Oral squamous cell carcinoma (OSCC) is a common malignancy for which there is poor prognosis and limited therapeutic options. The objective was to identify mRNA targets of dysregulated miRNAs in OSCC using integrated analysis and understand molecular abnormality in surgical margins. We used biopsies along the spatial axis from normal tissue defined by narrow band imaging (NBI) through conventional white light (WL) margins to tumour from 18 patients undergoing surgical resection for OSCC. Overall 119 miRNA and 4794 mRNA were differentially expressed along the adjacent normal tissue to tumour axis. Analysis of miRNA profiles demonstrated the NBI margins were molecularly distinct from both the tumour and WL margin. Integrated analysis identified 193 miRNA-mRNA interactions correlated to the spatial axis of NBI-WL-T. We used cross-validation analysis to derive a spatial interactome signature of OSCC comprising 100 putative miRNA-mRNA interactions between 40 miRNA and 96 mRNA. Bioinformatic analysis suggests that miRNA dysregulation in OSCC may contribute to activation of the oncostatin M, BDNF and TGF-β pathways. Our data demonstrates that surgical margins defined by NBI leave less potentially malignant residual tissue. The miRNA-mRNA interactome provides insight into dysregulated miRNA signalling in OSCC and supports molecular definition of tumour margins. Locoregional relapse continues to challenge the surgical management of oral squamous cell carcinoma (OSCC), occurring in 16–20% of cases and stalling survival rate improvement4. Despite surgeons’ best efforts to define surgical margins away from carcinoma and dysplasia, and confirmation of this by retrospective histopathology, undetected minimal residual disease (MRD) can be left in-situ, precipitating relapse5. Yet non-invasive techniques are available that identify potentially malignant perilesional tissue with improved sensitivity and specificity6. Recently we reported that use of Narrow Band Imaging for pre-operative OSCC assessment led surgeons to resect visually normal tissue that harboured significant molecular abnormalities without increasing the surgical field unnecessarily, and significantly decreased locoregional recurrence7. Similarly, molecular changes indicative of early tumour development have been demonstrated in histologically ‘normal’ surgical margins of tumours from the larynx, pharynx, and oral cavity10.The success of tumour resection lies in balancing disease removal with healthy tissue retention11. MicroRNA have huge influence on cellular metabolism, proliferation and differentiation, and are key to our understanding of tumorigenesis. Their link to cancer is strengthened by the clustering of miRNA loci within the genome, often at regions that have mutational association with cancer12. Although other studies have reported the importance of miRNA in head and neck SCC21, none have undertaken genome wide integration of microRNA and their target genes’ mRNA expression. The involvement of perilesional molecular abnormalities in MRD can now be studied through detailed assessment of the miRNA-mRNA interactions found at the tumour site compared to molecularly normal surgical margins, taking advantage of NBI to better determine clean surgical margins in situ. This coupling of research provides opportunity for more rigorous investigations into the role of miRNA in oral tumorigenesis as drivers of neoplastic transformation.MicroRNAs (miRNA or MIR) typically comprise 22 nucleotides with partial sequence homology to regions within mRNA molecules. They evoke post-transcriptional regulation of gene expression by promoting mRNA degredation and function as broad-acting regulatory hubs that may be impaired by mutationThe broad aim of our study was to characterise and integrate microRNA and mRNA expression patterns in OSCC using tissue samples along a spatial axis extending from the tumour to disease-free normal surgical margins. We prospectively sampled 18 patients with OSCC using biopsies of tumour, adjacent perilesional tissue , and distant (disease-free) tissue determined by NBI. Firstly, we sought to use molecular analysis of miRNA and mRNA to confirm and extend our previous findings that conventionally determined surgical margins can harbour molecular abnormality. We characterised this spatial gradient of dysregulation at the molecular level and through pathway and gene ontology analysis. As a consequence we aimed to create a miRNA profile of OSCC based upon improved definition of adjacent normal (non-diseased) tissue controls using NBI defined margins as opposed to the currently accepted delineation under white light. The other major objective of our study was to integrate the miRNA and mRNA data in order to select and validate an miRNA-mRNA signature for OSCC. Through this and analysis of ontology and pathways we aimed to derive biological insight into the role of miRNA in the perturbation of signalling in oral tumorigenesis.7. Briefly, patient age, sex and patterns of environmental risk factor exposure  were typical for the RBWH Head and Neck Cancer clinic. All tumour biopsies were negative for human papilloma virus (HPV)7. Tumour histology was predominantly moderate (n = 10) to well (n = 7) differentiated OSCC, with 1 case of verrucous carcinoma7. The most current follow-up data (up to 7 years post-surgery) shows that of the 18 patients, 1 patient (5.55%) declined follow-up. At 5 years follow-up, 14/17 patients (82.35%) were alive with no local recurrence, 1/17 patient (5.88%) had died from their disease, and 2 patients (11.76%) had died disease-free from other causes. In total, 16/17 patients (94.11%) who were followed for a minimum of 5 years were still alive and had not developed local recurrence.Full patient demographic and clinical details have already been published in tabular form together with photographs that describe the positioning of sample biopsiesGeneChip® Human Genome U133 Plus 2.0 Arrays provided 54675 mRNA probe sets, while SurePrint® G3 Human miRNA Microarrays (Release 16) provided 1205 human miRNA probes and 144 human-viral miRNA probes (not reported here). Whole genome mRNA and miRNA expression data from 54 Affymetrix mRNA arrays and 54 Agilent miRNA arrays were processed. Each dataset comprised 3 tissue samples, T, WL and NBI, from each of 18 patients. Quality control procedures eliminated data from 5 mRNA arrays and 10 miRNA arrays  were differentially expressed in at least one sample site Fig. . Overallles Fig. . Given dles Fig. .Figure 27. Consistent with the microRNA analysis, the largest number of statistically significant differentially expressed genes were found in the T vs NBI comparison  analysis of median expression values across the biopsy sites  identified 45 miRNA that were supported by a total of 193 target (mRNA) to effector (miRNA) correlations that were statistically significant (FDR < 0.05). The WL vs NBI paired comparison did not generate miRNA-mRNA associations (data not shown).In order to integrate miRNA and mRNA expression profiles we first used significantly DE miRNA then combined this data with experimentally verified interactions from miRTarBaseThe differential expression data  for the pair-wise comparisons, T vs WL and T vs NBI, were incorporated with the integrated miRNA-mRNA data. We used Circos plots to integrate the data from the miRNA and mRNA differential expression datasets, the miRNA-mRNA interactions and chromosomal mapping of individual targets as shown in Fig. 23.We next undertook additional analysis to develop and validate an integrated molecular signature of OSCC margins derived from the correlated miRNA-mRNA interactome shown in Fig. 7, and strongly supports our premise that resection to surgical margins determined by NBI rather than by WL examination leave less potentially malignant residual tissue and thereby increase surgical success and decrease locoregional recurrence.This study of 18 patients with intra-oral OSCC used both miRNA and mRNA expression profiling and bioinformatic analysis to evaluate the molecular divergence between tumour core and adjacent tissue judged negative for tumour using either conventional white light or Narrow Band Imaging. The miRNA expression profile alone provided unambiguous evidence that the surgical margins determined by NBI possess fewer molecular abnormalities than the more conservative surgical margins determined by WL examination. Pathway analysis of both the mRNA and miRNA dysregulated datasets also demonstrated greater level of biological distinction between NBI and tumour than for WL and tumour. These observations concur with our prior publication which examined mRNA expression profiles alone in the same patient cohort24. The reported accuracy of post-operative histopathology exceeds 95%25, yet around 30% of patients with histologically negative margins experience relapse, prompting concerns about its prognostic sensitivity28. Underlying local relapse in patients with clear surgical margins are two issues: minimal residual disease (MRD) and second primary tumour development from a field of pre-neoplastic cells1. By design, this study provides insight into both of these disease processes, but is primarily focused on the avoidance of MRD. Defining the dysregulation of biological signalling networks across the tumour margins enables the development of a more precise molecular definition of an effective margin.Microscopic tumour at surgical margins has been shown to increase 5-year mortality rates by 90% in a cohort of 707 intra-oral OSCC patients for whom 14.6% of surgical resections produced involved marginsThe three biopsy sites studied  comprise a spatial axis from disease-free histologically normal tissue, through non-cancerous tissue bearing evidence of abnormality, to tumour core. Initially, all of the miRNA and mRNA differential expression data was modelled collectively against this spatial axis and combined to form a putative miRNA-mRNA interactome. We strengthened our analysis by considering only experimentally validated miRNA-mRNA interactions in order to develop a more robust and less complex integrated signature but this approach is necessarily limited since many putative interactions are as yet unverified. This spatially referenced interactome is a “proper superset” of the three discrete paired comparisons , meaning that it includes miRNA-mRNA interactions that do not occur in any of the paired comparisons. Subsequent analysis identified which of the statistically robust miRNA-mRNA interactions were operative in each paired comparison, and could be correlated with the normal tissue – tumour spatial axis.30 although few studies have examined tumour margins spatially to the extent of integrated molecular detail used here.Integration of the putative miRNA-mRNA interactome with the paired comparisons of T vs WL and T vs NBI generated a complicated picture of miRNA-mRNA interaction Fig.  which wa23. Based upon recent comprehensive review of aberrant microRNAs in HNSCC23 of the 40 microRNA found in the integrated signature reported here 28 miRNA had been previously reported as dysregulated in OSCC34 and 7 miRNA had been reported in HNSCC23. The direction of dysregulation found in our own study was consistent with those reported for these miRNAs elsewhere. Thus, the miRNA profile we have developed through integrated analysis is both a consolidation and extension of these previous studies.As described, our integrated miRNA-mRNA signature included 5 unique miRNA  not previously reported in OSCC or head and neck squamous cell carcinoma (HNSCC) microRNA profiling35. Key oncogenic pathways were identified as perturbed across OSCC margins including oncostatin M, brain derived neurotrophic factor (BDNF) and TGF-β signalling pathways  has been identified as an important downstream mediator of Oncostatin M signalling40, and in our own data we found that TGM2 was upregulated in both T vs NBI and T vs WL. The oncostatin M, BDNF and TGF-β pathways have been demonstrated to co-operate to drive EMT and cancer stem cell expression signatures in other cancers. Recent evidence indicates that oncostatin M cooperates with TGF-β signalling to activate EMT via SMAD341. In our own data SMAD3 ranked highest when we used Enrichr to interrogate the database of transcription factor targets (TRANSFAC) with the integrated dataset (results not shown). Similarly in non-small cell lung cancer, STAT3 and BDNF signalling have been shown to cooperatively activate proliferative signalling42. While the contribution of STAT3 signalling to HNSCC progression are well recognised, the mechanisms underlying this activation are poorly understood37. Our data support a contribution by miRNA and their targets to this dysregulation with downstream effects upon multiple interacting oncogenic pathways. The ultimately derived putative miRNA-mRNA signature included interactions that had statistically supported correlation to the normal tissue–tumour spatial axis, as well as excluding all non-statistically robust interactions. Thus, the spatially correlated interactome remains a “proper superset” of the three discrete paired comparisons. Out of 100 interactions, it contains 13 which were exclusively identified by spatial integration including two miRNAs (miR-181a and miR-204) and five additional mRNA  that were not represented within the individual paired comparisons . These miRNA-mRNA interactions require further investigation but this supports the validity of our underlying premise that we could exploit the availability of the three biopsy locations to derive greater information regarding miRNA and mRNA dysregulation using spatial analysis.Enrichment analysis of dysregulation in the individual miRNA and mRNA data as well as the integrated miRNA-mRNA dataset provided further insight into the mechanisms and downstream consequences of this dysregulated signalling. Broadly, pathway enrichment of the miRNA and mRNA DE data was consistent with the findings of our study supporting increasing molecular dysregulation along the NBI to tumour axis. Focal adhesion was amongst the top 10 dysregulated pathways in both the miRNA and mRNA DE datasets Figs  and 4C bays Fig. . Oncosta11, but is a common finding in integrated analysis of miRNA-mRNA expression profiles44. Recent evidence suggests that such positive correlations may be a consequence of an adaptive response to changes in mRNA expression and reflect homeostatic mechanisms regulating miRNA expression44. Furthermore, we included non-reciprocal correlations in our network analysis because miRNA interactions with their targets may depend upon the topology of the signalling pathway45. For this reason, while we focussed upon negative correlations since they reflect direct miRNA regulatory networks, we did not entirely exclude non-reciprocal correlations from our analysis.In this study we observed a large number of non-reciprocal miRNA-mRNA correlations which is intuitively inconsistent with our current understanding of miRNA mechanisms of regulationThis study has shown that miRNA differential expression, putative miRNA-mRNA interactions and dysregulated biological signalling are greater in T vs NBI than in T vs WL. Our analysis across the three biopsy sites strongly supports our premise that resection of OSCC to surgical margins that are determined by NBI rather than by WL will leave less potentially malignant residual tissue and thereby increase the likelihood of surgical success. This is significant for surgical management of oral cancer but also has implications for molecular analyses of cancer genetic dysregulation using conventionally defined “normal” tissue observed under white light visualization. By stringent analysis we identified the specific miRNA-mRNA interactions that correlated with the normal tissue to tumour spatial axis. This signature is robust, statistically validated and is not conflicted by control “normal” tissue which may harbour molecular abnormality. These particular interactions are likely to include miRNA-mRNA interactions that accompany oral neoplastic transformation. Our integrated bioinformatic analysis suggests that miRNA dysregulation in OSCC could be a mechanism contributing to activation of the oncostatin M, BDNF and TGF-β pathways with signalling via STAT3 and SMAD3. Finally, our statistically robust miRNA signature in OSCC is a foundation for translational development of molecular assays which can be applied to the definition of surgical margins with greater fidelity than current methods.7. This study was run in strict accordance with the principles outlined in the Declaration of Helsinki (2008) after ethical review and approval from the Royal Brisbane and Women’s Hospital Human Research Ethics Committee (HREC/08/QRBW20 and HREC/10/QRBW336). All subjects provided written informed consent to participation in the study.Eighteen patients with intra-oral squamous cell carcinoma  who were scheduled for surgical resection were enrolled prospectively. Patient demographics, tumour characteristics and surgery have been described by us previously7.The NBI margin – 5 mm beyond the limit of tissue abnormality visible by NBI.The White Light (WL) margin –5 mm beyond the limit of tissue abnormality visible by White Light.The core of the primary tumour (T).Prior to surgery, primary OSCC sites were visualised under White Light (WL) and then by Narrow Band Imaging (NBI) using an Olympus NBI ENF-VQ nasendoscope with CLV-180 light source and processor . Upon completion of the OSCC resection which was taken to ≥5 mm beyond the NBI defined surgical margin, a 4 mm punch biopsy was taken for analysis from within the following zones as reported by us previouslylater  and subsequently stored at −80 °C. The NBI margin sample is histologically normal disease-free tissue adjacent tumour as previously reported by us7.The biopsies were immediately immersed in RNAfree Kit , RNA was purified by sodium acetate precipitation ; quality and quantity assessments used a NanoDrop spectrophotometer  and Qubit fluorometer . RNA integrity was assessed using an Agilent 2100 Bioanalyzer and RNA 6000 Nano kit .Frozen biopsies were ground in liquid nitrogen then incubated in 500 μL of lysis buffer , with 200 ng of Proteinase K  overnight with mixing at 37 °C. After incubation, 200 μL of lysate was used for RNA isolation with TRIzol Reagent  following the manufacturer’s protocol that was optimised to increase nucleic acid recovery by addition of 10 μg of RNase-free glycogen as a carrier and an overnight incubation at −20 °C. After DNase treatment with the TURBO DNA-This study utilised GeneChip® Human Genome U133 Plus 2.0 Arrays  and SurePrint® G3 Human miRNA Microarrays Release 16, 8 × 60 K  to derive whole genome mRNA and miRNA expression data from three samples  for each of 18 patients, giving a total of 108 expression profiles. 100 ng of total RNA was the input for target preparation for both mRNA and miRNA hybridisations using the GeneChip 3′ IVT Express Kit  and the miRNA Microarray System with miRNA Complete Labeling and Hyb Kit  respectively. Manufacturers’ protocols, reagents and spike-in controls were used throughout. The resultant labelled and amplified RNA (aRNA) was subject to quality control (QC) assessment of size distribution and yield using an Agilent 2100 Bioanalyzer prior to array hybridisation.affyAnalysisQC46 and simpleaffy47; miRNA via the AgiMicroRNA package48. Good library quality and consistent hybridization quality were evident, however some data from both mRNA and miRNA arrays were identified as outliers and excluded from further processing  were normalised by the Robust Multi-array Average (RMA) method without correction followed by quantile normalisation48. Bioinformatic data analysis was performed at the Queensland Facility for Advanced Bioinformatics (QFAB).An overview of the bioinformatics pipeline is shown in Fig. ng Table . The remLimma R package50 via 3 pair-wise comparisons: T vs WL; T vs NBI; WL vs NBI. Linear models were applied to the (log-transformed) mRNA and miRNA expression data, then analysed by paired t-tests adjusted for multiple comparisons via the Benjamini and Hochberg (BH) procedure51. The datasets were again filtered to remove probes that were not differentially expressed in at least one group (Padj < 0.01).After normalisation, preliminary filtering removed probes with coefficients of variation <0.1 across all mRNA or miRNA arrays. Next, differential expression of genes was tested using the mixOmics software package52. The PCA analysis used normalised expression data subject only to preliminary filtering that removed probes with coefficients of variation <0.1 across all arrays. Hierarchical clustering was performed using Cluster 3.053.Clustering of differentially-expressed genes was achieved by Principal Component Analysis (PCA) using the 54. All cross-mapping of mRNA targets to miRNA effectors used the experimentally validated interactions curated in miRTarBase database (version 4.5)22. Our analysis sought generalised correlation models and therefore used median cohort mRNA and miRNA expression values for T, WL and NBI. MicroRNA to mRNA correlations were established using sparse partial least-square (sPLS) analysis55 with concurrent parameter tuning to optimise the number of mRNA retained for each miRNA. Only data for biopsies where there were both mRNA and miRNA expression data were used for integrated analysis. The statistical significance of each correlation was tested using Pearson’s product-moment correlation coefficients corrected for multiple testing using the BH procedure51 to calculate adjusted p-values to estimate FDR. Circos plots for visualisation of mRNA and miRNA differential expression, and correlated interactions were generated using Circa (OMGenomics.com).Micro-RNA sequences, annotations and chromosome positions and clustering were identified using the miRBase database (version 16)56.Rotation estimation for each miRNA-mRNA correlation was achieved by leave one out cross validation (LOOCV) in order to establish the robustness of each putative interaction. For this, each patient’s data was omitted sequentially prior to repeating the mRNA to miRNA sPLS analysis. The number of times an mRNA target was selected (out of 18) is provided as Stability Rate, whereby a value of 1 implies perfect stability (mRNA selected 18 out of 18 times) whereas a value of 0.33 would indicate poor stability (mRNA selected 6 out of 18 times). The arbitrary threshold used here for inclusion in the final miRNA-mRNA signature was stability ≥0.5 (i.e. 50%). Circos plots for visualisation of the integrated miRNA-mRNA signature were generated using Circos Table Viewer v0.63–9https://ccb-compute2.cs.uni-saarland.de/mieaa_tool/)57. For pathway analysis of the differentially expressed mRNA genesets we used GSEA hosted at the Broad Institute (https://genepattern.broadinstitute.org/gp/)58. The KEGG (Kyoto Encyclopedia of Genes and Genomes pathway) and Reactome curated pathway genesets from Molecular Signature Database  were used. For GSEA gene differentially expressed gene lists were pre-ranked by logarithm transformation of p-value (base 10) and application of the sign of the fold-change . Overrepresentation analysis (ORA) of differentially expressed genes derived from the integrated miRNA-mRNA data for pathways and gene ontology terms was performed using the web based platform EnrichR (http://amp.pharm.mssm.edu/Enrichr)59 which permits interrogation of multiple databases. Up and down regulated gene lists were evaluated for significant enrichment against the following gene set libraries: GO Biological Process, GO Cellular Component, GO Molecular Function , the Wikipathways database (http://www.wikipathways.org/) and KEGG (http://www.kegg.jp/kegg) . Enriched annotations/pathways were selected/ranked based upon combined score which was calculated by the EnrichR platform following Z-score permutation background correction on the Fischer Exact Test p-value60. In order to facilitate the interpretation of GO analysis the selected output annotation lists were further consolidated and summarised using REVIGO61, a tool that removes redundant terms based upon similarity. Network analysis was performed using Cytoscape 3.5.162 and the Wikipathways plugin63.Gene set enrichment analysis of biological pathways associated with differentially expressed miRNAs was performed using the miEAA tool (Supplementary Information


Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient’s stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair  are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much higher than the 0.65% predicted for such a short time frame, based on ageing results for healthy individuals. Patients that undergo autologous hematopoietic stem cell transplant (aHCT) for the treatment of a persistent or relapsed/refractory Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) are at high risk of a secondary therapy-related myelodysplasia/acute myeloid leukemia (t-MDS/AML), which constitutes a fatal complication of aHCT –7. The mXRCC1—a protein involved in multiple repair pathways related to its involvement in dealing with single strand breaks  . We have shown previously that we can obtain consistent measurements of repair when starting from identical frozen aliquots for both types of samples using identical host-cell reactivation assays [To investigate DRC in individuals that underwent aHCT as a way to evaluate risk of developing t-MDS/AML, we used a systematic approach by first considering how to best measure interindividual differences in repair in healthy individuals. There is no absolute independent way to determine if any measurement accurately represent the DRC of a given individual, but assays that do measure interindividual differences in repair should consistently identify relative differences across cell types, if they are to represent the individual’s genetically-determined DRC rather than the specific cell type investigated. For aHCT patients in our cohort, we had access to cryopreserved peripheral blood cells, giving the possibility to analyze either directly primary lymphocytes , single-strand annealing (SSA), base excision repair (BER) and nucleotide excision repair (NER). We confirmed that primary lymphocytes are likely a better model to analyze interindividual DRC and then applied the finalized assays to investigate DNA repair in primary lymphocytes of lymphoma patients that were known to have later developed or not t-MDS/AML. Patient samples analyzed were cryopreserved immediately before as well as at some time point after aHCT.Use of human blood samples from healthy volunteers and patients undergoing aHCT was approved by the City of Hope Internal Review Board: IRB protocol #98117 entitled “The Molecular Pathogenesis of Therapy-Related Myelodysplasia/Acute Myelogenous Leukemia”. Patients and healthy volunteers provided their written consent.vs post-aHCT for the same individual or comparison of patients to healthy individuals).We have constructed a prospective cohort of patients undergoing aHCT for HL or NHL. Patients were followed longitudinally with collection of peripheral blood samples prior to aHCT, and serial peripheral blood samples until 5 years post-aHCT. This design allowed use of a nested case-control approach to compare DNA repair from “cases” that developed t-MDS/AML after aHCT with “controls” who did not develop t-MDS/AML after a period of follow-up that matched that of the corresponding index case. The matching criteria for control selection included underlying disease (HL or NHL), age at aHCT, race/ethnicity, and length of follow-up after aHCT (period of follow-up for the controls exceeded that of the patient cases under consideration). The selected individuals  were anaTo investigate DNA repair capacity in healthy individuals, blood samples (30-35ml in heparin tubes) were obtained from 16 healthy volunteers (numbered H33 to 48) and processed within 3h after being drawn. The blood was first diluted in 1 volume of Dulbecco's phosphate-buffered saline (DPBS) containing 2% heat-inactivated fetal bovine serum (FBS). The diluted blood was then transferred onto a SepMate-50 tube (Stem Cell Technologies) containing 15ml of HistoPAQUE-1077 (Sigma) or Lymphoprep (Stem Cell Technologies) and further processed as recommended by the tube manufacturer before freezing (see below).Peripheral blood of patients drawn before or after aHCT was prepared as to preserve all white blood cells (WBCs). The blood (30-35ml) was collected in heparin tubes and diluted in 1 volume of DPBS containing 2% heat-inactivated FBS. One volume of HESPAN Hetastarch  was then added to the diluted blood to aggregate erythrocytes. After 45min of incubation, the upper phase containing WBCs was then transferred to a new tube and washed once with DPBS and once with culture medium before freezing (see below).For all primary cell preparations , cells were frozen or thawed with the same protocol. Cells from ~2–3 ml of blood were resuspended in 1ml of Iscove’s Modified Dulbecco’s Medium (IMDM) containing 20% heat-inactivated FBS and 1ml of cold freezing medium  was added. Temperature was progressively decreased to -80°C in a Mr Frosty container  and tubes were transferred the next day to the vapor phase of a liquid nitrogen storage tank.For thawing, 30ml of IMDM (with 20% FBS) containing 4,000U/ml heparin and 62.5μg/ml DNase were added dropwise to a rapidly thawed aliquot and cells were allowed to recover for at least 2h in a 37°C incubator before a 15min spin at 200g and resuspension in RPMI 1640 medium containing 10% heat-inactivated FBS.EBV transformation of 16 healthy individuals’ B cells into LCLs  was perf6 total cells were resuspended in 50μl of cold isolation buffer (DPBS with 2% heat-inactivated FBS and 2mM EDTA) in a 2ml tube and 2.5μl of FlowComp Human CD3 antibody were added and incubated with the cells 10min at 4°C. Excess antibody was then washed out with 500μl of isolation buffer and cells were pelleted by centrifugation at 350g for 8min at room temperature. The cell pellet was then resuspended in 130μl of isolation buffer and 10μl of prewashed FlowComp beads were added and kept in suspension in the solution for 15min at room temperature by gentle agitation using a Hula Mixer (Life Technologies). Cells bound to the beads were then recovered by adding of 130μl of isolation buffer mixed well with 2–3 up and down pipet motions and then placing the tube on a DynaMag-5 magnet (Life Technology) for 2min. While still on the magnet, all buffer (containing unbound cells) was then removed from the tube and bead-bound cells were then washed a second time with 130μl of isolation buffer before being resuspended in 150μl of the release buffer provided with the purification kit (incubation 10min under gentle agitation with Hula Mixer at room temperature). T cells released from the beads were then counted using Trypan blue and washed by adding 500μl of isolation buffer and centrifugation 350g for 8min. Cells were then resuspended in an appropriate volume of culture medium (RPMI 1640 + 10% heat-inactivated FBS). When indicated, T cells were then induced to proliferate by adding 1:1 ratio of CD3/CD28 DynaBeads human T activator (Life Technologies) and 30U/ml of human recombinant IL-2 (PreproTech) as recommended by the manufacturer. Growth culture volume for the induction of proliferation was 500μl (24-well plate) for all healthy donor samples  or 100μl (96-well plate) for 5 x 104 T cells for patient samples.When indicated, T lymphocytes were purified either from PBMCs or all WBCs (hetastarch) samples using Dynabeads Flowcomp human CD3 kit (Life Technologies) as recommended by the manufacturer with the following modifications . Up to 5 x 10+ cells among viable (DAPI negative) transfected cells (tdTomato+) determined using a BD LSRFortessa cell analyzer with the FACSDiva software  as described previously [Plasmids pSF-tdTomato-END for NHEJ repair and pSF-tdTomato-HOM for SSA repair  were digeviously . NHEJ anPhotinus pyralis) luciferase (FL) reporter gene that is reactivated upon repair (in the pM1-Luc plasmid). The pRL-CMV plasmid expressing the renilla (Renilla reniformis) luciferase is used as an internal control to normalize FL activity levels for transfection efficiency (see below for description of the assay).BER and NER assays use the same plasmid as reporter  and diff4 pH 7.5 buffer was placed in a small weighing dish on ice and irradiated for 5min with a pre-warmed 150W incandescent bulb at a distance of 10.5cm in presence of 15μM methylene blue, which generated 8-oxoG damage. For the NER test plasmid, 250μg pM1-Luc in a final volume of 2.5ml in 10mM NaPO4 pH 7.5 buffer was placed in a small weighing dish on ice and irradiated with 120J/m2 with a germicidal (UVC) lamp to generate pyrimidine dimers. The irradiation time necessary to provide the UVC dose was determined based on the radiant incidence of the lamp measured that day using a UVX digital radiometer (UVP).To prepare the BER test plasmid, 250μg of pM1-Luc in a final volume of 2.5ml in 10mM NaPOth volume of 3M NaOAc pH 5.2 and 2.5 volumes of ice cold 100% ethanol. After a wash with 70% ethanol, DNA pellets were resuspended in 500μl H2O and the DNA concentrations verified with a Nanodrop spectrophotometer. Finally, the volume of H2O was adjusted to a final concentration of 400ng/μl. To verify the level of DNA damage present in BER and NER plasmids, we designed a method to estimate the level of inhibition of polymerase extension specifically in the coding sequence of the FL reporter gene  per template. As a consequence, a limited number of discrete repair events can be expected to result in increased expression levels, making the assays sensitive to low levels of repair.After exposure to DNA damage, both BER and NER plasmids were precipitated with 1/10ter gene . BamHI-dter gene . AlthougTo measure DRC, 400ng of either BER or NER template were co-transfected (see nucleofection protocol in previous section) with 100ng of pRL-CMV plasmid. Pre-mixes containing this ratio of BER+pRL-CMV or NER+pRL-CMV plasmids were prepared in advance to insure consistency in co-transfected amounts, eliminating pipetting error as a source of variation between transfections. After nucleofection, cells were transferred into 180μl phenol red-free RPMI 1640 medium with 10% heat-inactivated FBS and placed in an incubator for 8h. To quantify repair by BER or NER, cells were centrifuged in the 96-well culture plate and some of the excess culture volume was removed. Finally, 75μl of resuspended cells were used to measure the firefly over renilla luciferase relative activities for each transfection, using the Dual Glo Luciferase assay system (Promega) as recommended by the manufacturer. pRL-CMV plasmid alone and undamaged pM1-Luc+pRL-CMV were used as controls for 0% and 100% firefly luciferase activity, respectively. Two (patient samples) or 3  technical replicates were averaged to determine DRC, meaning separate test transfections of the same construct in the same experiment. The reproducibility among those replicates tended to be lesser in samples where more cell death was observed .Data obtained for all the assays are summarized .. PearsoPROC CORR, NPAR1WAY, GLM of SAS software, version 9.4  or GrapPad Prism 6 were used for analysis. Two-sided tests with p<0.0125 were considered statistically significant with Bonferroni correction for multiple comparisons.+ cells among transfected cells (tdTomato+), as determined by flow cytometry. EYFP expression is the result of NHEJ repair or SSA repair, depending on the design of the linearized plasmid transfected (To determine if transformed cells (LCLs) are a good proxy for DSB repair in primary lymphocytes of the same healthy individual, repair was evaluated using host-cell reactivation assays after transfection of damaged plasmids into both cell types using the same basic protocol. DSB repair capacity by NHEJ or SSA was measured by the proportion of EYFPnsfected .+) cells with a ratio of one bead per T cell. Experiment on the effect of induction were therefore performed on purified T cells rather than PBMCs as a whole, but we have verified that DSB repair is constantly the same for a given individual whether lymphocytes were purified or not prior to repair measurements  cells of two healthy individuals were either analyzed non-induced or induced to proliferate for up to 9 days. To determine the effect of induction on DSB repair, purified T . In the course of measuring DSB repair in non-induced lymphocytes and LCLs, we noticed that the two DSB repair pathways seemed to vary together for each cell type. in vitro prior to transfection (BER and NER repair activity were measured using the capacity to reactivate the firefly luciferase reporter expression that is inhibited by 8-oxoG or pyrimidine dimers damage generated sfection .+) cells  cells , but fou+) cells  could be+) cells .Therefore we compared BER and NER in the same early LCLs as previously, and in T cells induced to proliferate for 3 days and found there was no correlation between the two cell types for those pathways either . On the Overall, and although there is evidence that an individual’s genetic make-up contributes to the measured repair and that there are clear patterns to repair as determined in LCLs, it seems likely that primary cells allow more accurate measurements of interindividual differences in DNA repair capacity.+). After purification, a limited number of cells (5 x 104 T cells) were expanded using CD3/CD28 beads and IL-2 for 3 days for BER and NER assays, whereas the rest of the purified T cells were used non-induced for DSB repair assays  cells drawn prior to the transplant in order to determine if DRC could help in any way predict which individuals would later develop t-MDS/AML and 2) cells drawn for the same individuals at one time point post-aHCT but prior to any malignancy diagnosis, for DRC after transplantation . Cases . Table 4For 20 aHCT recipients (13 controls and 7 cases), we could obtain DSB repair data both before and after aHCT , allowinIn contrast to the results with NHEJ and SSA, BER and NER repair analyzed for 18 individuals (9 cases and 9 controls) was not significantly affected by aHCT. The results for NER and BER do not indicate that repair for individuals were actually identical before and after transplant, but rather that observed variations did not display a specific identifiable pattern .We wondered if a decrease in repair could be a manifestation of aging in immune system cells. To investigate if repair was affected by age, we analyzed again the repair results for healthy individuals for each type of repair pathway in function of age at blood draw  and founOn the other hand, neither BER nor NER were associated with age in healthy individuals . OverallWe wondered how pre-aHCT patients compared to our group of healthy individuals regarding their DRC. BER was lower in pre-aHCT patients as compared to healthy individuals (p = 0.0134) but NER was not (p = 0.4347). Especially the subgroup of control patients, those who never developed t-MDS/AML, showed a much lower BER capacity than healthy individuals  . After aIt is likely that differences in DRC influence an individual’s risk of t-MDS/AML after aHCT. Being able to measure DRC might therefore help in predicting who is at higher risk and how to adapt their treatment accordingly. DRC in peripheral blood cells can be investigated directly in primary (mostly T) lymphocytes, or in LCLs after EBV transformation. Before deciding which cell model to use for measuring DRC in aHCT patients, we first investigated what seemed the most reliable way to determine an individual’s DRC using samples obtained from healthy individuals.Unsurprisingly, the measurement of interindividual differences in BER and NER repair in primary lymphocytes was found to be possible only in lymphocytes that were induced to proliferate. This requirement was observed the first time a host cell reactivation assay was performed on primary lymphocytes to measure NER . Howevervs a homology-directed pathway that first requires strand resection (SSA). Those two systems are thought to be in competition with each other for the repair of the same breaks, the choice between direct end-joining and strand resection being thought to be predictive of preferential repairs in an error-prone or error-free manner, respectively . Altviews in . This coWhen we compared primary lymphocytes to the LCLs obtained from the same individual, no significant correlation in either NHEJ or SSA repair could be found . AlthougAn intriguing result from DSB repair measurements in non-induced lymphocytes of healthy individuals was that both NHEJ and SSA repair were inversely correlated to the age at time of blood draw of the individual . This explanation is unlikely, though, as the level of background luciferase expression was similar for all individuals in non-induced T cells for both BER and NER templates  either before or after aHCT. Identifying individuals that are most at risk before the transplant would be most relevant as the treatment could be adapted accordingly. We did not find any form of DRC that was significantly different in individuals that later developed t-MDS/AML (cases), as compared to matched individuals that did not (controls). There were some differences, though, that could be identified in pre-aHCT patients when compared to the group of healthy individuals. NHEJ was marginally higher in patients (cases and controls combined) than in healthy individuals after adjusting for age (p = 0.0149). Moreover, BER in control patients, but not in cases, was also lower than for healthy individuals (p = 0.0013). Maybe more meaningful, pre-aHCT patient samples did not show the correlation between NHEJ and SSA that was observed for every other group of samples analyzed, including the very same individuals at a time point post-aHCT . Moreove+ cells mobilized for aHCT in patients that later developed t-MDS/AML have been shown to present alterations in their DSB repair pathway as measured by microarray analysis [de novo mutations in those cells. As a result, the decrease in DSB repair after aHCT might reflect the necessity to replicate extensively to reconstitute the whole hematopoietic system from a limited number of stem cells, or it could be that the transplant itself could form a selection mechanism for cells with lower repair capacity, whether directly or indirectly, through the ability to mobilize stem cells and/or to recolonize the bone marrow after aHCT. Investigating DRC in stem cell donors and recipients post-HCT in the context of allogeneic stem cell transplantations might be able to isolate specifically the effect of the transplant itself on DSB repair from other potential changes associated with chemotherapeutic treatments that might be relevant to autologous transplant cases. Either way, the lower repair post-transplant seemed to be a stable feature among the samples investigated  when comparing the lymphocytes of the same individuals before and after transplant . The mecanalysis  and it ianalysis ,62. Patianalysis  and thisWe could not identify any difference in DRC existing prior to aHCT that could help predict which individuals are at higher risk of later developing t-MDS/AML. However, whether caused by the transplant itself and/or influenced by exposure to treatments, there is evidence that lymphocytes of patients post-transplant are greatly compromised in their ability to repair DSB. It remains to be determined whether the measured effect of transplantation on repair capacity is involved in making transplant patients, as a group, more susceptible for t-MDS/AML.S1 FileDNA repair capacities for each determination for different individuals using the assays described in the text.(XLSX)Click here for additional data file.S1 FigEach individual is represented by a code of 1 or 2 letter(s) (aHCT patients) or a combination H+number . Commas separate each individuals’ code name. The nature of the sample  used is indicated. All samples indicated have some data represented in the study but we did not obtain data for all of those samples and/or for all of the tests performed. In all cases, samples analyzed were taken prior to any t-MDS/AML diagnosis. For more details on the data used, including pairing of specific t-MDS/AML cases to specific controls, refer to the file presenting the raw data .(TIF)Click here for additional data file.S2 FigCGCAAATGGGCGGTAGGCGTG) using the LongAmp polymerase (New England Biolabs) on a BamHI-digested template. (A) Map of luciferase gene in pM1-Luc plasmid. (B) Cy5.5 signal after primer extension and 3.5% urea denaturating PAGE. The level of full length extension remaining on damaged templates (2.7kb) measures the proportion of plasmids undamaged in the luciferase coding sequence and can serve as quality control of damage level for each batch of plasmid generated for repair assays. The proportion of plasmid with blocking damage in the luciferase coding sequence can be inferred from the missing extensions as compared to the undamaged template control.Host cell reactivation assay plasmid pM1-Luc was treated with methylene blue + visible light (MB) or UVC (UV) to generate damage classically repaired by BER (8-oxoG) or NER (pyrimidine dimers), respectively. The damage frequency generated by the treatment in the transcribed strand of firefly luciferase is quantified using 5 cycles of primer extension from a Cy5.5-labeled CMV-F primer ((TIF)Click here for additional data file.S3 FigA) NHEJ or (B) SSA repair in lymphocytes analyzed unpurified (PBMCs in black) or after purification of the CD3+ cell subpopulation (T cells in gray) for 5 separate healthy individuals.((TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigA) BER and NER measure in the same 18 individuals  before and after aHCT (B) Repair post-aHCT normalized to pre a-HCT values for each individual. Mean value is indicated.((TIF)Click here for additional data file.S6 Fig95% confidence intervals and trend lines are indicated.(TIF)Click here for additional data file.


In this study, we assessed the possible mechanism of MSCexos action on SCI by investigating the tissue distribution and cellular targeting of DiR fluorescent labeled MSCexos at 3 hours and 24 hours after IV infusion in rats with SCI. The IV delivered MSCexos were detected in contused regions of the spinal cord, but not in the noninjured region of the spinal cord, and were also detected in the spleen, which was notably reduced in weight in the SCI rat, compared to control animals. DiR “hotspots” were specifically associated with CD206-expressing M2 macrophages in the spinal cord and this was confirmed by co-localization with anti-CD63 antibodies labeling a tetraspanin characteristically expressed on exosomes. Our findings that MSCexos specifically target M2-type macrophages at the site of SCI, support the idea that extracellular vesicles, released by MSCs, may mediate at least some of the therapeutic effects of IV MSC administration.In a previous report we showed that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) improved functional recovery after contusive spinal cord injury (SCI) in the non-immunosuppressed rat, although the MSCs themselves were not detected at the spinal cord injury (SCI) site . Rather, Previous studies have shown that intravenous delivery of bone marrow derived mesenchymal stem cells (MSCs) can promote functional recovery in rodent models of contusive spinal cord injury (SCI) , 3–6, asMSCs are multipotent cells capable of differentiating into cells of both neuronal and glial lineages –18, whicexos; see [exos have been shown to modulate immune function in vivo [in vitro, indicating that MSCexos might be capable of mediating many of the histological changes observed after intravenous MSC infusion in SCI animals. We therefore postulated that exosomes might be the secreted factors responsible for the therapeutic effects of IV infused MSCs on SCI recovery.In several experimental injury models, including stroke , myocardxos; see  for a re in vivo  as well  in vivo  and endo in vivo  in vitroexos could replicate some early therapeutic actions of IV delivered MSCs, but it was not clear whether MSCexos trafficked to the injury site or which cell types might be targeted.Exosomes are nanovesicles (30–150 nm) with bilaminar membranes. They are among a family of extracellular vesicles (EV) released by all cells, of all species, and are present in all biologic fluids examined –41. Exosexos, unlike IV infused MSCs, trafficked to contusive SCI sites, but not uninjured spinal cord. Furthermore, the IV infused DiR-labeled MSCexos, were specifically taken up by a subset of macrophages at the SCI site expressing the M2 marker CD206, but not by macrophages expressing the M1 marker iNOS. Importantly, the hotspots of DiR fluorescence within CD206+ M2-type macrophages strongly co-localized with staining for the characteristic exosome antigen CD63, supporting the identification of the fluorescence as clusters of exosomes, rather than nonspecific debris or dye accumulation. These findings argue that MSC derived exosomes may contribute to the therapeutic effects of IV administered MSCs on recovery from spinal cord injury by regulating the actions of macrophages within the lesion.In the current study, we report that IV infused DiR labeled MSCAll experiments were carried out in accordance with NIH guidelines for the care and use of laboratory animals, and the VA Connecticut Healthcare System Institutional Animal Care and Use Committee approved all animal protocols. Animals were sacrificed under sodium pentobarbital anesthesia (80mg/kg i.p.) prior to tissue harvesting.th or 5th passage (P4 or P5), MSC were washed 3 times in PBS and incubated with 10ug/ml DiR  (Molecular Probes) in DMEM for 10 minutes at 37°C in a culture incubator per manufacturer’s recommendations, or with RNA binding Syto RNASelect (Molecular Probes), or the fluorescent sphingolipid analogue BoDipy (Molecular Probes), for 30 minutes per manufacturer’s recommendations. Media containing the dye was aspirated and replaced with serum containing media for 3 hours to allow cells to recover before washing the adherent cultures in plain DMEM and transferring to serum free media for collection of exosome containing conditioned media.MSCs were isolated from the bone marrow of young adult Sprague-Dawley rats (150-200g) as previously described in , cultured in DMEM with 10% heat inactivated fetal calf serum (FCS), glutamate and penicillin/streptomycin and passaged when cells reached 70–80%. After the 46 P4 or P5 MSCs from 4 separate donors, incubating half of each cell suspension with 10 μg/ml DiR (Molecular Probes) for 10 minutes, and the other half in plain DMEM for the same length of time. Cells were washed once with DMEM and once with 10% fetal calf serum containing media, before resuspending in 2ml DMEM and counting cell samples with a hemocytometer. No reduction in the number of trypan blue excluding cells was detected in DiR treated samples from any of 4 separate tests (108 ± 4% of control (n = 4)). Cultures of MSCs were also examined before labeling and at the time of collection of conditioned media for exosome isolation. No reduction in densities of attached cells were noted in DiR labeled cultures compared to unlabeled cultures.Potential effects of DiR labeling on MSC viability were assessed by deplating 4 x 10Exosomes were isolated from 2-day serum free conditioned media from MSC cultures using the differential centrifugation methods described by Théry et al. (2006), and used previously by our lab . For one of the DiR labeled exosome transplant experiments, conditioned media was concentrated tenfold using Microsep 3K filter units prior to the final ultracentrifugation steps. Protein content of exosome fractions was assessed using a commercial Bradford protein assay kit (Thermoscientific) and Synergy HT plate reader (Biotek). Exosome numbers were assessed in two of the samples by nanoparticle tracking analysis (NTA) employing a NanoSight LM10  with data extrapolated to other samples based the Bradford protein assay. Morphology of exosome vesicles was verified for some samples by electron microscopy see . For EM Adult male Sprague-Dawley rats weighing 195-220g were deeply anesthetized with isoflurane (1–3%) inhalation anesthesia and subjected to contusion injury in the spinal cord using the Infinite Horizon (IH) impactor spinal cord injury device . Contusion injuries were induced by applying an impact of 2 Newton  at Th9 spinal cord level exposed by laminectomy as described previously [sexo (100 μg protein estimated to be 2.5 ×109 exosomes via NanoSight LM10in PBS) was infused (0.2 ml in PBS) into the saphenous veins of isoflurane anesthetized rats over the course of 1 minute. Control contused rats were infused with PBS only or PBS containing DiR.One week post-contusion, DiR labeled MSCexos (derived from approximately 50 x106 MSCs for each batch) were used for three separate infusion experiments. In each group, two animals were infused with DiR-MSCexos and one animal was infused with an equal volume of PBS. One DiR-MSCexos infused animal in each group was sacrificed at 3 hours and the other at 24 hours post infusion. The PBS infused control animals were sacrificed at 3 hours post- 24 post-infusion. A total of 6 contused animals were transplanted with DiR-MSCexos and 3 contused animals were infused with PBS. Two additional contused animals were infused with PBS containing DiR at the same concentration used for labeling MSCs.Three batches of DiR-MSCexos or control PBS infusion, rats were sacrificed under sodium pentobarbital anesthesia, perfused with saline, followed by 4% paraformaldehyde in Sorreson’s phosphate buffer. Spinal cords, spleens, livers, lungs and testes were removed and processed for standard 15 μm frozen sectioning. One centimeter longitudinal sections from both intact and lesioned areas of the spinal cord, as well as the spleen, liver, lung, kidneys, and testis were either mounted directly in DAPi imputing media or stained with primary antibodies directed against one or more of the following: Neurofilament , GFAP , RECA-1 , PDGFR-B , CD63 , OX-42 (1:100 BD Pharmingen 550299), CD206 (1:50 Santa Cruz Biotechnology Inc. sc-376108), iNOS (1:200 Abcam ab3523), CD4 , CD8 , and visualized with secondary goat anti-mouse, -rabbit, or -chicken IgG antibodies conjugated to Alexa Fluor 488, 546, 594, or 633 . Immunostained sections or unstained sections were counterstained with DAPi mounting media  and photographed with a Nikon A1R multiphoton confocal microscope with NIS Elements software.At 3 hours or 24 hours post DiR-MSCTo assess fluorescence of fixed red blood cells, blood was collected form one animal in a heparinized tube at the time of sacrifice, centrifuged at 6,000RPM for 30 seconds with a desk top microfuge, resuspended and washed in PBS before fixing with 4% paraformaldehyde in Sorreson’s phosphate buffer for 15 minutes and washed twice in PBS. One drop of resuspended cells was added to a glass slide and allowed to partially dry and adhere to the surface before mounting with DAPi mounting media , cover slipping.2 area) and quantifying the average fluorescence for each wavelength using NIS Elements software which ranked intensity on a scale from 0 to 4092. Lesioned areas were identified by mottled appearance in the 488, 595 wavelengths and altered distribution of nuclei as seen in the DAPI wavelength. Average autofluorescence in both the 488 nm and 595 nm wavelengths increased by an average of 159 and 215 units, respectively, in the contused region of the spinal cord, compared to the intact portion of the same spinal cord. By comparison, average autofluorescence in the 633 nm DiR wavelength was increased by only 54.For imaging and analysis of stained and/or transplanted tissues, thresholds for each wavelength were adjusted to subtract autofluorescence levels at the same wavelength in unstained tissue of the same type which was washed and mounted with DAPI mounting media, or processed with secondary antibodies only and mounted with DAPI mounting media. Effects of contusive injury on auto fluorescence of spinal cord tissue in the lesioned area at the 488, 595, and 633 nm wavelengths were assessed by mounting unstained sections of intact and contused spinal cord tissue from 2 PBS infused animals, photographing three fields in the center of each section at 20X , and 10 nonoverlapping fields at least 100 μm outside of the reactive astrocyte boundary within the same section as well as 10 nonoverlapping fields in sections of the spinal cord taken more than 1 cm rostral to the lesion. Three different sections were analyzed from two different contused and DiR-MSCTo evaluate relative sizes of DiR hotspots within contused spinal cord tissue at 3 hours and 24 hours post-infusion, compact areas of DiR fluorescence were measured at their longest diameter in 20X images of spinal cords harvested at 3 and 24 hours post-infusion using NIS Elements software. DiR hotspots were measured for all concentrated areas of DiR fluorescence, regardless of intensity or proximity to other hot spots, using the DiR channel only form the original image. A total of 260 and 524 hot spots respectively were measured for a total of 16 separate images of varying sizes from 3 and 24 hour post-contusion spinal cordsTo assess the gross effects of spinal cord injury on mobilization of circulating monocytes from the spleen, spleens from 1 week post SCI rats and age and sex matched controls were removed, washed with PBS, blotted to remove excess fluid and weighed.Spleen weights for control and spinal cord contused rats were compared using and one-way Analysis of variance (ANOVA) with a post-hoc test for multiple comparisons.All statistical comparisons were performed using Origin software , and one-way Analysis of variance (ANOVA) with a post-hoc test for multiple comparisons. Comparisons which are reported as statistically significant were significant at p<0.05 or lower.Electron microscopy confirmed the presence of high concentrations of 30–100 nm diameter vesicles, consistent with the morphology of exosomes within the isolated 100,000g pelleted exosome fraction from bone marrow mesenchymal stem cell (MSC) conditioned media . Exposinexos IV infused SCI rats revealed numerous hotpots of DiR wavelength fluorescence within lesion zones at 3 hours . Counts from ten sample fields of DiR hotspots within and outside of the contused areas found an average of only 0.66 ± 0.33 DiR hotspots outside of the lesion zones as compared to 44.33 ± 0 14.11 DiR hotspots within the lesion area (p<0.05 ANOVA). Counts were obtained from three 1 cm longitudinal sections from two separate contused rats.Spinal cord injury sites exhibited relatively high levels of autofluorescence in the 488 nm green  and 562  3 hours  and 24 h 3 hours . DiR hotNo DiR wavelength hotspots were detected in sections taken from spinal cord tissue greater than 1 cm rostral to the lesion site 0±0 (n = 3 sections) or in the lesioned area of an animal infused with DiR only without exosomes 0±0 (n = 3 sections). Unlike 488 nm (green) and 562 nm (red) wavelengths, fluorescence in the far red 640 nm wavelength of DiR was rarely observed in contused regions of the spinal cords or other tissues or organs from control PBS infused animals. When DiR wavelength hotspots were detected in control IV infused tissue, typically spleen  or lung exos IV infused rats. Large numbers of DiR fluorescence hotspots were detected within contused lesions at both 3 hours , with many cells at the 24 hr time point containing more than one hot spot.The pattern of DiR fluorescence distribution within spinal cord lesions suggested an increasing accumulation of DiR-MSC 3 hours  and 24 h 3 hours  post-infinfusion  than at infusion . In manyinfusion . Average+ macrophage rich areas strongly correlated with locations of DiR hot spots  were CD206+ and only 2 DiR hot spot containing cells (less than 1%) were iNOS+.Although only a fraction of CD206M2 cells . DiR fluB) failed to show convincing evidence of exosome uptake by cells other than macrophages. Although DiR hot spots were sometimes detected in close proximity to degenerating blood vessels , astrocytes (GFAP), endothelial cells (RECA-1) and pericytes  showed only small random specks of DiR wavelength fluorescence within the lesioned area  indicatiexos by cells in these organs.Diffuse DiR fluorescence was also observed in the livers (not shown) and kidneys  of animaexos infusion. Similar to the contused spinal cord, sizes of DiR hotspots in the spleen tended to be larger at 24 hours after DiR-MSCexos infusion , compared to 839 ± 23 mg (n = 8) for intact animals and 874 ± 30 mg (n = 14) for 10–12 weeks post-SCI animals .exos, unlike IV infused MSCs, traffic to the spinal cord injury site in non-immunosuppressed rats. Although the number of contused animals transplanted with MSCexos was relatively small , due to the need for large numbers of MSCs to produce a concentration of submicroscopic DiR-labeled MSCexos that could be reliably detected after cellular uptake in vivo, the localization or the DiR labeled MSCexos was extremely consistent. Specifically, DiR labeled MSCexos were taken up by M2 macrophages within the lesion site. These findings suggest that intravenously delivered exosomes derived from MSCs, target the spinal cord injury site and may contribute to the therapeutic effects of IV infused MSCs in spinal cord injury.The results of this study show that IV infused MSCexos used in this study were consistent with identification of the infused MSCexos samples and fluorescent “hotspots” within spinal cords as MSC derived exosomes. Sizes of individual vesicles in exosomal fractions observed with electron microscopy were consistent with size profiles of exosomes. Staining of exosomal fractions by prelabeling MSCs with Syto RNASelect, BODIPY, or DiR, and colocalization of DiR hotspots in tissue sections with CD63 staining also indicated that the presumptive exosomes contained the three characteristic components of exosomes: RNAs, sphingolipids, and tetraspanins, as well as nonspecific membrane lipids. The close correlation between DiR fluorescence hotspots and CD63 (tetraspanin) staining within macrophages is particularly significant as it argues that the fluorescence represented intact exosomes rather than residual dye from phagocytized particles. However, CD63 is also present on other internal vesicles [The morphology and staining of the MSCvesicles , 54 and exos to specific cells in the spinal cord demonstrates the feasibility of DiR labeling as a means of assessing the in vivo targets of systemically delivered exosomes, even in tissue with high endogenous autofluorescence, such as the macrophage rich areas associated with tissue injury. Endogenous fluorescence in the 640 nm wavelength for DiR was only observed in red blood cells, which could be readily identified by their distinct morphology and lack of nuclear staining. While others have demonstrated the feasibility of in vivo tracking of exosomes labeled with other membrane dyes such as PKH26 [Tracking of DiR labeled MSCas PKH26 , 55, 56 in vivo [B+ pericytes located within the central scar region of the lesion, but did not appear to be localized within these cells.The three-dimensional rotation of confocal images in this study allowed unambiguous localization of DiR labeled exosomes within M2 macrophages of contused spinal cords. Unlike earlier studies using two-dimensional imaging of labeled exosomes in vivo , 55–57),in vivo . In contexos were specifically localized to macrophages within the contused spinal cord, it is not clear whether the DiR-MSCexos were taken up by macrophages at the lesion site or whether macrophages migrated to lesion sites after taking up the exosomes. A circulating population of monocytes continuously cycles in and out of the splenic reservoir and migrates through blood vessels to sites of local inflammation [+ macrophages in the spinal cord exhibited DiR fluorescence, and many DiR hotspots were detected within the cells in the spleen, it is possible that DiR-MSCexos containing cells in both organs were derived from circulating monocytes, which took up exosomes in the blood stream and subsequently migrated into these organs. Alternatively, the cells may have taken up exosomes in the spleen and then migrated to the spinal cord.Although DiR-MSCammation , 59. Furammation , althougexos within the lesion site, rather than taking up exosomes only in the blood stream and retaining the same level of DiR-MSCexos accumulation after migrating into the contused spinal cord.The absence of DiR fluorescence in microglia outside of the lesion zone may indicate that exosome containing macrophages are not derived from resident microglia or that microglia/macrophages only take up exosomes after they become activated and/or migrate to the injury site. Although the blood spinal cord barrier (BSCB) becomes severely compromised immediately after SCI , the BSCexos uptake on macrophage function is uncertain. Exosomes produced by metastatic cancer cells have been shown to alter cell adhesion and promote neovascularization by endothelial cells [exos were only detected in CD206+ M2 macrophages, and not iNOS+ M1 macrophages, at both 3 hour and 24 hours post-infusion, it appears unlikely that uptake of MSCexos results in conversion of macrophages from a pro-inflammatory M1 to anti-inflammatory M2 activation state. Furthermore, preliminary counts did not show an increase in the ratio of M2/M1 macrophages at the injury site at this early time. However, it is possible that microRNAs or regulatory proteins within the exosomes increase the production of anti-inflammatory cytokines or block M2 macrophages from converting to an M1 pro-inflammatory activation state. Future studies assessing the ratio of M2/M1 macrophages at later time points will be important to assess the effects of exosomes on macrophage population subtypes over time.The effect of MSCal cells , 62, andal cells . But, inexos on spinal cord injury are due solely, or even primarily, to effects on macrophages. A very small number of DiR hotspots were also detected within astrocytic process endings (unpublished observation) and it is possible that exosomes were taken up by neurons or other cells as well, but at levels too low for us to detect. Hotspots of DiR fluorescence observed in this study almost certainly represent clusters of exosomes, since the sizes of individual exosomes (average 80 nm) are below the detection threshold for light microscopy. However, it is not known how many exosomes are required to produce a change in cell behavior and it is possible that cells may be influenced by DiR-MSCexos uptake even if the numbers of exosomes within the cell are too small to produce a detectable DiR hotspot. Previous studies have identified several cell types in the CNS as potential targets for MSCexos. Other investigators have reported that intraventricularly delivered PKH26 labeled MSCexos localized predominantly to astrocytes in diabetic mouse brains, but that some MSCexos were also associated with neurons and microglia [in vitro. These finding imply that MSCexos may have multiple cell targets in the CNS, and that the M2 macrophage population may simply be the most easily identifiable target in this injury model.It is also unclear whether the actions of DiR-MSCicroglia . Likewisicroglia . In addiicroglia  and tubeicroglia  in vitroexos to cells at the injury site supports the possibility of direct action of MSCexos at the injury site, it is also possible that MSCexos might influence SCI recovery by more indirect mechanisms. DiR hotspots were detected within the spleen over the same time course as their appearance at the contusion site, suggesting the possibility that MSCexos might influence recovery by modulating overall immune system function. Several studies have shown that spinal cord injury in the rodent results in distinct changes in the immune system within 1-week post-SCI [+ lymphocytes and an increase in CD4+ lymphocytes in the spleen and lymph nodes and an increased proliferative response to myelin basic protein [exos to the spleen in this study support the idea that systemic immune responses to injury and MSCexos infusion could play a role in SCI recovery. A recently published report [exos modulate immune cell function within the injured spinal cord, but similarly cannot rule out a systemic immune system response, rather than a locally targeted action.While the trafficking IV DiR-MSCpost-SCI , 65. The protein , as welld report  showing exos rapidly traffic to the injured, but not the uninjured spinal cord, and associate specifically with M2 macrophages. MSCexos were also detected in the spleen, which was notably reduced in weight in the SCI rat. Further studies will be needed to determine whether IV exosomes replicate the therapeutic actions of MSCs on SCI recovery as well as to elucidate the possible role of the spleen in SCI recovery.The results of this study indicate that IV delivered MSCS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Salmonella enterica serovar Enteritidis (SEn) in two independent cohorts consisting of age-matched individuals with and without CKD. Sera were evaluated for antigen-specific IgG titres and the functionality of antibody to SEn was assessed in a serum bactericidal assay. Surprisingly, patients with CKD and control subjects had comparable levels of IgG against TT and DT, suggesting preserved humoral memory responses to antigens encountered early in life. Lipopolysaccharide-specific IgG titres and serum bactericidal activity in patients with CKD were also not inferior to controls. CMV-specific IgG titres in seropositive CKD patients were similar or even increased compared to controls. Therefore, whilst responses to new vaccines in CKD are typically lower than expected, antibody responses to antigens commonly encountered prior to CKD onset are not. The immunodeficiency of CKD is likely characterised by failure to respond to new antigenic challenges and efforts to improve patient outcomes should be focussed here.Patients with chronic kidney disease (CKD) have an increased risk of infection and poorer responses to vaccination. This suggests that CKD patients have an impaired responsiveness to all antigens, even those first encountered before CKD onset. To examine this we evaluated antibody responses against two childhood vaccine antigens, tetanus (TT) and diphtheria toxoids (DT) and two common pathogens, cytomegalovirus (CMV) and  SignificReduction in eGFR or increase in albuminuria are both independently associated with all-cause mortality and progression to severe CKD–end stage renal disease (ESRD), which may require renal replacement therapy (RRT). , 4 ESRD Amongst the reasons for the observed increased susceptibility to infection is that public health measures to reduce infection, such as vaccination, are not as effective in CKD as in the general population. Studies have consistently shown that patients with ESRD exhibit a lower seroconversion rate, lower peak antibody titre and faster decline in protective antibody titres than healthy subjects. –12 RefleCollectively, these observations have led to a dogma that patients with CKD are immunodeficient and have a defective capacity to mount and maintain effective responses to antigens as compared to the general population. Host immune function has mainly been studied in the ESRD population and findings typically include lymphopenia and contraction of naïve T and B lymphocyte pools.  Lower nuSalmonella enterica serovar Enteritidis (SEn) and the viral pathogen cytomegalovirus (CMV), in two independent cohorts of patients with moderate/severe CKD not requiring RRT and age-matched controls. These antigens were chosen as epidemiological evidence suggests that they are all typically encountered by early adulthood. [The effect of less severe CKD on already established immunity to previously encountered antigens is not well explored. Our understanding in this area could be enhanced by assessing the level and functional activity of antibodies against commonly encountered pathogens and vaccines. In this study we have assessed levels of IgG against two vaccine antigens (tetanus toxoid (TT) and diphtheria toxoid (DT)), the bacterium ulthood. –23 Unexp2 (with majority of patients classified as CKD stage G4) and controls 81.5ml/min/1.73m2. The most common identified primary renal diagnoses were ischaemic/hypertensive nephropathy in 23%, polycystic kidney disease in 21%, diabetic nephropathy in 19%. Patients with CKD had a minimum of 12 months’ specialist nephrology follow-up at time of study entry. Just over one third of cohort 1 CKD patients had a diagnosis of diabetes mellitus (DM)– 35%, compared with none of the control subjects.43 patients with CKD and 39 age-matched controls were initially investigated (hereafter referred to as cohort 1) and their demographics and laboratory data are presented in In order to confirm our findings, we then evaluated a second independent cohort of 25 patients with CKD and 20 age-matched healthy controls (hereafter referred to as cohort 2) and their demographics and laboratory data are presented in Individuals from cohort 2 had similar levels of kidney function (eGFR) as cohort 1, but both the control and CKD arms were significantly older in cohort 2 (Mann Whitney 2-tailed p<0.01). Cohort 2 patients with CKD had a median of 2.5 years follow-up by specialist nephrology services (range 0.5 to 16 years) and the most common identified primary renal diagnosis was hypertensive/ischaemic nephropathy (40%), although a large number of individuals (40%) had uncertain CKD aetiology in the context of multiple comorbidities including diabetes, hypertension and cardiovascular disease. There was a significantly higher proportion of individuals with diabetes in cohort 2 CKD patients than in cohort 1 . None of the individuals in cohorts 1 or 2 had any known active malignancy, autoimmune/inflammatory disease  and were not receiving immunosuppressive therapies at the time of entry to the study. Although there was no known active chronic inflammatory disease in CKD patients from either cohort, these patients had significantly higher markers of inflammation than controls, including total white cell count (WCC), neutrophil count and serum high sensitivity C-reactive protein (hsCRP).Humoral responses to historical antigens in patients with CKD and healthy controls were evaluated by measuring serum anti-TT and anti-DT IgG using a Luminex assay.  None of No significant differences were observed in anti-TT or anti-DT IgG titres between healthy controls and patients with CKD in cohort 1 . Here, 3To confirm non-inferiority of long-term vaccine-specific antibody responses in patients with CKD, we then evaluated anti-TT and DT IgG levels in patients with CKD and age-matched controls from cohort 2 for whom the vaccination history was known (n = 22 and 19 respectively). There was no significant difference in the numbers of individuals with protective titres of anti-TT and anti-DT IgG between patients with CKD and controls (13 (59%) versus 15 (79%) and 2 (9%) versus 0, respectively) or their absolute anti-TT and anti-DT IgG titres . This waSera from study subjects were evaluated for CMV-specific IgG by an enzyme-linked immunosorbent assay (ELISA). This in-house assay was altered between testing sera from cohort 1 and those from cohort 2, therefore the absolute CMV-specific IgG titres are not directly comparable.The proportion of individuals seropositive for CMV (defined as a result greater than 10 arbitrary units) was not significantly different between CKD and control individuals in either study cohort (cohort 1: 29 patients with CKD (67%) and 22 controls (56%); 2-tailed Fisher’s exact p = 0.37; cohort 2: 21 patients with CKD (84%) and 13 controls (65%), Fisher’s exact 2-tailed p = 0.18). There were no significant differences in rates of CMV seropositivity in either patients with CKD or controls when the two study cohorts were compared (cohort 1 versus cohort 2).When cohort 1 CMV seropositive individuals were evaluated separately, patients with CKD were found to have significantly elevated IgG titres compared to controls , indepenThere were no significant correlations between CMV IgG titre and eGFR or ACR in seropositive patients with CKD from either cohort 1 (Spearman’s rank 2-tailed p = 0.36 and 0.10 respectively) or cohort 2 (p = 0.76 and 0.26 respectively). These findings suggest that renal impairment is not associated with a lower anti-CMV IgG response.Sera from all subjects were tested for anti-SEn LPS IgG by ELISA. Relative anti-LPS IgG titres were not significantly different between patients with CKD and healthy controls in either cohort 1 or 2 .10 kill of SEn at 180 minutes  were selected for testing in a serum bactericidal assay (SBA). This assay requires both functional antibody and complement for bacterial killing.  All sera minutes , which e minutes . All CKDThis study evaluates serological responses to several historical and persistent antigens in individuals with CKD not requiring RRT. Whilst many reports have evaluated humoral responses to newly encountered antigens, such as vaccines, the effect of CKD on already established humoral memory to previously encountered antigens is not well understood.After vaccination against hepatitis B, seasonal influenza and pneumococcus, patients with severe CKD consistently show reduced peak antibody titres and poor response longevity when compared to healthy controls. , 12, 28 Previous studies evaluating long-term B cell depletion therapy with Rituximab, an anti-CD20 monoclonal antibody, have shown no significant effects on vaccine-specific IgG titres, but a marked reduction in auto-immune antibody titres. , 31 ThusOur findings of comparable anti-TT and DT IgG titres in 2 independent cohorts of patients with CKD and their age-matched controls is consistent with renal impairment not adversely affecting well-established, long-lived plasma cell populations for antigens first encountered prior to the onset of CKD. This suggests that it is primarily the generation of immune responses to new antigens that is impaired. In support of this idea are the findings of a previous study involving subjects with ESRD that showed faster waning of anti-hepatitis B IgG following vaccination after ESRD was established as compared to historic natural infection .In addition to evaluating responses to protein vaccine antigens, we also examined responses to two pathogens, CMV and SEn. CMV, a betaherpesvirus, establishes life-long persistent infection in humans after primary infection, which often occurs in childhood, and reactivates periodically in response to stressors such as inflammation, infection or immunosuppression. , 33, 34 Salmonella serovars by the time of adulthood [Salmonella LPS antibodies provides a valuable measure of the immune response to a bacterial pathogen that is probably encountered repeatedly throughout life. [in vitro, [Salmonella and depends upon the presence of both intact complement and specific antibodies, which together combine to generate membrane attack complexes resulting in bacterial lysis. Therefore, we can conclude that patients with CKD are able to produce functional pathogen-specific IgG and have intact classical complement activation.The immunodominant antigen targeted by IgG after natural infection with SEn is the O-antigen of LPS. Virtually all adults in developed countries develop IgG antibodies to LPS from multiple dulthood  and in rdulthood  As such,ut life.  Unlike sn vitro, , 47 all Our study has several strengths including examination of humoral responses in two independent cohorts of age-matched individuals and the exclusion of dialysis therapy as a confounding factor, which is known to independently modulate immune function. , 49 LimiIn summary, our results indicate that the secondary immunodeficiency seen in CKD exhibits a relatively complex phenotype and does not simply reflect global immune failure. Indeed, humoral immunity to historical and persistent antigens is well maintained and may suggest that the cellular immune response to these antigens is also preserved. As such these findings suggest that future efforts to improve immune function in patients with CKD should focus on seeking to improve the adaptive immune response to new antigenic challenges, such as those observed during seasonal influenza infection or administration of novel vaccines. These efforts will be supported as a more complete understanding of the full phenotype and cause(s) of the secondary immunodeficiency seen in less severe stages of CKD is developed.A random sample of 60 subjects was selected from all study patients that had completed 18 month follow-up for the Renal Impairment In Secondary Care (RIISC) study  in AugusAll patients with a history of malignancy (except non-melanoma skin cancer) were then excluded from the sample, as was an individual with incomplete baseline clinical data. Clinical and laboratory data , together with baseline serum samples were sourced for the final sample of 43 RIISC patients. Blood samples from RIISC were collected as per previously published protocols. Clinical and laboratory data, together with serum samples from healthy control subjects recruited to a study investigating physical fitness in subjects with ANCA-associated vasculitis  were useA separate cohort of older patients (over 65 years) with CKD and age-matched controls was used to verify serological trends seen in cohort 1—baseline serum samples were tested from individuals taking part in the SONIC study  between 2015 and 2016 –a prospective observational cohort study utilising clinically recommended seasonal vaccinations to evaluate host immune function.Use of data and samples for this study falls under Ethical approvals granted to the above-named studies  and written informed consent was obtained from all patients in accordance with the Declaration of Helsinki.This assay was performed as previously described  using a Serum anti-CMV IgG titres were determined using a semi-quantitative in-house ELISA as previously described.  Briefly,An in-house ELISA was used to semi-quantitatively determine the presence of anti-LPS IgG in serum, relative to a single healthy donor positive control used as an internal standard. Briefly, a 96-well Nunc Maxisorp plate was coated overnight at 4°C with 5μg/ml LPS from SEn  in a carbonate/bicarbonate coating buffer . The plate was washed 3 times with PBS and blocked with PBS/1% bovine serum albumin (BSA) for 1 hour at RT, agitated on a plate rocker. After a further 3 washes with PBS, test serum added in single replicates of six 4-fold dilutions– 1:20, 1:80, 1:320, 1:1280, 1:5120, 1:20480, diluted in PBS/1%BSA/0.05% Tween 20. Each plate included a negative control (dilution buffer) and a positive biological control . The plate was incubated at RT for 1 hour on a plate shaker and then washed four times with PBS/0.05% Tween 20. Alkaline phosphatase conjugated secondary antibody  was added at 1:6000 and the plate incubated for a further 1 hour at RT on a plate shaker. P-nitrophenylphosphate  was then added and the plate incubated for 1 hour on a plate shaker at RT. The reaction was stopped using 3M sodium hydroxide and the plate read at an absorbance of 405nm.LPS titre from this assay is reported relative to the positive control serum and drawn from a single experiment. Sera from individuals with high/low relative LPS titres from this assay were run in 2 independent experiments to verify the trend. The intra- and inter-assay coefficients of variation for this assay were 2.11% and 8.11% respectively.7 colony forming units/ml and 5μl was added to 45μl neat sample serum in a 96 well plate, which was incubated on a plate rocker at 37°C, 5% CO2. Aliquots of 10μl were removed from each sample well at 45, 90 and 180 minutes and diluted in PBS into four serial 10-fold dilutions. Each dilution was pipetted in triplicate onto sterile Lysogeny agar plates, allowed to dry and incubated overnight at 37°C, 5% CO2. Colonies were counted the following day and the starting concentration of bacteria was determined by Miles & Misra serial dilution.This was performed as previously described  using SEAn increase in bacterial count from baseline denoted bacterial growth, whereas a decrease denoted bacterial killing. Each SBA contained one positive  and one negative control .  Normal sStatistical analysis of data was performed using Prism 5  and SPSS 22 . For continuous data, Normality has not been assumed and results are presented as medians with inter-quartile range (IQR) unless otherwise stated. The non-parametric Mann-Whitney test has been used to compare continuous numerical data and Fisher’s exact test to compare categorical data. Correlation analysis was performed using the non-parametric Spearman’s rank test. Multivariate linear regression was used to model predictors of CMV IgG titres in seropositive individuals and a repeated measures ANOVA was used to evaluate bacterial killing in the SBA.