopsy performed in the context of celiac disease follow-up between September 2008 and March 2012. The samples were the ones collected during patients standard care procedures and no extra sample was collected for this study. After collection, all samples were routinely kept frozen (-20°C) until use. All the biopsies were performed by upper endoscopy at the Georges Pompidou European Hospital Endoscopy Unit. The initial diagnosis criteria for CD patients were the presence of villous atrophy on an intestinal biopsy sample associated with positivity of anti-transglutaminase IgA in a clinical context compatible with celiac disease. For patients with IgA deficiency, IgG anti-transglutaminase were used. The GFD compliance of treated patients was assessed during standard care procedures by a single dietician specialized in CD follow-up. The compliance data was abstracted from the medical records for this study and linked to serological and histological data. The data was then anonymized and the correspondence table destroyed. Patient records/information was anonymized and de-identified prior to analysis, therefore no consent was necessary. This study was specifically approved by the local Ethics Committee of Georges Pompidou European Hospital (AP-HP)The characteristics of the serological tests used in this study are summarized in ELISA and immunoluminsescence are closely related techniques that measure the presence of specific antibodies by means of an antigen-coated plastic well, and a secondary antibody (anti-immunoglobulin) conjugated to an enzyme. The presence of the specific antibodies is measured by production of a colored (ELISA) or luminescent (immunoluminescence) substrate by the enzyme. While both techniques produce quantitative results, the luminescence technique has a greater dynamic range than classical ELISA.The positivity and titer of IgA anti-F-actin (named anti-actin IgA IF in this study) were evaluated semi-quantitatively by immunofluorescence with the commercial kit “F-Actin”  following the manufacturer’s instructions. Briefly, rat enterocytes fixed on a glass slide were incubated 30 minutes with patient serum diluted 1:5. After washing, a FITC-conjugated anti-human IgA antibody  was added for 30 minutes. Then, after washing and mounting, the slides were read immediately by 2 operators skilled in immunofluorescence reading. The fluorescence intensity of actin microfilaments was evaluated according to the following score: negative (0), weakly positive (1), positive (2), strongly positive (3). Discordant readings were reviewed by a third operator to reach a consensus.Samples were tested by ELISA techniques for IgG anti-transglutaminase cross-linked with deamidated gliadin peptides , IgA anti-transglutaminase , IgG anti-native gliadin , IgG and IgA anti-GAF-3x  and anti-Actin IgA . All tests were performed according to manufacturer’s recommendations. The procedures were realized manually for IgA anti-tTG, tTG-GP IgG, IgA anti-actin, IgG anti-gliadin or on a CARISμ instrument  for IgA and IgG GAF-3X. Results were given in arbitrary units per mL (AU/mL).Serum samples were evaluated for anti-DGP IgA and IgG (QUANTA Flash DGP IgG and IgA), anti-DGP Screen (detecting both IgA and IgG simultaneously) (QUANTA Flash DGP Screen), anti-transglutaminase IgA (named anti-tTG IgA BF for disambiguation) and IgG (QUANTA Flash tTG IgA and IgG) by a chemiluminescent method on a BIO-FLASH immunoanalyzer , according to manufacturer recommendations. Results were given in arbitrary units per mL (AU/mL).Slides from paraffin-embedded intestinal biopsy samples obtained from at least 5 different sites (1 at the bulb and 4 at distant duodenum) were all reviewed by a single pathologist (TM) skilled in intestinal pathology and scored according to Marsh-Oberhüber classification using an intraepithelial lymphocytes cut-off of 30/100. ReviewiIndependent groups of continuous data were compared using Mann-Whitney (2 groups) or Kruskal-Wallis tests (3 groups and more). Categorical data were compared using Chi-squared test. Correlations between continuous variables were calculated by Spearman rank correlation test. Trend analyses were realized by Cuzick test for trends. A p value of less than 0.05 was considered statistically significant. Hierarchical clustering was performed using Euclidian distance and complete clustering method with Genesis 1.7 Software . Statistical analyses were performed using GraphPad PRISM 5.0 (GraphPad Software inc.) and MedCalc 11.1 .The study cohort consisted of 100 serum samples paired with a concurrent small bowel biopsy from treated celiac disease patients who had been referred to a single specialized outpatient center for celiac disease follow-up. The median time between initiation of diet and gastroscopy procedure was 4.5 years (range: 0.5–29). The age, sex-ratio, GFD parameters and biopsy results for all patients are summarized in Two control groups were also constituted, consisting of untreated celiac patients (n = 10) and patients in whom CD had been excluded by intestinal biopsy (n = 12), to be used only as reference populations in antibody positivity pattern analysis . Demographical data of these groups did not differ significantly from the main patient group. All untreated CD patients presented a VA pattern, and all non-celiac patients had a normal mucosa except for two with mild VA. Two patients presented total IgA deficiency and were therefore excluded from analysis of IgA detecting tests.Currently available kits for serological diagnosis of celiac disease are mainly designed for screening previously undiagnosed patients. Consequently, manufacturer-provided cut-off values are calculated using untreated celiac patients as positive control population. To assess whether the analytical performance of tested antibodies would allow efficient discrimination of treated CD patients with or without persistent VA, we first verified if these cut-off values could be used for a treated patient population. A ROC (Receiver-Operating Characteristic) curve analysis was thus performed on our treated CD patient group for each test using presence of intestinal VA as the positivity criteria . Test peRecalculation of optimal cut-off values showed that differences could be observed between the manufacturers’ recommended cut-off and the calculated optimal cut-off for our population . We thusStatistical analysis of the ROC curves showed significant ability to discriminate VA-positive from VA-negative patients for all studied tests except for the two anti-actin IgA tests . As a reThese results show that serological tests for celiac disease are able to discriminate patients with persistent VA in a treated patient population. Of note, antibodies against deamidated gliadin peptides, particularly of IgG isotype, had the highest AUC in a ROC-curve analysis.Having determined that serological tests were able to discriminate patients with VA in a treated CD patient population, we assessed if there was a correlation between test results and intestinal damage. For this analysis, intensity of intestinal damage was measured by Marsh-Oberhüber classification as described in the Methods section .Antibody titers were found to differ significantly among the various intestinal damage groups  for all tests except tTG IgG test. Moreover, a strong positive correlation between the titers and VA intensity could be observed  versus partial atrophy (Marsh IIIa) . However, no significant difference could be observed between patients with normal versus subnormal biopsies (Marsh 0 versus Marsh I-II) except for DGP IgG test .Altogether, these results show that antibodies titers, with the exception of tTG IgG, are strongly linked with the severity of intestinal damage.The relevance of combined antibody detection in a single individual was then investigated by analyzing the test positivity profile for individual patients and correlating this profile with clinical parameters.First, the percentage of positive tests was assessed for each patient and plotted against patient’s clinical status . We obseHowever, the percentage of positive test results was significantly different between CD patients with partial atrophy versus subnormal biopsies (p = 0.003) and between CD patients with severe versus partial atrophy (p = 0.0006).s = 1.0, p = 0.017).Altogether, these data show that the number of positive tests for a single individual is strongly and positively correlated with the intensity of intestinal damage  consistent with previous reports in untreated celiac patients, and tTGThese efficacy figures may seem somewhat low as compared to the better performance of serology in untreated CD patients; however, treated patients are a much more complex population with great individual variation in diet compliance and treatment response. Accordingly, we believe that treated CD patient monitoring is an important aspect of clinical practice that needs to be addressed in serological studies.We found that the tests with the highest association with persistent VA in treated CD patients were deamidated gliadin-based tests, particularly of IgG isotype. This is in line with some reports on untreated CD patients, suggesting superiority of deamidated gliadin for CD diagnosis, 24, 25 The importance of the IgG isotype may seem surprising since IgA are usually considered the best markers for celiac disease. Indeed, IgA tTG clearly outperform IgG tTG for CD diagnosis; however, for deamidated gliadin antibodies, a slight advantage of the IgG isotype for CD diagnosis in untreated patients has been reported, though not much emphasized, in several studies, 28–30.Taken together, our results suggest that gliadin-derived tests, particularly of IgG isotype, are associated with VA persistence with a strong specificity and should be considered valuable tools in celiac disease follow-up despite their low sensitivity.While some previous studies on CD antibodies analyzed up to 6–7 different tests, 31, theTo analyze further these different serological profiles, we used hierarchical clustering, a classical approach in gene expression analysis, which allows sorting a population in an unsupervised manner in groups of patients with similar expression profiles. Here, using the same method, we observed that our treated patient population could easily be divided into groups with distinct antibody positivity profiles. These groups were clinically relevant since each group had distinct VA levels and GFD status. Furthermore, we suggest that this kind of analysis could be even more accurate than Marsh grading as far as CD follow-up is concerned. Indeed, most treated patients display partial atrophy (Marsh 3a) on their intestinal biopsy, with various degrees of GFD compliance, and consequently various degrees of actual intestinal damage. Here, our analysis of antibody positivity profile was able to define distinct groups of patients with significant differences in GFD compliance, even among those with the same level of intestinal damage. This suggests that antibody profile analysis could benefit treated CD patients by identifying more accurately those with greater intestinal damage and allowing them to be referred for diet reassessment, thus limiting the risk of subsequent severe complications. Altogether, we suggest that multiplex serologic analysis in CD represents a new and interesting tool with potential for improving personalized patient care.The concurrent measurement of 10 antibodies is currently costly and complicated in practice. Moreover, the sensitivity of each single test was not sufficient to warrant use in clinical practice on its own. We thus propose a set of 2 tests that could be helpful in serological screening of treated patients for persistence of VA. Several combinations seemed to perform adequately, all containing at least one anti-deamidated gliadin test. Interestingly, the best combinations reached a higher sensitivity than any single test. Moreover, most combinations contained both IgG and IgA tests, suggesting that in some treated patients the antibodies best associated with VA could be IgG and in others, IgA.The retrospective setting of this study led to some limitations. First, the time between diet initiation and follow-up gastroscopy differed between patients, as some came for a relapse after several years of diet while others came for systematic follow-up around 1–2 years after diet initiation. Second, assessment of gluten intake was not done using a standardized questionnaire and thus did not allow us to study groups with different levels of gluten exposure.To our knowledge, however, the present treated CD patient cohort with follow-up biopsy results and deamidated gliadin antibody measurements is the largest of its type published to date. With a greater number of patients, one could measure more precisely analytic performances of tests pairs on patient subgroups with distinct clinical parameters, like gluten intake. In the context of personalized medicine development, performance assessment of tests in specific clinical situations is of great importance, and we believe this question should be specifically addressed on a prospective setting.In summary, this study shows that several serum antibodies, in particular those against deamidated gliadin, are closely associated with biopsy results in CD follow-up. We suggest that cut-off values used for untreated celiac patients are not necessarily appropriate for patient follow-up and should be adjusted to the population being tested. Furthermore, we show that the use of several tests simultaneously could help to detect treated CD patients with persistent VA and more active disease.We believe that this preliminary work shows great potential for serology to significantly reduce the need for endoscopic procedure in treated CD patients and also provide additional and relevant information for diet assessment and patient follow-up.S1 STARD ChecklistSTARD checklist for reporting of studies of diagnostic accuracy. Required checklist for diagnostic study quality assessment.(DOCX)Click here for additional data file.S1 FigThis graph compares for each test the cut-off value provided by the manufacturer and the optimal cut-off value calculated with the ROC curve analysis on treated CD patient population .(TIF)Click here for additional data file.S1 TableThis table provides the p-values for the statistical analyses used for evaluating serological tests in (PPT)Click here for additional data file.