Micro RNAs (miRNAs) and piwi interacting RNAs (piRNAs), along with the more ancient eukaryotic endogenous small interfering RNAs (endo-siRNAs) constitute the principal components of the RNA interference (RNAi) repertoire of most animals. RNAi in non-bilaterians – sponges, ctenophores, placozoans and cnidarians - appears to be more diverse than that of bilaterians, and includes structurally variable miRNAs in sponges, an enormous number of piRNAs in cnidarians and the absence of miRNAs in ctenophores and placozoans.Amphimedon queenslandica, the ctenophore Mnemiopsis leidyi and the cnidarian Nematostella vectensis using a computational approach that clusters mapped small RNA sequences and annotates each cluster based on the read length and relative abundance of the constituent reads. This approach was validated on 11 small RNA libraries in Drosophila melanogaster, demonstrating the successful annotation of RNAi-associated loci with properties consistent with previous reports. In the non-bilaterians we uncover seven new miRNAs from Amphimedon and four from Nematostella as well as sub-populations of candidate cis-natural antisense transcript (cis-NAT) endo-siRNAs. We confirmed the absence of miRNAs in Mnemiopsis but detected an abundance of endo-siRNAs in this ctenophore. Analysis of putative piRNA structure suggests that conserved localised secondary structures in primary transcripts may be important for the production of mature piRNAs in Amphimedon and Nematostella, as is also the case for endo-siRNAs.Here we identify thousands of endo-siRNAs and piRNAs from the sponge Together, these findings suggest that the last common ancestor of extant animals did not have the entrained RNAi system that typifies bilaterians. Instead it appears that bilaterians, cnidarians, ctenophores and sponges express unique repertoires and combinations of miRNAs, piRNAs and endo-siRNAs.The online version of this article (10.1186/s12862-018-1274-2) contains supplementary material, which is available to authorized users. RNA interference (RNAi) evolved prior to the divergence of extant eukaryotic lineages, possibly in response to threats from parasitic double-stranded RNA species such as retroviruses and transposons . In contNematostella vectensis, while capable of bilaterian-like silencing by transcript destabilisation or translational inhibition , 6160, 6Drosophila show evidence of secondary structure while putative piRNA transcripts do not  from the originally annotated copy, and four new miRNAs in Nematostella, all of which are copies of previously reported miRNAs  structurally resemble canonical metazoan pre-miRNAs while the remaining three  are more similar to the eight previously described long-form miRNAs in Drosophila where evidence of a ping-pong biogenesis signature was only found in two of the 11 libraries, a bias for a 5′ uridine and an adenosine at position 10 was detected in all Amphimedon and Nematostella libraries and one of the two Mnemiopsis libraries . In contrast, unique endo-siRNA clusters in Mnemiopsis, as in Drosophila, show evidence of secondary structuring, as do multi-mapping endo-siRNA clusters in all four species  may constitute a new class or type of sRNA, these clusters were further investigated.In addition to putative endo-siRNA and piRNA clusters, a substantial proportion of gth Fig. . As the Mnemiopsis 25-mer clusters have a bias for a uridine at their 5′ end, as is common for endo-siRNAs but no ping-pong biogenesis signature was identified , Nematostella  and Mnemiopsis (NHGRI: ML005359a). Our analysis did not find any evidence of conserved piRNA cluster secondary structure in Drosophila or Mnemiopsis, however Amphimedon and Nematostella piRNA clusters do appear to be structured.Primary transcript secondary structure does not appear to be a requirement for piRNA biogenesis , althougescribed , 69. OrtAmphimedon, Mnemiopsis and Nematostella but not in those of Drosophila suggesting that sites of localised secondary structure within primary piRNA transcripts may be an ancestral feature of metazoan piRNA biogenesis , (ii) secondary structure is probably less important for the biogenesis of most unique endo-siRNAs and (iii) the most efficient targets of unique endo-siRNAs are likely found antisense to themselves, it follows that most unique endo-siRNAs are likely to be the products of cis-Natural Antisense Transcripts (cis-NATs) [Amphimedon [Given that (i) is-NATs) , 72 rathphimedon , 8133 arDrosophila also align to coding genes, although both unique and multi-mapping endo-siRNA clusters show evidence of forming secondary structures. Despite this, the 16% of genes that form cis-NAT pairs in this species account for 22% of all mature coding gene-mapping unique endo-siRNA clusters, suggesting that cis-NATs are the source of some uniquely mapping endo-siRNAs in Drosophila. Differences in the rate of cis-NAT endo-siRNA production observed between Drosophila cell types [Amphimedon. The more compact Amphimedon genome may also be responsible for a higher rate of overlapping antisense transcripts [Unique endo-siRNA clusters in ll types  may acconscripts , 74.Amphimedon, the ctenophore Mnemiopsis and the cnidarian Nematostella. The unexpected differences in the RNAi repertoire of bilaterians, cnidarians, ctenophores and sponges uncovered here, suggests that while the last common ancestor of extant animals employed miRNA, piRNA and endo-siRNA systems, these were not integrated into an ancestral gene regulatory system. This is in contrast to bilaterians, which appear to use a common RNAi system [The RNAi repertoires of non-bilaterian metazoans - sponges, ctenophores and cnidarians – differ both from each other and from the canonical RNAi repertoire of bilaterians. Although largely comprised of the same three major systems that constitute the bilaterian RNAi repertoire, the degree to which miRNAs, piRNAs and endo-siRNAs are expressed varies substantially between the sponge i system –16, althi system , 76. FolAmphimedon material was collected from Heron Island, Australia and RNA extracted using Tri Reagent (Sigma Aldrich). The adult small RNA library was prepared with the Illumina TruSeq Small RNA Sequencing Kit as per the manufacturer’s instructions. Pre-competent larval, competent larval and juvenile small RNA libraries were prepared with the Epicentre ScriptMiner Small RNA-Seq Library Preparation Kit as per the manufacturer’s instructions. Libraries were indexed and the four libraries were pooled with eight others unrelated samples giving a total of 12 samples. These were then split and sequenced over four lanes on an Illumina HiSeq 2000.Additional details on methods can be found in Supplementary Methods. Briefly, Mnemiopsis  [Nematostella  [Drosophila  [http://www.ncbi.nlm.nih.gov/sra) or from NCBI’s Gene Expression Omnibus .R039727)  and DrosR014367)  sRNA datAll fastq files were checked for quality with FastQC. 3′ adaptor sequences were removed with fastx_clipper from the FASTX-Toolkit (v0.0.13). Collapsed reads were mapped to their respective genomes with bowtie (v0.12)  allowingAmphimedon [p-value assigned to each cluster that describes the likelihood that the native sequence of that cluster will fold to form a secondary structure that is more stable than a randomised version of itself. This can be interpreted as the likelihood that the secondary structure predicted for a cluster has not occurred by chance and thus is likely functionally important.All sRNAs that map to annotated rRNAs were removed from the libraries and the remaining reads were clustered using bedCluster.pl . A 150 bphimedon  and in rphimedon . Only clphimedon  and snoRphimedon , and cluphimedon . For thophimedon  with 100Drosophila, 24, 25 or 26 nt were selected, reflecting the shorter length of piRNAs in this species [Mnemiopsis 25-mer clusters, 24, 25 or 26 nt peak clusters were also selected.Endo-siRNA, piRNA and 25-mer clusters were annotated based on the read length composition of their constituent sRNAs. For endo-siRNAs, clusters with peaks of expression at 20, 21 or 22 nt were first selected, reflecting the typical length of Dicer cleavage products. If the sum of the reads constituting the peak read length plus or minus one nucleotide was greater than the total number of reads of all other size classes, these were annotated as endo-siRNA clusters. For piRNA annotation, sRNA peaks of 26, 27 or 28 nt were required for the non-bilaterians while for  species . For MneThe four genomes were annotated according to their coverage by transposons or coding genes before being intersected with sRNA clusters. RepeatModeler and RepeatMasker  were useThe genomic context of endo-siRNA and piRNA clusters were assessed using overlapSelect from UCSC  to deterGene models for the four species were overlapped with themselves using overlapSelect . Gene moCircos plots  were conAdditional file 1:Demonstration of High Uniformity and Low Uniformity sRNA clusters. Two hypothetical hairpin RNAs demonstrating the difference between a high uniformity and a low uniformity clustering. In (a), a total of 16 reads composed of just three distinct reads map to a hairpin RNA giving a uniformity index of 5.3. In (b), 16 reads also map to a hairpin RNA but these are composed of 12 distinct reads resulting in a uniformity index of just 1.3. The high uniformity cluster (a) is composed of an equal number of reads to the low uniformity cluster (b) however these reads are less evenly distributed along the length of the source hairpin RNA. (PDF 304 kb)Additional file 2:Amphimedon juvenile library  show a trend towards increasing UI for high uniformity miRNA clusters as library depth increases (a). Dividing the UI by the library depth acts to normalise these values (b). Library depth normalised UIs can be more accurately compared between libraries. (PDF 138 kb)Effect of library depth on uniformity index. Random sampling of reads from the Additional file 3:Drosophila endo-siRNA and miRNA clusters. Endo-siRNA clusters (yellow) display a consistently lower uniformity of small RNA expression  in comparison to miRNA clusters (red) for both unique clusters (above) and multi-mapping clusters (below). (PDF 3341 kb)Uniformity of Additional file 4:Drosophila, Amphimedon, Nematostella and Mnemiopsis. Genomic loci of annotated miRNA, piRNA, endo-siRNA and 25-mer clusters in all four species. (GZ 1305 kb)Locations of annotated RNAi loci from Additional file 5:Supplementary Methods. Detailed methods (DOCX 66 kb)Additional file 6:Nucleotide biases of piRNA clusters. Nucleotide biases along the length of all sRNAs mapping to predicted piRNA clusters. sRNAs were anchored at their 5′ nucleotide and biases are displayed as a percentage the contribution of each nucleotide at each position. Of note is the tendency for a uracil at position 1 and an adenosine at position 10 in most libraries that together comprise the ping-pong piRNA biogenesis signature. Arrows indicate guanosine enrichments downstream of position 25. (PDF 323 kb)Additional file 7:Drosophila 1st instar larval libraries. (PDF 328 kb)Nucleotide biases of endo-siRNA clusters. Nucleotide biases along the length of all sRNAs mapping to predicted endo-siRNA clusters. sRNAs were anchored at their 5′ nucleotide and biases are displayed as a percentage of the contribution of each nucleotide at each position. Of note is the tendency for a uracil at position 1 which is present in all libraries except the Additional file 8:Amphimedon miRNA candidates. Wiggle plots and predicted secondary structures of mapped reads across the length of previously described miRNA miR-2016a, the newly identified miR-2016b and six novel miRNA candidates (aqu-mir-temp-1-6). For each cluster, the library with the most mapped reads to each loci was used to construct the graph. (PDF 483 kb)New Additional file 9:Nematostella miRNA candidates. Wiggle plots and predicted secondary structures of four newly identified miRNAs in the sea anemone. All four miRNAs are new copies of previously identified miRNAs. (PDF 205 kb)New Additional file 10:Amphimedon and Nematostella miRNAs. (XLSX 48 kb)New miRNA data. Sequence and genomic location data for the newly identified Additional file 11:Nematostella miRNA candidates. Results of miRDeep2 annotation of the newly identified miRNA candidates from Nematostella. (a) nve-miR-temp-1, (b) nve-miR-temp-2, (c) nve-miR-temp-3, (d) nve-miR-temp-4. (PDF 2157 kb)miRDeep2 identification of new Additional file 12:Amphimedon endogenous hairpin RNAs. Wiggle plots and predicted secondary structure of three long highly complementary endo-siRNAs from Amphimedon with unevenly distributed mapped sRNA populations. (PDF 348 kb)Additional file 13:Drosophila, all chromosomes are represented while for Amphimedon and Nematostella, the ten largest genomic scaffolds were used. Beginning with the developmental stage indicated by the arrow, the stages for Amphimedon, Nematostella and Drosophila are as per Fig. Amphimedon the emphasised stage is the pre-competent larvae, for Nematostella the female adult and for Drosophila, the female adult head. (PDF 23669 kb)Co-expression of multi-mapping endo-siRNA and piRNA clusters across development. Each plot is divided into groups of coloured scaffolds/chromosomes, each of which represents a developmental stage. For each plot, the earliest developmental stage is marked with an arrow indicating the chronological order of the following developmental stages. Links between scaffolds/chromosomes indicate co-expression from a particular endo-siRNA or piRNA cluster in the two linked developmental stages. For


PubMed, Medline, Embase and Google Scholar were searched for relevant research articles investigating the exogenous addition of OCN, FGF23 or LCN-2 to vascular smooth muscle or endothelial cells. Overall, these osteokines are directly vasoactive across a range of human and animal vascular cells. Both OCN and FGF23 have anti-apoptotic properties and increase eNOS phosphorylation and nitric oxide production through Akt signalling in human endothelial cells. OCN improves intracellular insulin signalling and demonstrates protective effects against endoplasmic reticulum stress in murine and human endothelial cells. OCN may be involved in calcification but further research is warranted, while there is no evidence for a pro-calcific effect of FGF23 in vitro. FGF23 and LCN-2 increase proliferation in some cell types and increase and decrease reactive oxygen species generation, respectively. LCN-2 also has anti-apoptotic effects but may increase endoplasmic reticulum stress as well as have pro-inflammatory and pro-angiogenic properties in human vascular endothelial and smooth muscle cells. There is no strong evidence to support a pathological role of OCN or FGF23 in the vasculature based on these findings. In contrast, they may in fact support normal endothelial functioning, vascular homeostasis and vasodilation. No studies examined whether OCN or FGF23 may have a role in vascular inflammation. Limited studies with LCN-2 indicate a pro-inflammatory and possible pathological role in the vasculature but further mechanistic data is required. Overall, these osteokines pose intriguing functions which should be investigated comprehensively to assess their relevance to cardiovascular disease and health in humans.Bone-derived factors that demonstrate extra-skeletal functions, also termed osteokines, are fast becoming a highly interesting and focused area of cross-disciplinary endocrine research. Osteocalcin (OCN), fibroblast growth factor-23 (FGF23) and lipocalin-2 (LCN-2), produced in bone, comprise an important endocrine system that is finely tuned with other organs to ensure homeostatic balance and health. This review aims to evaluate  The role of the skeleton has greatly evolved from its premise of being a functional tissue providing primary protection, support, and haematopoietic maintenance. It is now recognised as an endocrine organ providing exciting opportunities for cross-disciplinary research. One candidate thread of enquiry is the skeleton’s interaction with the vascular system. Bone is a highly vascularised tissue, which is important for growth, remodelling and repair. The endothelial cells lining blood vessels have an important influence on bone cells, often in a paracrine manner, establishing a ‘bone-vascular axis’ . This tr−∕− male mice were observed to be poor breeders due to decreased testosterone production and thus has been linked to fertility . These mice displayed a decrease in insulin sensitivity and secretion, through direct activity on pancreatic β-cells, and decreased glucose tolerance , fibroblast growth factor-23 (FGF23) and lipocalin-2 (LCN-2), comprise an important endocrine system that is finely tuned with other organs to ensure homeostatic balance and health . The endertility . Interesognition . FGF23 iognition . Brieflyd sodium , while iolerance . It has olerance .in vitro, and to highlight gaps in the literature which need addressing, for use by clinicians and researchers.While these investigations have drawn evidence for the skeletal control of energy metabolism, mineral homeostasis and much more, the effects of these hormones on the vasculature itself has also gathered interest. Conflicting observational data exists between OCN, FGF23 and LCN-2 and markers of cardiovascular disease in humans , which ain vitro i.e., studies that examined the endogenous over-expression of these osteokines were excluded, as the results would not be deemed to be bone-derived. Similarly, passive reporting of osteokines e.g., expression studies, were not evaluated as this approach excludes the possibility of bone-derived effects and we were interested in clarifying any direct, causal effects of ‘circulating’ osteokines. Only peer-reviewed, full-text articles were included, not conference abstracts or articles not in English.To retrieve articles concerning the exogenous administration of OCN, FGF23 and LCN-2 to micro- or macro-vascular cells, the following search terms were used for database searching : fibroblast growth factor 23, bone gamma-carboxyglutamic acid protein, osteocalcin, lipocalin 2, LCN-2, neutrophil gelatinase-associated lipocalin, NGAL, vascular cell, vascular endothelial cell, and vascular smooth muscle cell. Additionally, the reference lists of relevant review articles and included articles were hand-searched for further studies. Searches were carried out up until 20/09/2018. Retrieved articles were included if they described the exogenous addition of OCN, FGF23 or LCN-2 to vascular cells Osteocalcin (OCN) has been pioneered as a skeletal hormone for over a decade now, solidifying a role in influencing energy metabolism . The matThere is currently no consensus on which OCN fragments to measure, and there are no international or national standards . Total Oin vivo ucOCN treatments in mice and rats concluded that ucOCN protects vascular function and protects against markers that are commonly associated with the development of atherosclerosis . FGF23 is the first described bone hormone and original phosphotonin . FGF23 iex vivo degradation and exhibits diurnal variation, while cFGF2 concentrations can be influenced by iron levels, fibrous dysplasia or familial tumoral calcinosis  is the most commonly used measurement, while C-terminus FGF23 (cFGF23) assays can also be used which measure both iFGF23 and cFGF23 . iFGF23 lcinosis . In adullcinosis . Paediatlcinosis . In patilcinosis .A number of epidemiological studies in humans have reported associations between increased circulating FGF23 concentrations and vascular dysfunction , vasculaIn recent years, literature has emerged directed at elucidating the direct effects of FGF23 in the vasculature. In human aortic smooth muscle cells (HASMCs), 10 ng/mL of exogenous FGF23 has been shown to increase phosphorylation of ERK  after 10 min in serum-starved cells, which in turn increased production of Early Growth Response Protein-1 (EGR-1) . Also inIn microvascular endothelial cells, FGF23 did not increase ERK phosphorylation, nor in human umbilical vein endothelial cells did FGF23 increase hydrogen peroxide production . In HAECThese collated results begin to untie whether FGF23 may have any causal effects within the context of cardiovascular pathologies. A few conclusions can be made including: FGF23 initiates direct effects on vascular cells including SMCs and ECs but actions may be tissue specific; FGF23 appears to increase eNOS phosphorylation and NO production likely in an Akt dependent manner, and predominantly in a klotho-dependent manner ; however, FGF23 also appears to increase ROS and oxidative stress, which is counteracted by klotho; within calcification models, FGF23 increased proliferation of SMCs, but does not appear to increase calcification and indeed may be anti-calcific. Bringing these results together paints a conflicting but potential positive role of FGF23 in vascular function. The results presented encourages the hypothesis that the associations and correlations between FGF23 and CVD are most likely due to deleterious effects of altered phosphate and mineral metabolism and indeed that FGF23 levels likely follow, as oppose to direct, cardiovascular disturbances. It is a complex area, with further discrepancies such as variable physiological and disease states, deficiency of klotho, origin of secretion of FGF23 and klotho, and many more factors leaving the role of FGF23 in the vasculature not a simple question to be answered. Further confirmative examinations are necessary and investigations into whether N- and C-terminal fragments of FGF23 are additionally biologically active and if so, how they differ from intact FGF23. Interestingly, any direct pro- or anti-inflammatory properties of FGF23 on vascular cells have not yet been explored despite accumulating evidence in other tissues for a role of FGF23 in inflammation .in vivo as a monomer, homodimer, or heterodimer by forming a complex with matrix metalloproteinase-9 (MMP-9) which stabilizes MMP-9 by preventing autodegradation , also referred to as neutrophil gelatinase-associated lipocalin (NGAL) or 24p3, is the most recently identified osteokine. However, it is debatable whether LCN-2 can be strictly described as such as it is secreted by a wide variety of cell types, and has indeed been more widely described as an adipokine to date. Despite this, it has recently been reported to be produced in bone ten-fold more than in white fat, at least in mice, and it remains to be reported whether this is also the case in humans . As suchradation . LCN-2 hradation .LCN-2 is increased in obese individuals, which is presumed derived from increased adipose tissue and also increased expression from the liver, but may also be due to changes in skeletal homeostasis which is also altered in obesity . It has LCN-2 is a novel, debatable osteokine. Examining its effects on vascular cells when originating from osteoblasts presents a difficult task, requiring for example tissue specific knock-down studies. Further validation to characterise LCN-2 as an osteokine with more convincing human data is primarily needed. As LCN-2 has been associated with a stage-dependent contribution to atherosclerosis, promoting lipotoxicity within the vasculature in obese states, and causing endothelial dysfunction and cardiovascular complications, it is important to establish whether bone-derived LCN-2 is a key mediator that could be therapeutically targeted . LCN-2 iMost studies to date have focused on observational and epidemiological data on the involvement of LCN-2 in obesity and diabetic associated vascular complications. It is currently unfounded to speculate whether these effects are influenced by bone derived LCN-2. The limited investigations on the direct exogenous addition of this interesting protein in vascular cells are summarised below and in 2O2 induced apoptosis (2O2. This group went on to show that LCN-2 (10 ng/mL) in fact promoted endoplasmic reticulum (ER) stress and proliferation within HPASMCs via increased intracellular iron levels , LCN-2 at 10 and 20 ng/mL increased proliferation after 24 hrs . LCN-2 ppoptosis . They fun levels .HUVECs and human coronary artery smooth muscle cells (HCASMCs) treated with LCN-2  significantly increased secretion of inflammatory markers interleukin-8 (IL-8), IL-6 and monocyte chemoattractant protein-1 (MCP-1) in a dose-dependent manner . Within Despite the clinical associations between LCN-2 and vascular disturbances, the mechanisms at a cellular level by physiological concentrations of exogenous LCN-2 have been largely disregarded and research is warranted. The investigations to date have largely focused on LCN-2 in the context of pulmonary hypertension and the use of HPASMCs. Within this cell type LCN-2 is shown to have anti-apoptotic and pro-proliferative effects, while also decreasing ROS but promoting ER stress. It has also been demonstrated that LCN-2 may be pro-inflammatory in a number of cells types, which may be viewed as beneficial in the acute inflammatory response . Concentin vitro science that has been carried out to date. Thus, this review aimed to collate studies investigating the in vitro effects of OCN, FGF23 and LCN-2 to clarify whether there is support for a basic biological or pathological role within the vasculature. Overall, the evidence base points to direct vasoactive properties of the osteokines OCN, FGF23 and LCN-2 across human and animal cells. Both OCN and FGF23 have been demonstrated to be anti-apoptotic and increase eNOS phosphorylation and NO production through Akt signalling. Taken together, these findings suggest a role for these osteokines in supporting normal vascular functionality. OCN has been implicated in improving insulin signalling, and ensuing protectiveness against ER stress. OCN may be involved in calcification but further research is warranted, while there is no evidence at least of a pro-calcific effect of FGF23 in vitro. FGF23 and LCN-2 have been shown to increase proliferation in some cell types and increase and decrease ROS respectively. LCN-2 has been shown to have anti-apoptotic effects and may increase ER stress as well as have pro-inflammatory properties. LCN-2 has also been demonstrated to promote angiogenesis. There is no strong evidence to suggest a pathological role of OCN or FGF23 in the vasculature, however further research with LCN-2 needs to be completed in order to better understand its role. Despite the topical area, a number of preliminary questions remain to be answered. For example, the role of OCN and FGF-23 in inflammation, the role of OCN and LCN-2 in calcification, as well as their cellular receptors and related downstream signalling pathways in vascular cells. Various cleaved forms of the circulating proteins should also be investigated to assess their biological activity. It is clear that further, human data should be generated to understand the roles and mechanisms of actions of these osteokines in physiological and pathophysiological states.The effects of bone-derived factors on systemic health is an emerging, interesting branch of endocrinology research. As conflicting epidemiological reports have linked these bone-derived factors with multiple vascular pathologies such as atherosclerosis and calcification, there is interest in their therapeutic target potential. In order to derive any causal effects, it is important to review the basic


Despite the use of first-choice anti-epileptic drugs and satisfactory seizure outcome rates after resective epilepsy surgery, a considerable percentage of patients do not become seizure free. ANT-DBS may provide for an alternative treatment option in these patients. This literature review discusses the rationale, mechanism of action, clinical efficacy, safety, and tolerability of ANT-DBS in drug-resistant epilepsy patients. A review using systematic methods of the available literature was performed using relevant databases including Medline, Embase, and the Cochrane Library pertaining to the different aspects ANT-DBS. ANT-DBS for drug-resistant epilepsy is a safe, effective and well-tolerated therapy, where a special emphasis must be given to monitoring and neuropsychological assessment of both depression and memory function. Three patterns of seizure control by ANT-DBS are recognized, of which a delayed stimulation effect may account for an improved long-term response rate. ANT-DBS remotely modulates neuronal network excitability through overriding pathological electrical activity, decrease neuronal cell loss, through immune response inhibition or modulation of neuronal energy metabolism. ANT-DBS is an efficacious treatment modality, even when curative procedures or lesser invasive neuromodulative techniques failed. When compared to VNS, ANT-DBS shows slightly superior treatment response, which urges for direct comparative trials. Based on the available evidence ANT-DBS and VNS therapies are currently both superior compared to non-invasive neuromodulation techniques such as t-VNS and rTMS. Additional in-vivo research is necessary in order to gain more insight into the mechanism of action of ANT-DBS in localization-related epilepsy which will allow for treatment optimization. Randomized clinical studies in search of the optimal target in well-defined epilepsy patient populations, will ultimately allow for optimal patient stratification when applying DBS for drug-resistant patients with epilepsy. Epilepsy is a common chronic neurological disorder characterized by spontaneous recurrent seizures and affects around 70 million patients worldwide . Of thesIf the epileptogenic focus or network can be localized and if the benefits outweigh the risks, resective surgery is effective when compared to medication treatment alone , 89. A mHere, we present a review of DBS of the anterior nucleus of the thalamus in patients with drug-resistant epilepsy and discuss its rationale, clinical efficacy, safety, tolerability, and mechanism of action. Further, we will discuss future steps of identifying DBS as a third line treatment modality in drug-resistant epilepsy, within the spectrum of neuromodulation techniques.Literature for this review was identified searching Medline, Embase, and the Cochrane Library databases from the date of the first available article until September 2017. The following keywords were queried either individually or combined: deep brain stimulation, epilepsy, anterior nucleus of the thalamus, complications, and mechanism of action. The search was limited to studies published in English.The ANT is situated in the rostral end of the dorsal thalamus and is separated from the rest of the dorsal thalamus through a Y-shaped internal medullary lamina. The ANT consists of the anteroventral (AV), anterodorsal (AD), and anteromedial (AM) subnuclei. As part of the limbic circuit of Papez, the ANT receives input from the hippocampal subiculum either directly via the fornix or indirectly via the mammilothalamic tract from the mammillary bodies (MB). Other afferents to the ANT originate from the anterior and posterior cingulate cortex, retrosplenial cortex, and the inferior parietal lobule . Many ofThe recognition of the putative role of the ANT in epilepsy, emerged from several animal studies in the second half of the twentieth century. In a model of focal cortical epilepsy in Rhesus monkeys, ipsilateral lesions in the ANT led to a significant decrease in frequency and duration of seizure generalization . FurtherSubsequently, it was reported that specific electrical stimulation of the MB resulted in a protective effect against seizures . In agreThe first clinical case series with thalamic lesioning for the control of epilepsy date back to 1967 . Due to The pre-SANTE studies show a variable treatment efficacy, which may be explained by the significant differences between the studies, including seizure type, follow-up and ANT-DBS stimulation parameters. Regarding the latter, initial stimulation parameters were based on experimental evidence, experience with stimulation of the central median nucleus of the thalamus for epilepsy and STN-DBS for Parkinson’s disease , 60. Alln = 54, 145 Hz, 5 V, 90 μs, 1 min on/5 min off) or no stimulation . After the 3 months blinded phase, all patients received stimulation. At month 13, all patients entered long-term follow-up in which stimulation parameters and AEDs varied freely. The long-term (5 years) efficacy and safety of this trial was reported in 2015 [These encouraging results culminated with the publication of a randomized double-blind controlled trial of Stimulation of the Anterior nuclei of Thalamus for Epilepsy (SANTE) which enrolled 110 patients with localization-related epilepsy . One mon in 2015 .p = 0.0023). Secondary outcome measures: 50% responder rate, Liverpool Seizure Severity Scale (LSSS), and the Quality of life in Epilepsy (QoLIE-31) did not significantly differ at the end of the 3-month blinded phase. However, compared to baseline, all measures showed significant improvement at the end of the unblinded phase. At month 13 and 25, the median seizure frequency reduction was 41 and 56%, respectively, with corresponding 50% responder rates of 43, 54, and 67% at 37 months. Self-reported seizure severity decreased by 40% in the stimulated group compared to 20% in the control group (p = 0.047). Both LSSS and QoLIE-31 significantly improved at 13 and 25 months [At the end of the blinded phase, the stimulation group showed a relative greater estimated reduction of seizure frequency compared to the non-stimulated group with a difference of 29% (5 months . Long-te5 months . The aut5 months .Reported adverse events (AE) at any time after implantation were most commonly hardware related (22.7%) consisting of paresthesia (18.2%), implant site pain (23.6%), implant site infection (12.7%), and electrode misplacement (8.2%). Procedural related AE such as intracerebral hematoma occurred in 4.5% of the patients, none of which were symptomatic.There were no observed deaths during the operative month or 3-month double-blind phase. In total there were seven deaths during the study: one due to suicide, two definite, and two possible SUDEP, one due to cardiorespiratory arrest, and one died from liver cancer. None of which were considered to be device related by the authors.Of the 105 participants entering the long-term follow-up, 30 discontinuations were reported . Of these, 14 comprised device explants (implant site infection (2), device ineffectiveness (7), neuropsychological disorder (3), meningitis (1), and an undesirable change in stimulation (1)).Although neuropsychological test scores for mood and cognition did not differ between the control and stimulated groups at the end of the blinded phase, significantly more patients in the stimulated group reported on AE relating to depression (14.8%) and memory impairment (13%) compared to the control group . Depression related symptoms were reported in 32.7% and memory impairment in 27.3% of the patients during long-term follow-up. At 5-year follow-up several components of the neuropsychological examination showed gradual improvement from baseline including attention, executive function, depression, tension/anxiety, total mood disturbance, and subjective cognitive function. This paradoxical outcome regarding self-reported depression related symptoms and objective neurobehavioral testing was recently addressed by an in-depth and long-term analysis . During In the years after the publication of the SANTE trial, several case series further reported on the efficacy and safety of ANT-DBS in drug-resistant epilepsy, presented in Table Although the efficacy and safety of ANT-DBS in drug-resistant epilepsy patients was convincingly shown in the SANTE trial, questions remain about the variability of responsiveness to treatment. In addition, two patients displayed a paradoxical response to ANT-DBS: one patient in the SANTE trial suffered from 210 brief partial seizures corresponding to the on-off cycle of stimulation in the blinded phase and one patient had a 200% increase of seizure frequency reported by Krisna et al. The variability of the treatment effect of ANT-DBS may be partially explained by the localization of the seizure onset zone, as patients with a seizure origin in one or both temporal lobes showed a greater response to ANT stimulation when compared to extratemporal, or multiple seizure onset-zones , 60. AnoRecent proposed 3T MRI short tau inversion recovery and 1.5T T1 weighted magnetization prepared gradient echo (MPRAGE) images allow for visual delineation of the ANT. The imaging protocols are capable of clearly visualizing the anatomical boundaries of the ANT  , 54. As Despite its widespread clinical use, the exact mechanism of action of electrical stimulation on the central nervous system remains poorly understood. Initial hypothesis about the mechanism of DBS were based on the similarity between ablative procedures and high frequency stimulation with regard to treatment effect. High-frequency stimulation was therefore thought to function as a reversible lesion by inhibiting neurons near the stimulating electrode , 38, 63.Other mechanisms by which ANT-DBS may remotely modulate neuronal network excitability is through local molecular hippocampal alterations. Unilateral ANT stimulation in kainic acid (KA) induced seizures in rats provoked decreased levels of glutamate and aspartate and an increase of GABA concentration in the ipsilateral CA3 region of the hippocampus . This phAnother mechanism which may underlie the therapeutic effect of ANT-DBS is through influencing glucose metabolism. In patients with temporal and frontal lobe epilepsy, an ipsilateral thalamic and hippocampal interictal glucose hypometabolism is often observed on FDG-PET, and its severity is correlated with a prolonged course of epilepsy , 6, 76. In addition to the ANT, several other brain structures have been targeted with stimulation for epilepsy and have been addressed in randomized controlled trials (RCTs) –84, 90. To date, there are no trials comparing intracranial stimulation to either lesser invasive modalities such as vagus nerve stimulation (VNS), transcutaneous-VNS (t-VNS) and repetitive transcranial magnetic stimulation (rTMS) or best medical practice. Two RCTs report on a similar or slightly inferior treatment response for VNS (MD − 12.7% and − 18.4%) when compared with intracranial targets , 62. A dANT-DBS for drug-resistant epilepsy is a safe and well-tolerated therapy, where a particular emphasis must be given to monitoring of depression and memory function. ANT-DBS is an efficacious treatment modality, even when curative procedures or lesser invasive neuromodulative techniques failed. When compared to VNS, ANT-DBS shows slightly superior treatment response, which urges for direct comparative trials. Based on the available evidence ANT-DBS and VNS therapies are currently both superior compared to non-invasive neuromodulation techniques such as t-VNS and rTMS. Despite its clinical efficacy, ANT-DBS for drug-resistant epilepsy still faces great challenges. Optimization of the procedural DBS protocol including imaging techniques, surgical procedure, and algorithms for adaptation of stimulation parameters could aid to reduce the treatment response variability. Additional research will have to provide for better understanding of normal physiological neuronal networks compared to epileptogenic networks in order to gain more insight into the mechanism of action ANT-DBS in localization-related epilepsy. Ideally, further in-depth knowledge of epileptogenic networks may explain for the differential response of DBS of different anatomical targets in different seizure types , 69, 85.


Stunting is a common cause of early child developmental delay; Guatemala has the fourth highest rate of stunting globally. The goal of this study was to examine the impact of an intensive community health worker-led complementary feeding intervention on early child development in Guatemala. We hypothesised that the intervention would improve child development over usual care.A substudy from a larger individually randomised , parallel-group superiority trial, with blinding of study staff collecting outcomes data.Rural, indigenous Maya communities in Guatemala.210 stunted children (height-for-age z-score ≤−2.5) aged 6–24 months, previously randomised to usual care (106) or an intensive complementary feeding intervention (104). 84 in the intervention and 91 in the usual care arm agreed to participate.Community health workers conducted monthly home visits for 6 months, providing usual care or individualised complementary feeding education.The primary outcomes were change in z-scores for the subscales of the Bayley Scales of Infant Development (BSID), Third Edition.100 individuals were included in the final analysis, 47 in the intervention and 53 in the usual care arm. No statistically significant differences in age-adjusted scores between the arms were observed for any subscale. However, improvements within-subjects in both arms were observed (median duration between measurements 189 days (IQR 182–189)). Mean change for subscales was 0.45 (95% CI 0.23 to 0.67) z-scores in the intervention, and 0.43 (95% CI 0.25 to 0.61) in the usual care arm.An intensive complementary feeding intervention did not significantly improve developmental outcomes more than usual care in stunted, indigenous Guatemalan children. However, both interventions had significant positive impacts on developmental outcomes.NCT02509936.Results. Stunting is the most common cause of early childhood developmental delays, and nearly half of Guatemalan children are stunted, with higher prevalence among the Guatemalan indigenous Maya population.Stunting early in life has deleterious long-term impacts on educational attainment, intellectual outcomes and adult economic earning potential. Few investigations of these effects exist for the Guatemalan indigenous Maya population.Complementary feeding practices interventions are the cornerstone for stunting prevention and treatment; however, investigation of the effects of these interventions on developmental outcomes are limited.Both standard and augmented behaviour change approaches to improved complementary feeding practices by caregivers have important positive impacts on the development of stunted children.Current public malnutrition reduction strategies being implemented in indigenous Maya communities in Guatemala can improve childhood developmental outcomes.Developmental assessments can be feasibly adapted and contextualised to a rural, indigenous population at risk.In low-income and middle-income countries, 43% of children under 5 are at risk of not reaching their developmental potential, and 70% of the attributable risk for delays in early childhood development (ECD) is due to stunting, or low height-for-age.Early scientific research in Guatemala was critical to the international community’s understanding of stunting’s impact on human capital. The Institute of Nutrition of Central America and Panama cohort study—conducted from 1969 to 1977, with long-term follow-up—demonstrated the deleterious impacts of stunting on educational attainment, intellectual outcomes and adult economic earning potential.Given the gaps in adequate complementary feeding practices and diet quality, interventions to promote caregiver knowledge around infant-child feeding are an important strategy in Guatemala.In this study, we contribute by assessing developmental outcomes in indigenous Maya infants in rural Guatemala. We hypothesised that an individualised, intensive approach to caregiver complementary feeding education would improve outcomes over usual care. We individually randomised child-caregiver dyads  to 6 months of usual care, which included home-based growth monitoring and micronutrient and food supplementation, versus usual care augmented with individualised complementary feeding education, using monthly structured interviews and active goal-setting to promote incremental improvements.This study was conducted at Maya Health Alliance (MHA), a primary healthcare organisation working in rural Maya communities. At MHA, nutrition community health workers (CHW) provide home-based services to children aged 6–24 months with stunting.This was a planned substudy on developmental outcomes within a larger, individually randomised, two-arm trial comparing individualised complementary feeding caregiver education with usual care , described in detail elsewhere.Incaparina 900 g ). For the intervention arm, subjects received usual care and monthly individualised caregiver counselling focused on improving meal frequency and dietary diversity for the child,The study duration was 6 months. As described elsewhere,The primary outcome was a change in z-score for subscales of the BSID-III, calculated as described below. In preparation for use of the BSID-III, our team conducted a review of prior studies of psychometric testing with Guatemalan children, with the majority using previous versions of the BSID.In preparation for testing, our team  reviewed existing BSID-III English and Spanish materials.10.1136/bmjpo-2018-000314.supp1Supplementary file 1Nine psychologists  who had formal training in infant/child assessments as part of their education participated in a 1-week long training in rural data collection and multicultural competencies, including active BSID-III practice sessions. Five Maya interpreters  participated in a 1-week training with active BSID-III practice sessions. Senior study psychologist (MPG), with significant professional experience in infant/child assessments, supervised BSID-III training and data collection procedures. Testing was performed by a team of one psychologist and one interpreter, supervised by MPG and the principal study coordinator (BM).For the primary outcome, we calculated that enrolment of 72 subjects per arm would detect a change of 0.5 SD on the BSID-III composite score, with an α of 0.05, power of 80% and 15% lost to follow-up. All subjects recruited for the larger study were allocated by simple randomisation from a computer-generated list of random numbers, no further randomisation or allocation were performed for this substudy.2 or Fisher’s exact tests for categorical variables. Analysis was by intention-to-treat, except where loss to follow-up prevented collecting outcome data or baseline age-band BSID-III data were unavailable to calculate exit BSID-III z-scores.Descriptive statistics were calculated using Stata V.13. Family poverty scores (possible score range: 0–100) were calculated as described.Raw scores for each BSID-III subscale  were calculated as the sum of assessment items performed. BSID-III norms do not exist for Guatemala, and there are concerns about the validity of applying BSID-III norms from a different population. This is the case because, for example, some test items are most appropriate for urban, literate populations  and because some language items require adaptation to the syntactic structure of Mayan languages.We conducted an adjusted exploratory analysis to investigate the effect of prespecified covariates on the primary outcomes, including maternal parity and education, gender, number of under-5 children in the home, household socioeconomic status and age and length-for-age at enrolment. We constructed a hierarchical linear model for BSID-III subscales . Non-significant covariates were removed from the model using serial likelihood ratio tests.Eligible participants were recruited in rolling fashion from August to December 2015. Final participants exited in July 2016. A total of 210 children were eligible for the substudy . Eighty-four children in the intervention and 91 in the control arm received BSID-III evaluation at study entry. Baseline characteristics of participants in the two study arms were well balanced, except for children in the intervention arm having greater minimum dietary diversity .The cumulative incidence of loss to follow-up was 18% (n=15) in the intervention and 14% (n=13) in the usual care arm . One chi10.1136/bmjpo-2018-000314.supp2Supplementary file 210.1136/bmjpo-2018-000314.supp3Supplementary file 3The analysis of primary outcomes was by intention-to-treat for subjects younger than 25.5 months at study exit in this substudy, including one child who discontinued treatment. One hundred subjects were included in the primary analysis  z-scores) and the intervention (mean change of 0.45 (95% CI 0.23 to 0.67) z-scores) arms. These improvements remained highly statistically significant after controlling for important covariates  and occuNo statistically significant difference in improvements was observed between the study arms, suggesting that both usual care and intensive approaches were equally effective in promoting development. Important limitations of our study lead to two alternative explanations for this finding. First, when designing the trial, usual care was to be delivered by an existing public sector rural programme. However, widespread closures of these public services happened during study preparation.Our study also has two important strengths. First, despite extensive work on stunting in rural indigenous Maya populations, there have been few contemporary efforts to investigate developmental outcomes in this population.Our study contributes to the literature on complementary feeding interventions, a cornerstone of stunting prevention and treatment efforts in low-income and middle-income countries.Our study has two important implications for child nutrition policy in Guatemala. Guatemala’s indigenous Maya population has some of the poorest nutritional outcomes in the world and, despite a recent resurgence public interest in this problem,Future research priorities for our group include large-scale well-powered investigations of complementary and responsive feeding interventions, as well as more comprehensive, integrated nutrition and ECD interventions by CHWs in rural Guatemala. This latter point is especially important, since it is increasingly recognised that comprehensive, multisectorial interventions are most likely to generate sustained positive impact. Design and evaluation of comprehensive wrap-around interventions is also most in-line with the Nurturing Care Framework for childhood recently put forth by WHO and Unicef.


Mesenchymal stem cells (MSCs) have emerged as a promising candidate for tissue regeneration and restoration of intra-articular structures such as cartilage, ligaments, and menisci. However, the routine use of MSCs is limited in part by their low numbers and the need for methods and procedures outside of the joint or surgical field.To demonstrate feasibility of a technique in which minimally manipulated synovial MSCs can be mobilized during knee arthroscopy, thereby showing proof of concept for the future evaluation and clinical use of native joint resident MSCs in single-stage joint repair strategies.Descriptive laboratory study.Patients (n = 15) undergoing knee arthroscopy who were free from synovitis or active inflammation were selected. Three samples of irrigation fluid were collected from each patient at inception of the procedure, after an initial inspection of the joint, and after agitation of the synovium. MSC numbers were evaluated by colony forming unit–fibroblastic assay. The phenotype of synovial fluid resident and synovial-mobilized MSCs was determined by flow cytometry, and their functionality was determined by trilineage differentiation. Adhesion of culture-expanded mobilized MSCs to fibrin scaffolds was also evaluated to ascertain whether mobilized MSCs might concentrate at sites of bleeding.P = .001; n = 15). The mobilized cellular fraction contained viable MSCs with proliferative potential and trilineage differentiation capacity for bone, cartilage, and fat lineages, and cultured daughter cells exhibited the standard MSC phenotype. Following culture, mobilized synovial MSCs also adhered to various fibrin scaffolds in vitro. The technique was simple and convenient to use and was not associated with any complications.Normal irrigation during arthroscopy depleted resident synovial fluid MSCs . Numbers of MSCs mobilized through use of a purpose-made device were significantly higher (105-fold) than those mobilized through use of a cytology brush (median of 5763 and 54 colonies, respectively; Numbers of functional MSCs can be greatly increased during arthroscopy through use of this technique to mobilize cells from the synovium.This study highlights a novel, single-stage technique to increase joint-specific, synovial-derived MSCs and thereby increase the repair potential of the joint. This technique can be undertaken during many arthroscopic procedures, and it supports the principle of integrating mobilized MSCs into microfracture sites and sites of bleeding or targeted repair through use of fibrin-based and other scaffolds. The repair of cartilage, however, is limited given its avascular, aneural, and alymphatic nature. Consequently, management of isolated articular cartilage defects poses numerous challenges, and these defects, if left untreated, are risk factors for developing osteoarthritis (OA) in later life.23 Mesenchymal stem cells (MSCs) have been proposed as an alternative source of reparative cell owing to their ease of harvest and their proliferative and differentiation capacity.37 MSCs are multipotent and are internationally recognized by their characteristic adherence to plastic; colony-forming capacity; expression of cell surface markers CD73, CD90, and CD105; lack of expression of CD14, CD34, CD45, and HLA-DR; and ability to differentiate in vitro into osteoblasts, chondrocytes, and adipocytes.10 Several clinical studies using MSCs to treat OA46 have shown positive results; however, similar to ACI, many MSC methods incorporate tissue or cell extraction (from either bone marrow or adipose tissue) before culture expansion and reimplantation. Intra-articular injection of culture-expanded MSCs has also been hampered by both cost and loss of potency.40 Other strategies being explored include the combination of various techniques such as scaffolds and fibrin glue for implantation and retention at sites of injury, which appeared promising at second-look arthroscopy.45Advanced treatment of cartilage defects is available in the form of autologous chondrocyte implantation (ACI). However, ACI is a 2-stage procedure; the first stage requiring harvesting of healthy tissue followed by tissue processing and cellular expansion outside of the surgical field, and the second stage entails reimplantation into the defect site. However, long-term follow-up results of ACI are no better than those of microfracture, a more conservative, much less expensive, single-stage procedure.35 However, for single-stage cartilage repair and OA, joint cavity MSCs including synovium and synovial fluid (SF) are of particular interest as these populations of MSCs have superior chondrogenic capacity and may be more readily and conveniently manipulated since they are already in the surgical field.39 It is now recognized that the knee joint cavity has comparatively abundant reservoirs of MSCs,30 and such MSCs might play a role in joint homeostasis and repair. These MSCs are naturally shed from the synovium20 and are elevated with joint injury and early OA,41 but their repair capacity may be limited due to interactions with SF hyaluronan that limit their adhesion to cartilage.4 Current arthroscopic procedures may inadvertently remove these cells , and procedures such as microfracture to encourage MSCs to percolate from the bone marrow may be inefficient as a result of low and variable numbers of MSCs in bone marrow.29A major drawback with the aforementioned strategies involving cellular therapy is the need for more than one operative procedure. Recent single-stage strategies for joint repair include the use of adipose tissue stromal vascular fraction, which contains MSCs.In this study, we hypothesized that arthroscopic procedures likely wash away resident SF-MSCs imbued with reparative potential . Our priPatients included in the study were treated arthroscopically for a spectrum of knee conditions  includinNormal saline was used to inflate the knee joint to adequately visualize all joint compartments. To achieve this, gravity-fed saline was irrigated through the capsule to maintain a sufficient intra-articular pressure to obtain arthroscopic views. Irrigation fluid was collected from each patient at 3 stages during knee arthroscopy. The first sample contained resident SF-MSCs and was collected by aspirating the initial saline used to irrigate the joint cavity . A second sample of fluid was then collected after inspection of the knee compartments to evaluate the extent to which native SF-MSCs were being “washed away” during surgery . This sample was collected before any procedure that breached the subchondral bone, in settings where this was undertaken. Each sample was placed into a sterile container before transport to the laboratory.The feasibility of mobilizing MSCs from the synovium during arthroscopy was investigated by collecting a third sample of irrigation fluid after agitation of the synovium to mobilize MSCs. To do this we used a cytology brush  routinelThe cytology brush was difficult to introduce into the knee portal due to its inherent flexibility and was limited in the range of synovium that could be agitated. To further improve the capability to mobilize synovial MSCs, we used a purpose-made device fabricated from medical-grade acetal copolymer, the stem cell mobilizing device STEM device) , D and EM device Clinical data for all patients receiving synovial agitation via the STEM device were evaluated retrospectively by a senior soft tissue knee fellow (A.A.T.). At the time, this fellow had completed training and had passed the FRCS examination. The surgeon gave his opinion regarding the likely effect that mobilizing MSCs would have on the expected progression of each participant for his or her indicated procedure . Clinica2.Colony forming unit–fibroblastic (CFU-F) assays were used to determine the number of viable MSCs in each sample. In brief, samples of joint irrigate were centrifuged to pellet cells before resuspending in 10 mL of StemMACS expansion media (Miltenyi Biotec). Duplicate 100 mm–diameter dishes (containing 2 mL of the cell suspension) for each sample were cultured for 14 days in StemMACS expansion medium with twice-weekly media changes, before both dishes were fixed in 3.7% formalin and stained with 1% methylene blue (Sigma-Aldrich). All colonies containing more than 50 cells (verified under a microscope) were counted. The remaining sample was either expanded for further assays or frozen. For expansion, cells were cultured in StemMACS expansion medium with twice-weekly media changes until cells reached approximately 90% confluency before being passaged. MSCs were expanded for 3 or 4 passages, all cells were incubated at 37°C and 5% CO10 through use of an LSRII 4-laser flow cytometer (BD Biosciences). The following antibodies and appropriate isotype controls were used: anti-CD34-allophycocyanin-cyanine (APC), anti-CD19-phycoerythrin (PE), anti-CD45-phycoerythrin-cyaine (PE-Cy7), anti-CD14-fluorescein isothiocyanate (FITC), anti-CD73-PE, anti-CD90-PE-Cy7, and anti-CD105-PE . Dead cells were discriminated by use of 4′,6-diamidino-2-phenylindole . At least 10,000 live cell events were collected for each antibody combination.Flow cytometry was performed on matched, culture-expanded SF-MSCs and Sm-MSCs (n = 3 donors) using a standard panel of markers to define cultured MSCs36 Osteogenic differentiation was assessed with alkaline phosphatase and Alizarin red staining on days 14 and 21 of culture, respectively. The medium used for osteogenic differentiation contained Dulbecco’s modified eagle medium (DMEM) and 100 mg/mL penicillin-streptomycin (Gibco); 10% fetal calf serum (FCS) (Biosera); and 100 nM dexamethasone, 10 mM β-glycerolphosphate, and 50 µM ascorbic acid . Quantitative analysis of calcium deposition was performed on day 21 according to the manufacturer’s instructions . Adipogenic differentiation was assessed with Oil Red-O staining and quantified using Nile red fluorescence on day 21 of culture. The medium used for adipogenic differentiation contained DMEM and 100 mg/mL penicillin-streptomycin (Gibco); 10% FCS (Biosera); 10% horse serum (Stem Cell Technologies); and 500 µM hydrocortisone, 500 µM isobutylmethylxanthine, and 60 µM indomethacin . Pellet cultures were used to evaluate chondrogenesis on day 21 of culture; the medium contained high-glucose DMEM supplemented with 100 mg/mL penicillin-streptomycin and 100 µg/mL sodium pyruvate, 40 µg/mL L-proline, 50 µg/mL L-ascorbic acid 2-phosphate, 1.25 mg/mL bovine serum albumin, ITS+ , 100 nM dexamethasone, and 10 ng/mL recombinant human transforming growth factor-β3 . For quantitative analysis, glycosaminoglycan production was measured according to the manufacturer’s instructions . Toluidine blue staining was used for qualitative analysis of frozen sections.Trilineage differentiation of donor-matched, culture-expanded SF-MSCs and Sm-MSCs (n = 5 donors) was performed by use of standard protocols.g for 10 minutes. Platelets were further concentrated from the supernatant by centrifugation at 1500g for 10 minutes, and the pelleted platelets were resuspended in one-fifth of their original volume using donor matched serum. FG scaffolds were formed with bovine purified fibrinogen (23.7 mg/mL) resuspended in StemMACS expansion media. All scaffolds including WB were formed after coagulation by the addition of 100 mM calcium chloride (CaCL2) and 50 U/mL thrombin (Sigma-Aldrich). To each scaffold and each time point (in triplicate), 10,000 Sm-MSCs were added and allowed to adhere for 10, 30, or 60 minutes before the supernatant was removed and any nonadherent cells were transferred to tissue culture well. These cells were allowed to attach to the culture well before fixing, staining with 1% methylene blue, and counting. A standard curve using a known number of cells was used to interpolate counted cells as a percentage of initial cell number.Culture-expanded Sm-MSCs were used to assess their ability to adhere to various biological scaffolds. Scaffolds were formed from whole blood (WB), platelet-rich plasma (PRP), or fibrin glue (FG). Blood from healthy volunteers was collected in sodium citrate tubes. PRP was prepared after centrifugation at 250U test. The confidence level for each was set at 95%. All statistical analysis was performed with SPSS version 21 (IBM).Due to the limited number of samples, all data were assumed to be nonparametric. As such, paired samples were analyzed with Wilcoxon single rank test and nonpaired samples with Mann-Whitney P = .01, n = 15) in the initial irrigate  compared with the second sample . Samples of subsequent irrigation fluid indicated that MSC numbers on average decreased 4-fold over the course of surgery, suggesting that standard orthopaedic practice depletes the joint of stem cells.CFU-F numbers (a measure of viable MSCs) varied between donors , with MSResident SF-MSCs were collected as above, and these served as a baseline to assess the ability of the cytology brush to mobilize MSCs from the synovium. The number of resident SF-MSCs in these donors (n = 7) ranged from 3 to 1235 . After sP = .007; median, 531; range, 1-1675). In comparison with the cytology brush, the STEM device mobilized 105-fold greater viable MSCs from the synovial lining during arthroscopy (P = .001). Finally, we retrospectively examined follow-up clinical data for those patients who received MSC mobilization with the STEM device. From these data, none of the patients appeared to fare worse than would be expected for their primary surgery had they not undergone MSC mobilization with this device.Intraoperative use of the cytology brush mobilized low numbers of synovial lining MSCs. This could be attributable to the limited area of synovium that the cytology brush could access and because this type of device is intended to trap and remove cells from the body. To overcome this limitation and to further increase MSC number during surgery, we used a purpose-made device  increases the number and availability of stem cells (compared with those resident before and during arthroscopy); further, if left in the joint, these cells occupy a more suitable biochemical environment, being free from high-molecular-weight hyaluronan, which can limit their interaction with cartilage.15 Our results using the STEM device are encouraging, supporting the potential of this technique and the synovium as a robust and convenient source of MSCs. Although colony numbers expressed here are low in comparison with the number of chondrocytes or MSCs currently used in the clinical setting, these Sm-MSCs have lost none of their proliferative or differentiation capacity as a result of culture expansion.16 Direct comparisons between these colony numbers and those obtained from fresh bone marrow aspirate are difficult owing to variations in aspiration technique. However, when an optimized aspiration procedure is used, in which small draw volumes are taken from multiple sites, this would be equivalent to up to 80 mL of bone marrow.13 For nonoptimized bone marrow aspirates in which larger volumes (~60 mL) are drawn before being concentrated,11 we can equate the number of colonies mobilized with the STEM device as being equivalent to up to 380 mL. More research and clinical studies are required to determine whether these Sm-MSCs will lead to clinically significant results. However, the idea is to achieve cartilage repair with a single procedure and thus avoid the need for 2-stage harvest and cell expansion. This will in turn reduce the burden and risks to the patient and the cost to the health care provider.One of the concerns of stem cell–based cartilage repair is cartilage hypertrophy, which leads to uneven articular surface. Synovium-derived MSCs exhibit superior chondrogenic capacity with limited potential to hypertrophy compared with MSCs derived from adipose and bone marrow.We recognize that synovial MSC mobilization was performed on a limited number of patients with a range of injuries. These injuries and the time between injury and surgery may affect the numbers of MSCs and the ability to mobilize them into the joint cavity. The primary purpose of the study was to determine to what extent synovial MSCs could be mobilized during a range of routine arthroscopies. In this study, the stem cells were removed from the joint for laboratory analysis, and so there was no expected benefit to the patient.Here we show proof of concept that a potent source of joint-specific mesenchymal stem cells can be accessed and those cells can be mobilized during routine arthroscopy. This technique can reliably and rapidly repopulate the knee with synovial MSCs that are viable, with trilineage potential in a single-stage procedure. Further studies are needed to ascertain whether this leads to better joint repair across the spectrum of arthroscopic procedures for damaged cartilage and other joint structures.


We report human T-cell lymphotropic virus type 1 infection associated with self-flagellation in 10 UK residents. These persons were heterosexual men from Pakistan, India, and Iraq. One person showed seroconversion in adulthood; 1 was co-infected with hepatitis C virus. No other risk factors for bloodborne virus acquisition were identified. Onward sexual transmission has occurred. Human T-cell lymphotropic virus type 1 (HTLV-1) is transmitted sexually, by contaminated blood products, by organ transplantation, or from mother to child. The estimate of 5–10 million global infections excludes 85% of the general population, for which testing has not occurred, and is probably an underestimate . The patient was of Indian origin and had lived in the United Kingdom since early adulthood. He provided no history of receiving blood products, tattoos, or injection drug use. No family history was suggestive of HTLV-1 infection. In 2008, he donated blood in the United Kingdom that was tested for HTLV-1; he was seronegative. His wife of 10 years was also negative for HTLV-1. He had engaged in zanjeer voluntarily during childhood outside the United Kingdom and continued this practice. In the United Kingdom, the blades were soaked in a bucket containing an over-the-counter antiseptic solution, along with the blades of other men conducting the practice simultaneously. In the previous few years, his practice had also involved striking his forehead with a knife, which was subsequently shared by other men. Physical examination showed widespread scarring on his back  and the We provide epidemiologic findings and HTLV-1 proviral load  for this case-patient and 9 other asymptomatic HTLV-1 carriers of similar demography who reported a history of self-flagellation in Iraq, Pakistan, India, or the United Kingdom . Most reAll 10 patients were given a diagnosis of infection with HTLV-1 through screening programs since 2013. Nine patients had strongly positive Western blot results and positive PCR results. One patient had indeterminate Western blot results and negative PCR results but an HTLV-1/2 enzyme immunoassay sample/cutoff ratio >80, which is consistent with HTLV infection  do not ask about the practice and do not screen for HTLV. We propose that self-flagellation be added to the list of risk factors that result in testing for bloodborne viruses, including HTLV-1. Blood transfusion services might screen for this practice when assessing potential blood donors; for 6 men in this study, blood donation was the route of diagnosis. However, screening blood donors for HTLV-1 infection is not universal. Absence of screening, particularly in regions where self-flagellation is practiced, could accelerate dissemination of this infection. The seroprevalence of HTLV in Pakistan is unknown, but a recent study reported a prevalence of 0.19% among low-risk blood donors , which places them at risk for HTLV-associated disease. In all but 1 case-patient, HTLV-1 was the only bloodborne virus detected. Within heterosexual populations, transmission of HCV is commonly associated with using contaminated injection equipment. HTLV-1 predominance in our cohort suggests the presence of a pool of monoinfected persons and spread within this international community.All patients have been advised by medical practitioners not to share implements during self-flagellation and to encourage fellow practitioners of flagellation to be tested for bloodborne viruses. Our visits to communities in which these practices occur to discuss risk elimination, raise awareness, and promote testing facilities have been favorably received, and risk reduction has been implemented.


To compare the Esterman Disability Score (EDS) obtained with Goldmann perimetry (GP) testing and the Humphrey field analyzer (HFA) in low vision Japanese subjects. Subjects were also divided into groups by diagnosis to examine how disease influences EDS measurements.The EDS was obtained using GP (GP-EDS) and the built-in testing program of the HFA (HFA-EDS). Tests were performed within 3 months of each other. Regression analyses were used to examine the relationship between GP-EDS and HFA-EDS.A total of 128 visually impaired subjects were included in this study. Subjects had low vision because of glaucoma (57 subjects), age-related macular degeneration , retinitis pigmentosa , and other causes (37 subjects). The GP-EDS obtained was well-correlated with HFA-EDS  and it was possible to estimate HFA-EDS from GP-EDS. The GP-EDS was significantly lower than the HFA-EDS in eyes with glaucoma and RP. There was no significant difference between EDS values in eyes with AMD or other disease.The GP-EDS correlated well with the HFA-EDS. However, the relationship between the EDS measured with the two different testing modalities varies by disease. The extent of visual field (VF) impairment widely varies and is dependent upon which region is affected and the severity of the VF impairment that has occurred. In some cases, VF impairment considerably limits movement and the ability to walk, inevitably leading to a deterioration in vision-related quality of life (VRQOL).Previous studies have reported that both VRQOL and health-related QOL decreases with increasing VF impairment severity,2. DiffiGoldmann perimetry VF results are often used in Japan to qualify patients for a formal visually impaired certification. As previously proposed by the American Medical Association (AMA), the sum of angles of an existing VF on 8 meridians in the GP map is used to evaluate VF impairment . HoweverEsterman noticed another limitation of the AMA scale. Mainly, central vision and lower hemianopia were not weighted properly in the overall score. Therefore, Esterman developed his own scoring system, which is now known as the Esterman disability score (EDS)–11. EsteThe official visual impairment grade is determined in Japan using visual acuity measurements and VF impairment severity. Therefore, it is important for Japanese low vision individuals to include peripheral VF examinations when evaluating VF impairment. We consider the EDS to be more suitable for evaluating VF impairment than either the AMA or FVS scales. Furthermore, the EDS was recently made the official method for evaluating VF impairment in Japan. The current study compares the EDS scores obtained with GP and HFA in Japanese patients. Study subjects were also subdivided by disease type to better understand how disease influences the correlation between GP and HFA EDS measurements.This study was conducted at the Department of Ophthalmology at the University of Tokyo Graduate School of Medicine. The study protocol was reviewed and approved by the University of Tokyo Hospital Institutional Review Board and all study conduct adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all patients after the study purpose, experimental procedures, and risks/benefits of study participation were carefully and thoroughly explained.All subjects underwent GP at the University of Tokyo Hospital ophthalmology clinic between June 2015 and February 2016. Subjects were considered for inclusion in this study if all of the following were true: (1) measurements with the Goldmann III-4e isopter could be obtained, (2) they had undergone EDS testing with the HFA within 3 months of enrollment, and (3) they met the VF impairment criteria using the official Japanese visual impairment grading system.The EDS was calculated from GP measurements using the Esterman grid for dynamic perimetry . If a paData are presented as mean ± standard deviation where applicable. A comparison between EDS values obtained with GP (GP-EDS) and HFA (HFA-EDS) was made in Japanese subjects with visual impairments. Subjects were striated by disease and the correlation between GP-EDS and HFA-EDS values was re-examined using Spearman correlation coefficients. Regression and residual analyses of GP-EDS and HFA-EDS were also performed. All statistical analyses were performed using PASW Statistics  and statistical significance was defined as P < 0.05.A total of 128 consecutive Japanese subjects  with a mean subject age of 63.4 ± 15.1 years  were included in this study. Nearly all patients had a stable VF and had been certified as visually impaired individuals in Japan. The causes of vision loss were glaucoma , retinitis pigmentosa , corneal disease (n = 10 [7.8%] subjects), diabetic retinopathy , or other causes (n = 18 (14.1%) subjects).2 = 0.75) was:There was a significant correlation between GP-EDS and HFA-EDS when all subjects were examined . However, in subjects with concentric contraction of the VF, GP-EDS was significantly smaller than HFA-EDS. Glaucoma patients also had a significantly smaller GP-EDS than HFA-EDS. Unfortunately, the HFA does not evaluate fixation and when fixation is poor, EDS will be high.The EDS has been previously shown to be correlated with a variety of disabilities due to visual impairment in glaucoma patients. All studies,15, inveThere are 2 ways to obtain the EDS. One method uses dynamic perimetry and manual calculations, while the other method uses static perimetry and automated software calculations. Both methods have advantages and disadvantages. In dynamic perimetry, EDS is calculated from a binocular VF that is created using a composite of VF measurements from both eyes. However, static perimetry stimuli better mimic everyday life visual stimuli. The time it takes to calculate EDS is shorter for static perimetry than for dynamic perimetry. Lastly, when VF impairments are advanced, especially when central vision is poor, fixation losses can influence static perimetry results. In these cases, examination with dynamic perimetry is more suitable than examination with static perimeter. Patients are required to place their chin in the center of the chin support during HFA measurements. Therefore, patient fixation cannot be checked in the monitor. In summary, poor central visual acuity, concentration losses, and comprehension problems all make dynamic perimetry a better choice than static perimetry.2 = 0.72):The correlation between GP-EDS and HFA-EDS is somewhat controversial. Yamagata et al reportedder et al reportedLangelaan et al investigIn conclusion, the relationship between GP-EDS and HFA-EDS is linear and HFA-EDS can be used to estimate GP-EDS. In practice, simply adding 4.0 points to the obtained HFA-EDS value will be sufficient for estimating GP-EDS. This result increases our confidence in using the HFA to quantify VF impairment. However, the influence of disease-related VF contraction, as in glaucoma and RP, should not be ignored.S1 Data(XLS)Click here for additional data file.


Gemcitabine/capecitabine ) and gemcitabine/nab-paclitaxel ) resulted in a longer median OS and fluorouracil/oxaliplatin/irinotecan ) resulted in a shorter median OS than previously reported. Fluorouracil/oxaliplatin ) and best supportive care ) could not be benchmarked against any RCTs. The degree of protocol adherence explained differences between real-world outcomes and the respective RCTs, while exposure to second-line treatments did not.Clinical outcomes of chemotherapy for patients with advanced pancreatic adenocarcinoma in a real-world setting might differ from outcomes in randomized clinical trials (RCTs). Here we show in a single-institution cohort of 595 patients that median overall survival (OS) of patients who received gemcitabine alone ( Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the European Union . It is eFor the vast majority of patients, who have an unresectable tumor or metastatic disease at diagnosis, and for those who relapse after surgery, cytotoxic chemotherapy remains the best available treatment. Monotherapy with gemcitabine is generally well-tolerated, even by patients with reduced performance status. It alleviates disease symptoms but only marginally improves survival outcomes ,4. CombiGemcitabine and nab-paclitaxel show an improved OS of 8.5 months compared to 6.7 months for gemcitabine . The maiFor patients, whose cancer progresses after first-line treatment, various approaches have been tested, but no consensus has been reached . Best stWe hypothesized that clinical outcomes of patients with pancreatic cancer in routine clinical care differ from those in RCTs. We used a single-institution cohort to evaluate overall survival according to first-line chemotherapy compared to the pivotal RCTs for the respective regimen. To determine which underlying factors are important for differences between RCT and real-world outcomes, we assessed secondary clinical outcomes, protocol adherence, different sequences of first- and second-line treatment regimens, and adverse events.We included 595 patients in our analysis and observed a total of 535 deaths during the follow-up period between January 1, 2013, and April 4, 2018. Data from 60 patients were censored with a median follow-up of 404 days (range 61–1506 days). Patient characteristics indicated substantial heterogeneity across groups that received different first-line treatments . The pron = 27), gemcitabine/nab-paclitaxel (n = 23), 5-FU/oxaliplatin (n = 53), 5-FU/irinotecan (n = 19), or 5-FU or capecitabine monotherapy (n = 13) were used as second-line treatments . We analyzed how the choice of first-line chemotherapy correlated with overall survival a. PatienWe stratified patients by the presence or absence of metastases to assess the effect of disease stage on OS. Differences between patients with local vs. metastatic disease were greatest among those who received gemcitabine , gemcitabine/capecitabine , or 5-FU/oxaliplatin , and thus were greatest for regimens where the RCT used for comparison included patients with both disease stages or where, in the case of 5-FU/oxaliplatin, no RCT was available for comparison . Only siMonotherapy with gemcitabine is used as the control arm in RCTs of combination regimens in PDAC, and we assessed the potential survival benefits of drug combinations over gemcitabine in our cohort a. When wBecause the proportional-hazard assumption appeared violated, we also estimated HRs and 95% CIs derived from flexible parametric survival models . DynamicWe analyzed the time to treatment failure (TTF), the clinical evaluation at the end of treatment, and protocol adherence to evaluate the implementation of different regimens in our routine-care setting to better understand the differences in OS and survival benefit between our cohort and previously reported RCT.Patients with advanced PDAC were generally treated with cytotoxic chemotherapy until progress or unacceptable toxicity. With the exception of gemcitabine/nab-paclitaxel , TTF was approximately three months in all groups . When co2 = 90.9%) and gemcitabine/nab-paclitaxel  groups had the highest cumulative adherence to the protocol. Cumulative adherence to gemcitabine  was reduced and it was low among patients treated with 5-FU/oxaliplatin/irinotecan  and 5-FU/oxaliplatin . We assessed the degree to which treatment protocols were followed in clinical practice by comparing the number of administered courses of chemotherapy over the treatment period to a hypothetical ideal protocol adherence, i.e., the amount of chemotherapy that would have been given if patients had been treated without any complications or delays . Patientn = 75; 24.1% of all patients who had received one of the three first-line regimens) or according to treatment with 5-FU/oxaliplatin/irinotecan or 5-FU/oxaliplatin followed by gemcitabine or gemcitabine/nab-paclitaxel .Patients who were eligible for second-line treatment typically received a fluoropyrimidine-based protocol after gemcitabine-based treatment or vice versa. We grouped similar protocols together and explored the possibility that crossing over to an effective second-line treatment affected the observed OS when patients were stratified by first-line therapy. We identified 102 patients who could be clustered according to treatment with gemcitabine, gemcitabine/capecitabine, or gemcitabine/nab-paclitaxel followed by 5-FU/oxaliplatin, 5-FU/irinotecan, 5-FU, or capecitabine (The median OS measured from the end of first-line treatment a was thep = 0.008). The univariate HR for death among these patients was 1.91 . When we adjusted the HR as above, the multivariate HR was 3.78 . Over the whole course of treatment, however, we observed a median OS of 12.8 months  for patients who received a fluoropyrimidine-based second-line protocol b. For pan = 94; 22.1%) and grade 2 ; more severe grade 3 AEs  were still common but life-threatening grade 4 AEs were relatively rare . We recorded 16 (3.8%) AEs with fatal outcomes. Among the most frequently recorded AEs were hematological AEs, general malaise, gastrointestinal problems, and severe infections. We observed significant differences across treatment groups, but the only identifiable pattern was the restriction of peripheral sensory neuropathy to patients treated with gemcitabine/nab-paclitaxel or 5-FU/oxaliplatin with or without irinotecan . In total, 298 of 389 patients (76.6%) had AEs of any grade during first-line chemotherapy. Among these patients, we retrospectively identified 426 individual AEs that were categorized into 76 different items according to the Common Terminology Criteria for Adverse Events (CTCAE) 4.03. The majority of AEs were grade 1  at the initial visit. Serum levels of the tumor marker CA 19-9, shown to have prognostic and predictive value in PDAC, measured closest to the initial contact or relapse date were used . InsertiWe registered chemotherapy regimens according to our local protocol library. Different protocols for the same drug or drug combination were pooled, and capecitabine could substitute for 5-FU as monotherapy or in doublets with oxaliplatin or irinotecan according to institutional practice. Single drugs or combinations given to fewer than ten patients were summarized as “other”.For patients with unresectable or metastatic disease at primary diagnosis, OS was measured from the initial physician visit related to the patient’s diagnosis of PDAC until the date of death recorded in the Swedish population register. For patients who had undergone tumor resection, OS was calculated from the date that relapse was documented in the medical record or was brought to our attention if the patient was referred. Patients who were alive and receiving treatment at the hospital or best supportive care (BSC) were censored at the date the record was accessed. Patients who were not reported deceased but with no current information available were considered lost to follow-up and were censored at the time of last contact. TTF was calculated from the initial visit at our department for newly diagnosed unresectable cases or the first return visit for relapse until the visit at which discontinuation of treatment was decided.Any cycle of chemotherapy of which at least one dose was administered was counted. For gemcitabine-based protocols with weekly application for three weeks followed by one week of rest, two partially administered cycles could be pooled into one if they were administered within four weeks. We registered dose-reduction, temporary discontinuation (≤1 cycle) of one drug in combination therapies, and partial application of any cycle except for the last one as protocol modifications. To examine protocol adherence over time, we plotted TTF against the total number of cycles that each patient had received and fitted a linear regression model. For comparison, we also plotted a line that corresponded to an ideal 28-day schedule for gemcitabine-based protocols or an ideal 14-day schedule for 5-FU-based protocols. We then expressed protocol adherence as the relative difference between the slopes of the two lines.Adverse events (AE) were assessed per the treating physicians’ notes at follow-up visits and regular blood work. They were initially recorded in a non-standardized fashion, as is routine practice. We used the CTCAE version 4.03 for retrospective classification, and AEs were assigned to the respective treatments during which they occurred .p < 0.05.We compared basic characteristics and categorical outcomes across treatment groups with ANOVA, Kruskal–Wallis, and chi-square tests depending on the scale of measurement of the variable. The rates of AEs were compared using the chi-square test. The Kaplan–Meier method and log-rank test were used to analyze OS and TTF. We calculated the HR for death according to treatment protocol with a Cox proportional-hazards model and used multivariate Cox regression to adjust for covariates. We tested the proportional-hazard assumption using the Schoenfeld residuals test. If the assumption appeared to be violated, we estimated HRs and 95% CIs that were derived from flexible parametric survival models, which allowed HRs to change over time. Analyses were performed with SAS v9.4 , Stata v14.1 , and SPSS, v25 . Statistical significance was defined as All data handling and analyses involving human subjects were approved by the Ethical Review Board in Stockholm . We were exempt from obtaining informed consent for this retrospective study of chart data. The register was reported to the hospital’s Data Protection Officer as mandated.All data are available from the corresponding author if written approval from the data custodian  and the relevant Swedish Ethical Review Board has been obtained. Approvals must be obtained within five years of the establishment of the datasets after which the data will be destroyed according to the originally approved research plan. Chemotherapy for patients with advanced pancreatic cancer in a clinical real-world cohort can achieve survival benefits similar to those described in RCT. The survival benefit associated with gemcitabine/capecitabine was on par with gemcitabine/nab-paclitaxel and greater than previously reported. While evidence from a RCT supports the use of gemcitabine/nab-paclitaxel as the preferred gemcitabine-base first-line combination therapy, our findings warrant reconsideration of gemcitabine/capecitabine for selected patients, e.g., if other combination therapies are no option. 5-FU/oxaliplatin/irinotecan triple therapy remains a good option for well-selected patients, but our findings highlight the importance of good protocol adherence. 5-FU/oxaliplatin did not appear to be an alternative to other combination therapies. Gemcitabine should be chosen in cases where other drugs cannot be used. Our findings do not support that second-line treatment can compensate differences between the outcomes associated with different first-line therapies.