NEBs produce the bioactive amine, serotonin (5-HT), and a variety of peptides with multiple effects on lung physiology and other organ systems. NEBs in mammals appear prominent and numerous during fetal and neonatal periods, and decline in the post-natal period suggesting an important role during perinatal adaptation. The naked mole-rat (NMR), Heterocephalus glaber, has adapted to the extreme environmental conditions of living in subterranean burrows in large colonies (up to 300 colony mates). The crowded, unventilated burrows are environments of severe hypoxia and hypercapnia. However, NMRs adjust readily to above ground conditions. The chemosensory NEBs of this species were characterized and compared to those of the conventional Wistar rat (WR) to identify similarities and differences that could explain the NMR’s adaptability to environments. A multilabel immunohistochemical analysis combined with confocal microscopy revealed that the expression patterns of amine, peptide, neuroendocrine, innervation markers and chemosensor component proteins in NEBs of NMR were similar to that of WR. However, we found the following differences: 1) NEBs in both neonatal and adult NMR lungs were significantly larger and more numerous as compared to WR; 2) NEBs in NMR had a more variable compact cell organization and exhibited significant differences in the expression of adhesion proteins; 3) NMR NEBs showed a significantly greater ratio of 5-HT positive cells with an abundance of 5-HT; 4) NEBs in NMR expressed the proliferating cell nuclear antigen (PCNA) and the neurogenic gene (MASH1) indicating active proliferation and a state of persistent differentiation. Taken together our findings suggest that NEBs in lungs of NMR are in a hyperactive, functional and developmental state, reminiscent of a persistent fetal state that extends postnatally.The pulmonary neuroepithelial bodies (NEBs) constitute polymodal airway chemosensors for monitoring and signaling ambient gas concentrations (pO Heterocephalus glaber; NMR) has been gaining increasing attention by biologists since it has been recognized to possess several remarkable and often unusual properties compared to other rodents and humans The subterranean dwelling naked mole-rat  Finally, NEB cell immunophenotyping suggests that in the NMR NEBs, these cells exist in a fetal-like transitional developmental state which may underlie their adaptive plasticity. These observations highlight the value of NMRs as a potential model for the study of human relevant chemosensory functions.Heterocephalus glaber) at postnatal day 5–3 months were embedded in polyethylene glycol (OCT medium) . Wistar rats (n = 4), used as a conventional animal control, at postnatal day 3–8 months, were obtained from Charles River  and housed in the Hospital for Sick Children lab animal facilities. All animal procedures were in accordance with Canadian Council of Animal Care guidelines and were approved by the Animal Care and Use Committee of the Hospital for Sick Children. Adult rats were sacrificed by lethal injection of euthanyl and neonates by cervical dislocation. Dissected lungs were washed three times in CO2-independent medium and embedded in OCT and then snap frozen on dry ice. All tissue blocks were sealed and stored at −80°C until use.Lung tissues from naked mole-rats (n = 8)  was performed on sections permeabilized with 1% Triton X-100 in PBS for 10 min and then blocked in 20% normal donkey serum in 4% BSA plus avidin/biotin blocking solution for 60 min at RT. Following several brief washes slices were incubated with selected pairs of primary antibodies (e.g. mouse anti-SV2 mAb mixed with rabbit anti-pannexin1 pAb or rabbit anti-synaptophysin mAb mixed with mouse anti-MASH1 mAb) observing different host species (rd Gen IHC Detection Kit (Invitrogen).Cryosections of the medial segment of the middle lobe from NMR and Wistar rat (WR) lungs were cut at 60–80 µm under low working temperature (−12 to −15°C). The sections were immediately transferred to a dish with zinc formalin fixative  at RT. After three changes of fresh fixative (10 min each at RT), the sections were washed in PBS and stored at 4°C for 2 weeks maximally. For immunoflourescence labeling studies a variety of primary and secondary antibodies were used with the type, source and dilutions listed in  species , at diluFluorescent immunolabeling images of PNEC/NEBs, airway nerves, and smooth muscle in the double-stained whole mount slices were obtained with a Leica confocal laser scanning microscope (model TCS-SPE) and LAS-AF software. The variable excitation wavelengths of the krypton/argon laser were 488 nm for FITC, 568 nm for Texas Red and 695 nm for RedDot 2 (nuclear staining).3 of lung tissue based on calculated volume of three 10 µm frozen sections. To determine the ratio (%) of serotonin positive cells among cells staining for pan-neural markers SV2/SYP marking NEBs; 5-HT positive cells and SV2/SYP stained cells were manually counted in all 45–50 µm thick sections dual immunolabeled confocal images. The individual ratios of 5-HT positive cell numbers to total SV2/SYP positive cells from two size NEB groups (>40 µm and <40 µm) were calculated For quantification of NEBs in NMR lungs we used a method similar to that for mouse lung as previously reported One-way analysis of variance (ANOVA) with repeated measures was used for statistical analysis of NMR lungs and rat lungs with respect to the different stages in development. One-way ANOVA tests with repeated measures were also used for comparison of NEB numbers and integrated density of immunostaining in NMR lung and rat lung. All data are expressed as means (+/−) standard error of the mean (SEM).Neuroendocrine markers were used to identify PNEC/NEBs in NMR airways, and the antibodies were used to delineate structural similarities and differences are listed in 2 airway, were significantly more numerous by ∼2 fold over that in WR  . Anti-Cx2 sensing NADPH oxidase chemosensory complex at the apices of NEB cells 2 sensing complex and the degree of expression. We found a very uniform and robust expression of gp91phox and p22phox by NMR NEB cells , and found CAII expression in NMR NEB cells occurred to a lesser degree than that in the surrounding epithelium  and that expression of this transcription factor diminishes and disappears as NEBs mature It was not possible at this time to determine if the cells not staining for PCNA were also negative for MASH1 suggesting that these could be functionally terminal. Taken together with the other observations it would appear that the NMR NEB cells, being in a postnatal advanced age, occupy a developmental state seen in the neonatal and prenatal period in other species.This first exploration of the pulmonary neuroendocrine cell system in the NMR has highlighted several unique differences between the NMR and other rodents and interestingly has indicated that epitopes recognized by the different antibodies more closely align with those in humans. Here we show that antibodies that do not readily work in other rodents easily worked with NMR lung tissues. We found that the NMR NEB cells were more variably compact in their organization compared to WR and other species, and probably communicate via the gap junction protein Cx43. On the other hand, NCAM, a homophilic binding protein, and ZO-1, the tight junction protein, appeared to be expressed minimally, and perhaps only on interspersed non-NEB cells. Barring possible alterations in NMR of unique detectable epitopes, this finding supports the idea that the neuroendocrine cells within NMR NEBs maintain a weaker cell-cell association which might permit greater ability to reorganize functionally. This then could be surmised as potential developmental plasticity leading to adaptability.The robust expression of SV2, CGRP, synaptophysin and 5-HT essentially maximized on the expression levels for these markers as seen in NEBs in general, but were equally abundant in the NMR NEB cells. By comparison, whereas mouse only expresses 5-HT at a low to moderate level, human and rabbits have relatively high levels of 5-HT 2 chemosensing proteins, gp91phox and p22phox, at appreciably high levels, suggests that O2 chemosensing in these cells is equivalent to other species and thus supports the idea of a highly conserved mechanism 2 chemosensing is equivalent cannot be determined, although we do show the presence of one key cytoplasmic CA, CAII. Since we have evidence that NEBs in other species are responsive to hypercapnia by releasing 5-HT, it will be interesting to determine if NMR NEB cells have similar sensing characteristics, or given their natural microenvironment, an alternate threshold for CO2 sensing. It is possible that NMR NEBs could be more insensitive to hypercapnia.The finding that NMR NEB cells are competent in expression of the expected OWe also studied the expression of the hypoxia sensitive transcriptional machinery that involves the hypoxia inducible factors HIF1α and HIF2α The neurogenic transcription factor ASH-1  is obligatory for PNEC/NEB development but then diminishes during postnatal and then into adult life 2 sensitive interacting protein that leads to degradation of HIF1α 2 sensor complex in NMR NEBs , all localized at the apical plasma membrane. We therefore surmise that NEB cells in the NMR are robustly functionally attuned to the hypoxic/hypercapnic state of their natural environments. In tune with this heightened sensory state we found NMR NEBs to be innervated by the multiple efferent and afferent fibers, cholinergic, purinergic and nitrergic, as seen in rat NEBs 2×2 cells were noted in the NEBs supporting the idea of functional sub-specialization. Along this line the finding that only some cells within a NMR NEB turn on HIFs also supports this idea. Thus it would appear that NMR NEBs constitute a mix of specialized cells serving individual specific sensor functions. This then begs the question whether these different NEB cells within the complex subserve specific functions and, if they then communicate via Cx43 positive gap junctions to permit functional coordination.The observation of increased size and number of NMR NEBs relative to WR also corresponds to the hyperplasia seen in humans and other animal species whether due to high altitude, lung disease with build up of hypoxia, or in a mouse model deficient in PHD-1, the OA recent study suggests that PNEC cells are functionally receptive to odorants 2 chemosensors or as pain sensors Although there is still debate on the physiological functional significance of NEBs, whether as O


DiGeorge syndrome (DGS) presents with a wide spectrum of thymic pathologies. Nationwide neonatal screening programs of lymphocyte production using T-cell recombination excision circles (TREC) have repeatedly identified patients with DGS. We tested what proportion of DGS patients could be identified at birth by combined TREC and kappa-deleting element recombination circle (KREC) screening. Furthermore, we followed TREC/KREC levels in peripheral blood (PB) to monitor postnatal changes in lymphocyte production.TREC/KREC copies were assessed by quantitative PCR (qPCR) and were related to the albumin control gene in dry blood spots (DBSs) from control (n = 56), severe immunodeficiency syndrome  and DGS (n = 13) newborns. PB was evaluated in DGS children (n = 32), in diagnostic samples from SCID babies (n = 5) and in 91 controls.All but one DGS patient had TREC levels in the normal range at birth, albeit quantitative TREC values were significantly lower in the DGS cohort. One patient had slightly reduced KREC at birth. Postnatal DGS samples revealed reduced TREC numbers in 5 of 32 (16%) patients, whereas KREC copy numbers were similar to controls. Both TREC and KREC levels showed a more pronounced decrease with age in DGS patients than in controls (p<0.0001 for both in a linear model). DGS patients had higher percentages of NK cells at the expense of T cells (p<0.0001). The patients with reduced TREC levels had repeated infections in infancy and developed allergy and/or autoimmunity, but they were not strikingly different from other patients. In 12 DGS patients with paired DBS and blood samples, the TREC/KREC levels were mostly stable or increased and showed similar kinetics in respective patients.The combined TREC/KREC approach with correction via control gene identified 1 of 13 (8%) of DiGeorge syndrome patients at birth in our cohort. The majority of patients had TREC/KREC levels in the normal range. The embryopathy is most commonly caused by a deletion on the 22nd chromosome, called del22q11, but can be caused by other mutations as well DiGeorge syndrome is a complex disorder caused by an embryopathy; it presents with a number of clinical symptoms arising from the disturbed development of the 3The degree of thymic pathology correlates with various degrees of T-lymphocytic dysfunction Thirty-two children (age 1.5–18 years) with DiGeorge syndrome with verified deletion of 22q11 and macroscopically missing/dysplastic/hypoplastic thymus were investigated. Del22q11.2 was verified either via FISH (fluorescent in-situ hybridisation) using the DiGeorge/VCFS TUPLE 1/22q Deletion Syndrome LPU004 probe  or by using Salsa MLPA (Multiplex Ligation Probe Amplification) kits P-245-B1 or P-250-2B (MRC Holland). Twenty-eight of the 32 DGS patients underwent cardiac surgery. In 10 patients the thymus was reported as missing in the surgery protocol; in 4 patients the thymus was found to be hypoplastic and was subtotally resected; and in 3 patients only subtotal thymus resection was reported. In 10 patients, there was no information about thymic appearance; however, most of the remaining tissue was presumably also resected during surgery. Four patients had no surgery and were investigated and genetically verified based on their clinical symptoms. As controls, residual blood samples after routine clinical evaluations due to medical conditions that do not affect the lymphocyte count were used, or samples from healthy siblings of patients investigated at our department were used providing that they signed a written informed consent for the use of residual material . Samples from five consecutively diagnosed patients with SCID in 2011–2013 in the Czech Republic (age of 2–4 months) were also evaluated. Archived neonatal DBSs were examined for 13 patients with DiGeorge syndrome and 10 patients with SCID. All patients or their parents provided written informed consent for the use of residual PB or DBSs in accordance with the Declaration of Helsinki. Fifty-six control DBSs were randomly chosen as controls, with an attempt to cover a DBS storage period similar to those of the patients. The birth weight of control newborns was recorded and subsequently the control DBSs were anonymised. The study was approved by the Ethics Committee of 2nd Medical School in Prague, Czech Republic.DNA was isolated from 200 µl of peripheral blood using QIAamp DNA Blood Mini Kit . From each DBS, a circle with a diameter of 3.2 µm was cut and DNA was eluted at 99°C for 1 hour in a shaker (500 rpm) using 100 µl of Generation DNA Elution Solution  supplemented with 100 µg/ml of yeast tRNA .The albumin gene level was quantitatively detected in isolated samples using qPCR www.r-project.org).The distribution of variables between groups was assessed using the chi-squared or Fisher's exact test. The Mann-Whitney test was used to estimate the significance of the differences in continuous values. These statistical analyses were performed using StatView version 5.0 . For evaluating TREC (KREC) dependence on age in patients and controls, a linear model was developed using R-package 3.1.1. . First, we established ranges for normal and abnormal TREC and KREC levels. Most current approaches use either TREC alone or combined TREC/KREC detection without correction for DNA quality. Therefore, the numerical TREC/KREC values are not directly comparable between patients. We adopted an approach presented by Kamae et al Of the patients with DiGeorge syndrome, only one had completely negative TREC levels with normal KREC levels. This patient with complete athymia and the clinical picture of CHARGE syndrome received two DLIs from an unrelated donor at the age of six months The monitoring of TREC/KREC postnatally is used in research studies, but it is not routinely used in clinical examination of patients with immunodeficiency. We collected TREC/KREC values in a cohort of 91 control PB samples from children aged 0–18 years. Although there is a published trend of both TREC and KREC decrease postnatally In the group of 32 children with DiGeorge syndrome aged 1.5 to 18 years, the TREC values were under the normal range in 5 of 32 (16%) patients . Even afThe four patients with TREC values under the normal range had repeated respiratory infections in infancy. All of them later developed allergies and/or autoimmunity in a form of autoimmune thyroiditis or thrombocytopenia, autoimmune conditions typically associated with DiGeorge syndrome. Their status, however, is not markedly different from that of other patients with diGeorge syndrome.In 12 patients, it was possible to monitor TREC/KREC development from birth to the present. Neonatal screening for SCID is not yet performed in the Czech Republic. During the period of this study, we investigated five patients with suspected SCID (age 2–4 months) based on clinical findings and flow cytometry results. The TREC/KREC analysis confirmed the SCID diagnosis in those patients  within tIn the retrospective part of our study we measured TRECs in newborn dry blood spots from patients with clinically diagnosed and genetically verified DiGeorge syndrome. However, we were only successful in using this method to detect DiGeorge syndrome in a patient with complete athymia and the clinical picture of CHARGE syndrome Decreased T-lymphocyte counts during the critical period of the first few years of age in DiGeorge syndrome have been demonstrated in previously published studies Our method uses correction for albumin gene levels for all the patients in the first-line test, which is different from most nationwide screening programs. This approach is more expensive (estimated costs ∼12 USD per sample) than the “TREC-only” approach with estimated cost of 4.22 USD per sample. Recently, a newborn SCID screening study was performed by a British group using the commercial TREC EnLite kit (Perkin Elmer), and it successfully detected all SCID cases in DBS KREC levels were normal in most patients at birth, which is consistent with a previous report One of the major achievements of our study was establishing a robust platform for TREC/KREC testing not only in newborn screening but also for rapid postnatal differential diagnostics of severe immunodeficiency. One has to keep in mind that normalised TREC/KREC numbers in postnatal blood can be skewed in cases with clonal expansion of mature lymphocytes, e.g., in response to infections. Indeed, the TREC/KREC levels did not correlate well with relative T (B) lymphocyte counts . We alreIn our study, TREC levels only identified one severe case with deep lymphopaenia, which corresponds to 8% of the limited DiGeorge syndrome cohort. Most children with incomplete DiGeorge syndrome had TREC/KREC levels in the normal range both at birth and postnatally. Nevertheless, the decline of both TREC and KREC levels with age was more significant in DGS patients than in controls. Thus, the assay complements routine investigations and follow-up of DiGeorge patients and might reflect age-related immune changes based on the del22q11 background.Figure S1TREC and KREC numbers expressed as copies per one µl of dry blood without control gene correction for controls, patients with DiGeorge syndrome and patients with severe combined immunodeficiency (SCID).(TIF)Click here for additional data file.Figure S2Changes in TREC (A) and KREC (B) values between birth and the present in children with DiGeorge syndrome (dotted lines). The levels in control children are depicted by empty circles. Grey areas represent the abnormal TREC/KREC range.(TIF)Click here for additional data file.Figure S3The correlation of TREC levels with relative numbers of T lymphocytes (A) and correlation of KREC levels with relative numbers of B lymphocytes (B) for DiGeorge syndrome patients (full diamonds) and controls (empty diamonds).(TIF)Click here for additional data file.


Despite single gene and whole genome duplications on the largest evolutionary timescales, and the fact that most gene duplications eventually result in loss of one copy, it is surprising that all jawed vertebrates (gnathostomes) have retained two paralogous VKOR genes. Both VKOR paralogs function as entry points for nutritionally acquired and recycled K vitamers in the vitamin K cycle. Here we present phylogenetic evidence that the human paralogs likely arose earlier than gnathostomes, possibly in the ancestor of crown chordates. We ask why gnathostomes have maintained these paralogs throughout evolution and present a current summary of what we know. In particular, we look to published studies about tissue- and developmental stage-specific expression, enzymatic function, phylogeny, biological roles and associated pathways that together suggest subfunctionalization as a major influence in evolutionary fixation of both paralogs. Additionally, we investigate on what evolutionary timescale the paralogs arose and under what circumstances in order to gain insight into the biological  VKORC1 and VKORC1L1  to vitamin K hydroquinone (KH2) . In. Inet alpe mouse . Expresset al. (2015) have assessed Vkorc1 and Vkorc1l1 expression by qPCR analysis during larval development and in adult tissues . T. TVKORC1e Note 4 ,82. Furtunctions .VKORC1 genes mostly cause in vivo warfarin resistance phenotypes [VKORC1 mutations have been comprehensively reviewed elsewhere [VKORC1L1 coding region mutations, we surveyed the NCBI dbVAR database, which includes combined whole genome sequence data for thousands of individuals, and found evidence for 21 total coding region SNPs in living adult humans of which seven are non-synonymous, one premature termination that shortens the C-terminus, and six synonymous variants that do not alter translated VKORC1L1 protein sequence. To date, no human in vivo phenotypes have been reported for VKORC1L1 non-wild type variants.Naturally occurring coding region mutations for human, rat and mouse enotypes ,74,102, lsewhere . To gainet al. (2004) first noted that VKORC1L1 primary sequences are considerably more conserved among mammalian orthologs than the respective VKORC1 sequences [Here we summarize various conclusions drawn from the studies reviewed in this article and propose a novel hypothesis to explain why two VKOR paralogs with apparently identical enzymatic functions have persisted in vertebrates over ~400 million years of evolution. Rost et al. 004 firstequences . PhylogeAddressing this theme, Robertson (2004) commented, “…If this duplication follows the neofunctionalization model of gene duplication Ohno, 1970) , then th70 , thende novo reduction of K vitamins acquired from dietary sources, in addition to recycling oxidized forms of K vitamins to the respective reduced hydroquinone forms in the vitamin K cycle, evolutionary selection pressure has apparently maintained unique physiological functions for both paralogs by a tissue-specific “division of labor” under independent expression and regulatory controls.In this review, we have attempted to comprehensively summarize published results concerning structural and functional similarities and differences for VKORC1 and VKORC1L1 paralogs in extant metazoan genomes and to relate these to a rationale that explains why these proteins are evolutionarily maintained when their enzymatic functions are virtually identical. While it is presently clear that both enzymes are responsible for In order to address important questions that remain about these paralogs, it will be necessary to more deeply investigate regulation of their expression with respect to cell and tissue type and developmental stage, to identify their functional intracellular protein partners, and to comprehensively identify and characterize new VKD proteins and the extent of the VKD proteome in individual species. We hope this review will stimulate discussion and cooperative investigation among researchers already engaged in vitamin K-related research areas as well as encourage researchers new to the field with expertise in complementary research methods.


Burkholderia pseudomallei (Bp) causes melioidosis, with septic patients attaining mortality rates ≥40%. Due to its high infectivity through inhalation and limited effective therapies, Bp is considered a potential bioweapon. Thus, there is great interest in identifying immune effectors that effectively kill Bp. Our goal is to compare the relative abilities of murine macrophages and neutrophils to clear Bp, as well as determine the importance of serum opsonins and bacterial capsule. Our findings indicate that murine macrophages and neutrophils are inherently unable to clear either unopsonized Bp or the relatively-avirulent acapsular bacterium B. thailandensis (Bt). Opsonization of Bp and Bt with complement or pathogen-specific antibodies increases macrophage-uptake, but does not promote clearance, although antibody-binding enhances complement deposition. In contrast, complement opsonization of Bp and Bt causes enhanced uptake and killing by neutrophils, which is linked with rapid ROS induction against bacteria exhibiting a threshold level of complement deposition. Addition of bacteria-specific antibodies enhances complement deposition, but antibody-binding alone cannot elicit neutrophil clearance. Bp capsule provides some resistance to complement deposition, but is not anti-phagocytic or protective against reactive oxygen species (ROS)-killing. Macrophages were observed to efficiently clear Bp only after pre-activation with IFNγ, which is independent of serum- and/or antibody-opsonization. These studies indicate that antibody-enhanced complement activation is sufficient for neutrophil-clearance of Bp, whereas macrophages are ineffective at clearing serum-opsonized Bp unless pre-activated with IFNγ. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis.Infection of susceptible hosts by the encapsulated Gram-negative bacterium  Burkholderia pseudomallei is the causative agent of melioidosis, which is a major cause of septic death in endemic areas of Southeast Asia and northern Australia. This range now appears to be expanding, and with the increased incidence of diabetes, which is a major predisposing factor for B. pseudomallei infections, it is believed these cases will continue to increase. This organism is also categorized as a Tier 1 select agent with great potential for misuse as a bioweapon. Thus, there is great interest in developing vaccines and other immunotherapies for melioidosis. To better design these treatments, it is essential to understand which immune cells are capable of controlling B. pseudomallei, particularly during early stages of infection, and which immune mediators are required for these cells to optimally eradicate these bacteria. In this work, we identified that both macrophages and neutrophils were capable of internalizing and killing B. pseudomallei, but required different immune mediators to allow these effects. Complement and antibody-mediated opsonization was sufficient to promote uptake and killing of B. pseudomallei by neutrophils, whereas these opsonins could only enhance uptake by macrophages; macrophage-mediated killing required IFNγ. These findings suggest that successful therapies must elicit complement-activating antibodies and IFNγ to eradicate/prevent melioidosis. Burkholderia pseudomallei (Bp), is an encapsulated, motile, Gram-negative bacillus found free-living in soil The causative agent of melioidosis, Although Bp can thrive as a free-living saprophyte in moist environments, it can also successfully persist within both phagocytic and non-phagocytic cells, including neutrophils, macrophages, dendritic cells, epithelial cells and endothelial cells The importance of macrophages in Bp pathogenicity is somewhat unclear. Depletion of macrophages from BALB/c or C57BL/6 mice significantly increases mortality during experimentally induced melioidosis compared with untreated mice in vivo findings suggest that neutrophils are important for controlling melioidosis. Depletion of neutrophils from mice resulted in an acute and more rapidly lethal Bp infection in vivo reports suggest the importance of neutrophils in controlling melioidosis, but the mechanisms of how neutrophils affect Bp infection are not clear.In contrast, a number of in vitro to detect the effectiveness of macrophages and neutrophils in clearing Bp, using various culture conditions and producing mixed results Burkholderia thailandensis (Bt) species, thus allowing for delineation of capsular versus other genetic effects on resisting phagocyte clearance. The findings of these studies will contribute to the understanding of the immune mediators required for cellular clearance of Bp, as well as provide a roadmap of the type(s) of immunity that need to be elicited by Bp-directed preventative therapies.A number of studies have been performed Burkholderia pseudomallei (Bp) 1026b Burkholderia thailandensis (Bt) E264 Escherichia coli strain K12  was used in certain experiments as a positive control for complement- or phagocyte-mediated killing. All bacterial strains were cultured on tryptic soy agar (TSA) plates aerobically at 37°C for 16–18 h prior to use. Bacterial suspensions were prepared in PBS by scraping the bacteria from TSA plates. Bacterial stocks were enumerated using a spectrophotometer at OD600 and the bacterial numbers were confirmed by dilution plating. All studies involving Bp were performed in the CDC-certified BSL3 laboratory at the University of Toledo Health Sciences Campus.All of the bacterial strains used in this study are listed in Female C57BL/6 and BALB/c mice were obtained from the National Cancer Institute . Mice were housed in the Department of Laboratory Animal Research at the University of Toledo Health Sciences Campus, and all procedures were in accordance with and approved by the University of Toledo Institutional Animal Care and Use Committee (IACUC).Animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Toledo Institutional Animal Care and Use Committee .Burkholderia strains over time, 105 bacteria were incubated in macrophage co-culture media  containing different concentrations of kanamycin for a total of 24 h. Bacterial viability was assessed at the indicated times by serial dilution and plating onto TSA plates. Bacterial numbers were enumerated as colony forming units (CFU) per ml.To determine the relative antibiotic sensitivities of the different 5 cells/well in complete RPMI. C57BL/6-derived macrophages were used for all studies unless otherwise indicated.Bone marrow from the femurs and tibias of C57BL/6 (B6) or BALB/c mice was cultured as previously described 5 cells/ml. After allowing the cells to adhere to the particular assay plate and eluting non-adherent cells, the remaining cell population contained ≥90% pure neutrophils. C57BL/6-derived neutrophils were used for all studies unless otherwise mentioned.Primary neutrophils were isolated from murine bone-marrow as previously reported Guinea pig serum used as a complement source was purchased from MP Biomedicals (Cat# 642831). Guinea pig serum shows enhanced complement-dependent bactericidal activity compared to mouse serum 2 and 4.25 mM MgCl2 (PBS++) at 37°C for 30 min. Guinea pig serum inactivated by heating at 56°C for 30 min  was used as a negative control. For antibody-mediated opsonization, bacteria were incubated with heat-inactivated mouse antiserum for 10 min at 37°C before adding guinea pig serum (i.e. complement source) for 30 min. The antisera were generated by immunizing groups of mice i.p. with 107 heat-killed Bp or Bt in complete Freund's adjuvant, followed by two similar boosting immunizations in incomplete Freund's adjuvant at 3 week intervals. Bacteria-specific antibodies were initially confirmed by Western blot and ELISA using sonicated Burkholderia sp., and antibody levels were quantified by comparison to standards produced using known amounts of purified Ig of the appropriate isotype to wells coated with rabbit anti-mouse IgG+IgM+IgA antibodies per our previously published protocol Before opsonization, bacteria from overnight cultures on TSA plates were suspended in PBS and enumerated by spectrophotometry. These suspended bacteria were incubated with different concentrations of guinea pig serum in PBS containing 1.2 mM CaClBacteria were opsonized with serum as described above. Opsonized bacteria were washed and then stained using 1∶500 Fluorescein isothiocynate (FITC)-conjugated polyclonal anti-guinea pig C3 antibody , then washed and fixed with 2% paraformaldehyde. Labeled bacteria were analyzed by flow cytometry using a BD FACSCalibur and results reported as mean fluorescence intensity (MFI).8) were incubated with different concentrations of guinea pig serum in PBS++ at 37°C. At the indicated times, samples were serially diluted and plated onto TSA plates to determine the colony forming units (CFU)/ml.Bacteria (102 before infecting with unopsonized or opsonized bacteria at a multiplicity of infection (MOI) = 1 in duplicates. Plates were centrifuged at 300× g for 5 min to synchronize infection and incubated at 37°C in 5% CO2 for 1 h. Monolayers were washed 3 times with media to remove extracellular bacteria before replenishing with complete RPMI containing 500 µg/ml of kanamycin to kill extracellular bacteria. After 2 h of incubation in kanamycin-containing medium, the cells were washed thoroughly to remove the residual antibiotic. At this time (3 h post-infection), the macrophages were either lysed with 0.5% saponin in PBS and dilution plated on TSA to determine CFU/ml  or fresh media was added and infected cells were incubated until 10 h post-infection . Bacterial uptake is reported as the internalization index, which is the percentage of bacteria recovered at 3 h compared to the bacterial input (0 h). Bacterial survival is represented as the survival index, which is the fold-change in bacterial numbers at 10 h compared to 3 h .Bone marrow-derived macrophages were seeded in 24-well plates in complete RPMI. Cells were allowed to attach overnight at 37°C in the presence of 5% COTo determine if macrophages pre-stimulated with IFNγ showed enhanced killing of opsonized compared to unopsonized bacteria, macrophages were pre-activated with 100 U of murine IFNγ (R&D Systems) for 16–18 h prior to infection. Intracellular growth assays were then performed as described above, and internalization and survival indices were determined.5 cells per well in 48-well plates containing complete RPMI with 10% FBS and incubated at 37°C in 5% CO2 for 20 min. Non-adherent cells were removed by washing before infecting with the bacteria at MOI = 1. Intracellular growth assays with neutrophils were performed under two conditions: 1) No blockage of reactive oxygen species (ROS) production when treated with dimethylsulfoxide , and 2) blockage of ROS production when treated with diphenyleneiodonium (DPI), which is a potent inhibitor of NADPH oxidase Murine neutrophils were seeded to obtain 4×10g for 5 min to synchronize infection and incubated at 37°C in 5% CO2 for 10 min before washing 3 times with media to remove extracellular bacteria. At 10 min post-infection, neutrophils were lysed using 0.5% saponin solution, and internalized bacteria were enumerated by dilution plating to determine bacterial uptake. Parallel co-cultures of neutrophils were lysed at 2 h post-infection and bacteria again dilution plated to determine bacterial killing. Bacterial uptake was reported as the internalization index, which is the percentage of bacteria recovered at 10 min compared to the bacterial input (0 h). Bacterial survival was represented as survival index which is calculated as the fold-change in bacterial numbers at 2 h compared to those internalized at 10 min post-infection. The 10 min and 2 h time points were chosen to determine internalization and survival in neutrophils, respectively, due to faster activation kinetics and short life span of neutrophils compared to macrophages Neutrophils (+/−10 µM DPI or DMSO) were infected with bacteria at MOI = 1. Plates were centrifuged at 300× 6 cells per well in a 6-well plate for 2 h. Neutrophils were then re-seeded at 2×105 cells per well in a 96-well plate in PBS++ in the presence or absence of DPI (10 µM). Neutrophils were pretreated with luminol , horseradish peroxidase (50 U/ml in sterile water) and cytochalasin B (5 ug/ml) at 37°C in 5% CO2 for 20 min before infecting with differentially opsonized bacteria at MOI = 5. Cells were immediately analyzed on a FLUOstar Omega plate reader (BMG Labtech) with luminescence detection every 20 s for 10 min. The time required to start detection of chemiluminescence post-infection (40 s) was considered as the starting point in the data figures.Luminol-based chemiluminescence assays were performed to assess the reactive oxygen species (ROS) production by neutrophils. Isolated murine neutrophils were cultured in complete RPMI at 2×10P-values of ≤0.05 were considered statistically significant.Graphpad Instat  was used for statistical analyses of all datasets using one-way ANOVA with the Tukey's post-hoc test. Because intracellular growth assays were used in the majority of these experiments, it was important to determine the optimum antibiotic concentration required for killing extracellular Bp and Bt in the minimal amount of time. Both Bp and Bt required relatively high kanamycin concentrations for efficient killing within 1–2 h incubation , althougIntracellular growth assays were performed in the presence/absence of relatively low residual kanamycin concentrations to determine whether it affects intracellular growth assays. In the presence of 20 µg/ml, which is >25× less than the levels needed to efficiently kill either strain in media and elicits little to no killing of Bp or Bt , no effeE. coli strain K12 by 10 h post-infection, indicating that the macrophages were functional . Two of the best-known opsonins include complement system components and certain antibodies isotypes that can assist both macrophages and neutrophils in phagocytosis. To begin addressing the importance of these opsonins, initial studies were performed to quantify differences in complement deposition and antibody binding between the different bacterial strains in vitro. Flow cytometric analyses indicated that acapsular Bt incubated in normal serum displayed similar levels of complement component C3 deposition as the positive control (E. coli) at all serum concentrations, whereas Bp demonstrated significantly reduced C3 deposition at 5% and 20% serum concentrations compared to Bt and E. coli  by macrophages . When inTo determine if complement opsonization can affect uptake and killing by neutrophils, Bp and Bt were pre-incubated with normal serum and assessed in the presence/absence of the ROS-inhibitor DPI. Without blocking ROS (DMSO vehicle control), complement opsonization had no effect on bacterial uptake . HoweverTo more directly assess the role of capsule in serum-mediated activation of neutrophils, similar experiments were performed comparing Bp with an acapsular mutant. Again, DMSO-treated neutrophils displayed similar low uptake of Bp and the acapsular mutant irrespective of serum opsonization . AltogetBecause major differences in bacterial uptake were seen within 10 min only for neutrophils treated with DPI, experiments were performed to confirm if complement-opsonization elicited a rapid ROS response that was responsible for this killing using a luminol-based chemiluminescence assay. Co-culture of neutrophils with Bp or Bt opsonized with ≤1% or heat-inactivated serum did not elicit ROS production .in vitro.To address whether antibody binding could enhance complement-mediated effects on macrophages, intracellular uptake and killing assays were performed with the indicated combinations of antisera and complement sources. Macrophages co-incubated with Bp or Bt opsonized with either specific antibodies or complement alone displayed significantly increased uptake compared to unopsonized bacteria  infections can be highly lethal due to its infectivity through multiple routes, existence of various predisposing conditions, variability of symptoms displayed, and lack of effective therapies. Categorization of Bp as a potential bioterrorism agent and the spread of its endemic locations have increased its importance as a global threat. Thus, there is a great need to develop better preventive and curative strategies for Bp infections. Because Bp can persist within multiple host cell types, it is important to better understand the immune response elicited during Bp infection and identify mechanisms to enhance their protective properties. The innate immune responses are critical for containing Bp infections, particularly as acute disease can prove lethal within a few days of infection and before the development of the adaptive responses. In these studies, we sought to delineate the bactericidal capacities of two important immune cell types, macrophage and neutrophils, and whether opsonization of Bp with complement or antibodies can enhance these protective abilities. Additionally, the direct effects of the bacterial capsule on evasion of these immune mechanisms were assessed for both cell types.Listeria monocytogenes that the presence of even low levels of antibiotics in macrophage medium leads to increased antibiotic concentration within these cells due to their pinocytic properties, allowing an artificial intracellular killing effect In the absence of any serum opsonins, Bp, Bt, and a Bp acapsular mutant were phagocytosed by both macrophages and neutrophils at similar rates, and were subsequently able to efficiently persist within both cell types at similar levels. This indicated that the capsule produced by Bp is not anti-phagocytic and is not essential for evasion of intracellular killing mechanisms elicited by either phagocyte under these conditions, which is different from previous speculation Burkholderia sp. This difference does not appear to be due to the absolute number of bacteria ingested, since antibody- and/or complement-opsonization of the same number of bacteria elicits similar internalization of Burkholderia sp.  produced by Bp is essential for allowing infection in susceptible hosts in vivo, either due to direct suppressive properties of the capsule of its ability to harness host complement regulatory proteins. While antibodies did not display synergistic or additive activation in vitro, it is possible that the observed antibody-mediated enhancement in complement deposition is necessary to achieve the minimum threshold stimulatory levels on Bp in vivo, whereas Bt attains these activating levels even in the absence of specific antibodies and are thus avirulent even in immunologically naïve hosts. This is supported by reports that passive administration of capsule-specific monoclonal antibodies can provide some protection from even aerosol challenge with Bp, but does not provide sterilizing immunity This study represents the first side-by-side comparison of macrophages and neutrophil responses to Bp, and delineates the immune mechanisms that are required to allow these early immune cell responders to efficiently recognize and kill Bp. The innate activation of the complement cascade appears to have the potential to opsonize Bp sufficiently to eradicate these pathogens, but only if enough deposition occurs to elicit ROS activity by neutrophils. It is possible that this minimal activation level is not normally achieved


Based on this prediction we investigated whether early retinal abnormalities in streptozotocin-induced diabetic rats are alleviated by preventing the rods from dark adapting. Diabetic rats and their non-diabetic littermates were housed in a 12:12 hour light-dim light photocycle (30 lux during the day and 3 lux at night). Progression of early retinal abnormalities in diabetic rats was assessed by monitoring the ERG b-wave and oscillatory potentials, Müller cell reactive gliosis, and neuronal cell death, as assayed by TUNEL staining and retinal thickness at 6 and 12 weeks after diabetes induction. Maintaining diabetic animals in a dim-adapting light did not slow the progression of these neuronal and glial changes when compared to diabetic rats maintained in a standard 12:12 hour light-dark photocycle (30 lux during the day and 0 lux at night). Our results indicate that neuronal and glial abnormalities in early stages of diabetes are not exacerbated by rod photoreceptor O2 consumption in the dark.The aetiology of diabetic retinopathy (DR), the leading cause of blindness in the developed world, remains controversial. One hypothesis holds that retinal hypoxia, exacerbated by the high O Diabetic retinopathy (DR) is a serious complication of both type 1 and type 2 diabetes and a leading cause of visual impairment and blindness222 profiles in cats diabetic for 6 yearsThe initial steps leading to DR have not been fully elucidated. One hypothesis of the aetiology of DR holds that compromised blood flow in early stages of the disease results in retinal hypoxia. Although the evidence for hypoxia in DR is controversial, some studies support the hypothesis. In psychophysical experiments on diabetic patients, reduced contrast sensitivity and reduced colour vision perception is restored when patients breath 100% OGB Arden has suggested that the high metabolic demand of photoreceptors in the dark will exacerbate retinal hypoxia and may be a primary cause of DR8Diabetic retinopathy has traditionally been viewed as a disease of the retinal vasculature. However, there is strong evidence that neuronal and glial dysfunction precede overt vascular lesions and play a role in the development of DR1210We have now assessed whether preventing dark-adaptation reduces early neuronal and glial changes in the diabetic retina in a rat model of type 1 diabetes. We find that preventing dark-adaptation by maintaining diabetic rats in dim light during the night does not slow the progression of early retinal abnormalities, as assessed by changes in the ERG, Müller cell reactive gliosis, and neuronal cell death, assayed by TUNEL staining and retinal thickness.We tested the hypothesis that preventing dark adaptation slows the progression of early retinal abnormalities in diabetic rats using the streptozotocin (STZ) animal model of type 1 diabetes. Sprague-Dawley rats were treated with a single injection of STZ or citrate buffer and serum glucose was assessed weekly. STZ-treated rats became hyperglycaemic within 3 days after injection, and glucose concentrations remained elevated throughout the study . Rats weRats housed under dim-adapting conditions were maintained in a room with an ambient light intensity within the cages of 3.0 ± 0.9 lux during subjective night to prevent rod photoreceptor dark adaptation. We assessed whether 3 lux illumination was sufficient to light-adapt the rods by measuring changes in the ERG under different background illumination levels.Flash ERGs were recorded in control (non-diabetic) rats in the dark (0 lux) and at three background light intensities . Both the a-wave and b-wave were significantly reduced with background light levels as low as 0.3 lux a–c, demoWe evaluated the progression of diabetes-induced retinal abnormalities in rats housed under standard and dim-adapted lighting conditions. Changes in the ERG, Müller cell reactive gliosis, neuronal apoptosis, and reduced retinal thickness, a measure of retinal cell loss, were measured.The ERG b-wave, reflecting bipolar cell function, and the oscillatory potentials (OPs), primarily reflecting the activity of amacrine cells12However, there were no differences in ERG responses between diabetic rats housed under standard and dim-adapted lighting conditions. Changes in b-wave amplitudes and in OP delays were similar in the two groups of animals.Müller cell reactive gliosis is a hallmark of retinal disease and is observed in diabetic patients and in animal models of diabetes5Müller cell gliosis, evidenced by an upregulation of GFAP, was apparent at 6 weeks of diabetes in the central and mid retina, and at 12 weeks in the peripheral retina . ControlNeuronal cell death, indicated by TUNEL staining, and the resulting decrease in retinal thickness, is observed in both diabetic patients and in STZ-treated diabetic ratsDiabetic rats housed under standard and dim light conditions showed similar increases in TUNEL-positive cells and similar decreases in retinal thickness. The one exception was retinal thickness at 6 weeks, where rats housed under dim light conditions showed a decrease in retinal thickness while rats housed under standard light conditions did not. This result might be due to light damage caused by the 3 lux dim-adapting light. At 12 weeks, reduced retinal thickness was apparent in both diabetic groups.We have evaluated whether preventing dark adaptation with a dim adapting light slows the development of early neuronal and glial abnormalities in diabetic rats. Our results indicate that the development of these early diabetes-induced retinal abnormalities is not slowed, at least in our animal model of diabetes.We found that diabetic rats housed under dim-adapting lighting conditions at night to prevent rod dark adaptation showed a similar degree of retinal abnormalities as rats housed under standard (0 lux) lighting conditions at night. This was true at both 6 and 12 weeks following diabetes induction and for multiple measures of retinal function and morphology, including ERG alterations, Müller cell reactive gliosis, and cell death, as assayed by TUNEL staining and retinal thickness. It should be noted, however, that neuronal cell loss in diabetes occurs predominately in the inner retina172. Accounting for the 0.3% transmittance of the human eyelid for green lightThere are several possible reasons why light adaptation failed to slow the progression of retinal abnormalities. The 3 lux adapting light that was used to prevent rod dark adaptation at night may not have been bright enough to sufficiently reduce photoreceptor metabolism. This is unlikely, however, as a 0.3 lux background light was sufficient to cause significant reductions in retinal sensitivity, as measured by ERG a- and b-wave amplitudes. In addition, the 3 lux adapting light we used was similar in intensity to the adapting light used by Arden in his clinical trials on diabetic patients8Negative results may also have been obtained because the maximal diabetic duration of 12 weeks used in our study may have been too short to detect differences between experimental groups. In addition, the adapting light we used might not have reached the rat retina due to eye closure or burrowing. This is unlikely, however, because rats are active during the subjective night, even when exposed to bright background light202 also complicate the interpretation of our results. Minimum retinal PO2 in the dark is lower in primates than it is in rodents22The focus of our study was to evaluate neuronal and glial abnormalities in the retina in early stages of diabetes. Vascular changes, which occur in diabetic rats only after many months, were not followed. It is possible that the light adaptation protocol we used to reduce retinal hypoxia might have been beneficial in limiting the vascular complications of DR, as vascular abnormalities might be caused by different mechanisms than neuronal and glial abnormalities. Species differences in retinal POThe dim-adapting light used in our experiments would be expected to reduce oxidative stress and the production of free radicals by photoreceptors, which may contribute to the development of DR2 consumption does not slow the development of early neuronal and glial abnormalities in diabetic rat retinas. This may occur because the diabetic rat retina may not, in fact, be hypoxic252 in diabetic rats at 12 weeks following diabetes induction indicate an increase, not a decrease in PO2Our results indicate that preventing dark adaptation and thus reducing retinal O2 consumption, the results do not rule out the dark adaptation-hypoxia hypothesis of DR. The validity of this hypothesis will only be resolved by additional experimentation. Ultimately, large scale clinical trials will determine whether dim-adapting light therapy is beneficial in slowing the progression of DR. One such clinical study is currently being conductedAlthough our results indicate that early diabetes-induced neuronal and glial abnormalities are not caused by retinal hypoxia exacerbated by rod OAll experimental procedures were approved by and adhered to the guidelines of the Institutional Animal Care and Use Committee of the University of Minnesota (Protocol ID: 1304-30570A). Two-month-old Sprague-Dawley male rats received a single intraperitoneal (i.p.) injection of streptozotocin  or vehicle alone. Successful induction of diabetes, defined as a blood glucose level ≥250 mg/dl, was confirmed 3 days later. Non-fasting blood glucose and weight were monitored weekly with a OneTouch Ultra Glucometer  and, if required, diabetic rats received insulin  at a sub-therapeutic dose of either 1.5 U (glucose range of 250–400 mg/dl) or 2.5 U (above 400 mg/dl) to prevent excessive weight loss and a catabolic response.Rats were randomly assigned to two experimental groups. One group of animals was housed under a 12:12 light-dark photocycle with illumination of 30 lux during the day and 0 lux at night (standard lighting). A second group was housed under a 12:12 light-dim light photocycle of 30 lux during the day and 3 lux at night (dim-adapting lighting). Dim illumination conditions were achieved with two 4 Watt compact fluorescent light bulbs (colour temperature 2700 K). These housing conditions were introduced three days before induction of diabetes and maintained for the duration of the experiment. Light levels were measured with a digital illuminance meter  in the centre of the polycarbonate cages used to house the animals. The 3 lux night illumination was used to prevent dark adaptation. This intensity corresponds to a luminance level in the mid-mesopic rangeIn addition, five untreated male Sprague-Dawley rats  were used to assess the effects of background illumination on the ERG. Rats from this group were housed under standard lighting conditions.2/76% N2), with an Espion testing system and ColorDome LED/Xenon-full field stimulator . Pupils were dilated with 1% atropine  and the corneal surface anesthetized with 0.5% proparacaine HCl . Eyes were lubricated with 2.5% Hypermellose . After completing a scotopic intensity series, photopic flash responses were recorded in presence of an adapting white light . Flash ERG records were band pass filtered at 0.3 and 500 Hz. Oscillatory potentials were band pass filtered at 50–200 Hz. Each record represents an average of 2–6 responses. Rats were killed immediately following ERG measurement and eyes were harvested for histological evaluation.Rats were maintained in complete darkness overnight prior to an ERG recording session. Preparations for recordings were conducted under dim red illumination and were followed by a 20 minute period of complete darkness to ensure a dark-adapted state. The full-field flash ERG was recorded using custom DTL fibre electrodes from isoflurane-anesthetized rats  and Aquamount (Lerner Laboratories). Twelve-μm-thick cryostat sections were cut and mounted using Vectashield containing DAPI (Vector Laboratories). Sections were imaged with confocal microscopy (Olympus FluoView 1000), using either a 20X oil  or a 40X oil  objective. Offset, laser power and gain parameters were held constant for control and diabetic samples.To assess Müller cell gliosis, transverse retinal sections were double-labelled with antibodies to glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), a Müller cell-specific marker, using procedures modified fromTotal thickness in the central region of the retina was evaluated in DAPI-stained cryostat sections that crossed the optic disc. Although histological artifacts such as dehydration and shrinkage can contribute to changes in the structure of histological sections, these processes will affect the measurement of retinal thickness equally in all experimental groups.In Situ Cell Death Detection Kit, Fluorescein  according to the protocol provided by the supplier. Briefly, fixed sections were permeabilized in PBS containing Triton X-100 (Sigma) and then incubated in the reaction mixture for 60 min at 37 deg C. TUNEL-positive cells were counted manually in each of two sections running through the optic disc and analysed as the number of positive cells per length of the retinal section.Apoptotic cells were imaged with TUNEL labelling using the Experimental values are reported as mean ± s.e.m. Statistical analysis was performed using GraphPad Prism software. Standard and repeated measures ANOVA was used to compare experimental groups across time points and housing conditions. Post hoc multiple comparisons were performed when appropriate using Tukey’s method. Two-sided t-test was used to determine pairwise differences. All analyses were performed with significance at P < 0.05.How to cite this article: Kur, J. et al. Light adaptation does not prevent early retinal abnormalities in diabetic rats. Sci. Rep.6, 21075; doi: 10.1038/srep21075 (2016).


Considerable effort has been made to categorise the bacterial composition of the human gut and correlate findings with gastrointestinal disease. The infant gut has long been considered sterile at birth followed by rapid colonisation; however, this view has recently been challenged. We examined first-pass meconium from healthy term infants to confirm or refute sterility.Bifidobacterium; Bacteroides-Prevotella; Lactobacillaceae/Enterococcaceae; Enterobacteriaceae; Streptococcaceae; Staphylococcaceae and Enterococcaceae. Absolute counts of all bacteria and proportional identification of each bacterial group were calculated. Confirmation of bacterial presence by PCR was undertaken on FISH-positive samples.Healthy mothers were approached following vaginal delivery. First-pass meconium stools within 24 hours of delivery were obtained from healthy, breastfed infants with tight inclusion/exclusion criteria including rejecting any known antibiotic exposure - mother within 7 days preceding delivery or infant after birth. Stools were processed in triplicate for fluorescent in-situ hybridisation (FISH) with 16S rRNA-targeted probes including 4 cells/g with a mean of 15.4 x 104 cells/g. (The limit of detection for automated counting is 106 cells/g). Cell counts were too low to allow formal diversity analysis. Amplification by PCR was not possible despite positive spiked samples demonstrating the feasibility of reaction. One baby was dominated by Enterobacteriaceae. The others contained 2-5 genera, with Bifidobacterium, Enterobacteriaceae, Enterococcaceae and Bacteroides-Prevotella the most prevalent. There was no association between bacterial counts and rupture of membrane duration, time to passage of meconium or time to lab.The mothers of 31 newborn infants were recruited, 15 met inclusion/exclusion criteria and provided a sample within 24 hours of birth, processed in the lab within 4 hours. All babies were 37–40 weeks gestation. 8/15 were male, mean birth weight was 3.4kg and mean maternal age was 32 years. Meconium samples from 10/15 (66%) infants had evidence of bacteria based on FISH analysis. Of these, PCR was positive in only 1. Positive FISH counts ranged from 2.2 - 41.8 x 10This study provides evidence that low numbers of bacteria are present in first-pass meconium samples from healthy, vaginally-delivered, breastfed term infants. Only two-thirds of meconium samples had detectable bacteria, though at levels too low for automated counting or for reliable confirmation by PCR. This study suggests that gut bacterial colonisation is extremely limited at birth and occurs rapidly thereafter. There has been a recent explosion of interest in the potential role of resident gut bacteria (the gut microbiota) in the causation or alleviation of human disease, however comparatively little attention has been paid to the process of early life colonisation. The long-standing dogma of gut sterility at birth has recently been challenged by suggestions that early meconium samples , the plaThe simple question of whether or not the gut is sterile at birth is a fundamental one in understanding human microbial colonisation, particularly as the gut harbours the most diverse bacterial community of the human body . Data gaHealthy mothers following a normal vaginal delivery at term in Aberdeen Maternity Hospital were invited to participate in the study. Primigravida and parous mothers were considered equally. The first meconium nappy, passed within 24 hours of delivery, was collected and processed in the laboratory within 4 hours. Although the method of feeding was not thought to be of significance during this short period after birth, only infants exclusively breastfed up to the point of first meconium were included.th- 90th centiles); vaginal delivery; healthy infant; breastfed only to point of sample collection. Exclusion criteria: significant background maternal health concerns; significant maternal health issues during pregnancy; perinatal antibiotic exposure (mother within 7 days preceding delivery or infant after birth); premature delivery (<37 weeks); post-term delivery (>42 weeks); small weight for gestational age (weight <10th centile); large weight for gestational age (weight >90th centile); Caesarean section delivery; instrumental delivery; prolonged rupture of membranes prior to delivery (>24 hours); meconium passed in liquor; maternal pyrexia>38°C during labour; neonatal health concerns sufficient to warrant admission to neonatal unit; formula milk fed at any point prior to sample. The period prior to delivery within which maternal antibiotic exposure was deemed an exclusion criterion (7 days) was decided on a pragmatic basis. Data on inclusion and exclusion criteria were collected for each recruit on a study proforma sheet and cross-checked during analysis.Tight inclusion and exclusion criteria were applied to approximate normal physiology as closely as possible within a hospital setting. Inclusion criteria: healthy mother; healthy pregnancy; term at delivery (37–42 weeks inclusive); appropriate weight for gestational age  into a sterile universal container, placed immediately into a fridge in the maternity unit then transferred to the laboratory and processed within 4 hours of collection. This same initial approach is used routinely in the collection of multiple clinical samples for hospital microbial analysis, is in keeping with standard practice. Samples were then received into Category 2 microbiology laboratories and processed.Firstly, 0.5g samples were taken from the meconium specimens and placed into lysing matrix tubes from the FastDNA SPIN Kit for Soil  then stored at -80°C until DNA extraction was performed in batches using the same kit within 8 weeks of collection. Secondly, a 0.5g sample was fixed in a 1/40 solution for fluorescent in-situ hybridisation (FISH) following the stool processing protocol . BrieflyBifidobacterium genus), Bac303 (Bacteroides-Prevotella group), LAB158 (Lactobacillaceae and Enterococcaceae), EntD (Enterobacteriaceae), Streptococcaceae (Strep), Staphylococcaceae (Staphy) and specifically Enterococcaceae (ENC221). These probes have all been validated previously and hybridisation was carried out using standard methods [Streptococcus species was observed. The low bacterial load in the samples meant that automated counting could not be done and each field of view had to be examined and counted manually.16S rRNA-targeted fluorescent probes were used to detect the predominant groups of human faecal bacteria. Absolute counts of all bacteria and proportional identification for each bacterial group were calculated. The probes used were Eub338 , Bif164  as the total time from rupture of membrane to time of birth, plus time to passage of meconium, plus time to laboratory processing and have used this in analysis.Ethical approval was granted by North of Scotland Research Ethics Service (10/S0801/16) and written informed consent was obtained from the parents of all subjects.The mothers of 31 infants were identified, approached and consented for the study immediately post-partum. Of these, 18 ultimately met the inclusion/exclusion criteria and 15 provided a meconium sample within 24 hours, which was subsequently collected and processed in the laboratory within 4 hours. All babies were 37–40 weeks gestation. 8/15 were male, mean birth weight was 3.4kg and mean maternal age was 32 years. The final cohort (n = 15) is described in Tables 4 cells/g, with the mean of positive samples being 15.4 x 104 cells/g. (Limit of detection for automated counting is 106 cells/g). These cell counts were too low to allow formal diversity analysis, however a representation of bacterial composition in positive samples is shown in Enterobacteriaceae. The others contained 2–5 genera. Bifidobacterium, Enterobacteriaceae, Enterococcaceae and Bacteroides-Prevotella were the most abundant bacteria identified.Following FISH analysis, 10/15 (66%) infants had evidence of bacteria present. Positive FISH counts ranged from 2.2 to 41.8 x 102 = 0.023), however interestingly four of seven samples with an MCI <500 minutes were sterile compared with only one of eight above this threshold.PCR amplification using generic bacterial primers was only possible in DNA extracted from one sample. The remaining 9/10 FISH positive samples failed to give any amplification products, despite positive spiked samples demonstrating the feasibility of a reaction. There was no clear association between “meconium colonisation interval” (MCI) with bacterial counts  as the period between externalisation of the amniotic sac contents (by rupture of membranes) and processing of the meconium sample in the laboratory. Assuming sterility of the amniotic sac and fetus, the start of this period would coincide with the commencement of fetal microbial colonisation. We demonstrated no correlation between MCI and bacterial cell counts in this study, albeit with limited subject numbers and consequently limited power to demonstrate such an association. Around one third of first-pass meconium samples in our cohort appeared sterile on triplicate microscopy analysis by FISH, and four of these five samples had an MCI time <500 minutes. No meconium sample was obtained in this study with an MCI time <200 minutes (3 hours 20 minutes), a considerable gap during which microbial changes may well occur. Addressing the MCI in the question of gut sterility at birth is a challenge for studies in this field and we would suggest that recording these times and analysing microbial data using MCI may help in future studies exploring early colonisation. This is a particular difficulty when addressing premature neonatal colonisation as delayed passage of meconium is commonplace with prematurity and often takes a number of days . MicrobiEnterococcus faecalis, Staphylococcus epidermidis and Escherichia coli. No sample was entirely sterile. Makino and colleagues recently demonstrated a dearth of Bifidobacteria colonisation in first-pass meconium, but did not extend the analysis beyond this single genus [et al recently published a further amplicon sequencing study on meconium samples from both term and preterm infants, and could find no DNA evidence of bacteria in 8/17 (47%) of meconium from infants >33 weeks gestation, with a greater proportion of preterm infants positive (74% vs 53%) [Comparing our study against other such investigations in the term neonate is challenging, as most utilise a “catch-all” approach to recruitment, rather than our selective approach, and presume all first pass samples are equivalent. The genre-defining culture-based paper on this subject from Jiménez and colleagues looked at early-passed meconium (within 2 hours of birth) and processed half of these immediately and half within 4 days . No datale genus . Hu and le genus . The ext vs 53%) . PositivIn summary, we present here a clinically considered study on a select group of infants, selected for “normality” as much as possible, demonstrating that although ~2/3 of first-pass meconium samples contain bacteria, this appears less common when colonisation factors and timings are considered, and the number of different bacterial species detected in positive samples appears extremely limited. Further targeted work on the earliest aspects of microbial colonisation is required to fully address the sterility question, and we propose use of the MCI and its constituent timings as a potentially important aspect of data gathering for such studies. The seminal study to date on early neonatal colonisation demonstrated a lack of differentiation across neonatal body sites and inferred that this was a feature of early colonisation . The imp


Inflammatory, autoimmune and metabolic disorders have been associated with alterations in osteopontin (OPN) serum levels. Furthermore, elevated serum levels of OPN were reported from a small cohort of patients with sepsis. We therefore analyzed OPN serum concentrations in a large cohort of critically ill medical patients.A total of 159 patients  were studied prospectively upon admission to the medical intensive care unit (ICU) as well as after 3 days of ICU treatment and compared to 50 healthy controls. Clinical data, various laboratory parameters as well as investigational inflammatory cytokine profiles were assessed. Patients were followed for approximately 1 year.We found significantly elevated serum levels of OPN at admission to the ICU and after 3 days of treatment in critically ill patients compared to healthy controls. OPN concentrations were related to disease severity and significantly correlated with established prognosis scores and classical as well as experimental markers of inflammation and multi-organ failure. In the total cohort, OPN levels decreased from admission to day 3 of ICU treatment. However, persistently elevated OPN levels at day 3 of ICU treatment were a strong independent predictor for an unfavorable prognosis, with similar or better diagnostic accuracy than routinely used markers of organ failure or prognostic scoring systems such as SAPS2 or APACHE II score.Persistently elevated OPN serum concentrations are associated with an unfavourable outcome in patients with critical illness, independent of the presence of sepsis. Besides a possible pathogenic role of OPN in critical illness, our study indicates a potential value for OPN as a prognostic biomarker in critically ill patients during the early course of ICU treatment.The online version of this article (doi:10.1186/s13054-015-0988-4) contains supplementary material, which is available to authorized users. Sepsis and septic shock represent major causes of mortality in patients referred to intensive care units . Sepsis Osteopontin (OPN) represents a phosphorylated acidic glycoprotein that is involved in a broad variety of physiological and pathological processes such as cancer, fibrosis, inflammation and heart disease –7. RegarDespite the emerging roles of OPN in the regulation of inflammation and immunity, its functional involvement in systemic infections remains to be elucidated. Moreover, the diagnostic and prognostic value of OPN measurements in critically ill patients is currently unclear . We ther2 at ICU admission (prior to any treatment) were defined as obese. Patients with a medical history of type 2 diabetes and a concomitant intake of diabetes-related medication were defined as diabetics. As a control population, we analyzed 50 healthy blood donors with normal values for blood counts, C-reactive protein (CRP) and liver enzymes.A total of 159 consecutive patients  that were admitted to the general Internal Medicine ICU at the University Hospital RWTH Aachen were enrolled into this study . Written informed consent was obtained from the patient, his or her spouse or the appointed legal guardian.Serum was obtained at admission to the ICU before therapeutic intervention. For 65 patients, follow-up measurements were available at day 3 and day 7 of ICU treatment. All samples were immediately placed on ice, centrifuged, and the serum samples were stored at −80 °C. Interleukin 6 (IL-6), IL-10, TNF, procalcitonin (PCT), soluble urokinase plasminogen activator receptor (suPAR), NTproCNP, ghrelin, hyaluronic acid, a proliferation-inducing ligand (APRIL) and micro ribonucleic acid (miRNA)-133a were measured as described previously –29. GlomOsteopontin (OPN) serum concentrations were analysed using a commercial enzyme immunoassay according to the manufacturer’s instructions .U test, and multiple comparisons between more than two groups have been conducted by Kruskal-Wallis ANOVA and Mann–Whitney U test for post hoc analysis. Box plot display a statistical summary of the median, quartiles and extreme values. The whiskers extend from the minimum to the maximum value excluding outside and far out values, which are displayed as separate points. An outside value (indicated by an open circle) was defined as a value that is smaller than the lower quartile minus 1.5 times the interquartile range, or larger than the upper quartile plus 1.5 times the interquartile range. A far out value was defined as a value that is smaller than the lower quartile minus three times the interquartile range, or larger than the upper quartile plus three times the interquartile range. All values, including “outliers”, have been included for statistical analyses. Correlations between variables have been analysed using the Spearman correlation tests, where values of p <0.05 were considered statistically significant. The prognostic value of the variables was tested by univariate and multivariate analysis in the Cox regression model. Kaplan-Meier curves and log-rank test calculations were performed to display the impact on survival. Receiver operating characteristic (ROC) curve analysis and the derived area under the curve (AUC) statistic provide a global and standardized appreciation of the accuracy of a marker or a composite score for predicting an event. ROC curves were generated by plotting sensitivity against 1-specificity. Optimal cutoff points were calculated with the highest Youden Index, also positive likelihood ratios (LHR+), negative likelihood ratios (LHR-) and diagnostic odds ratios for this cutoffs, as previously described by Ray et al. [All statistical analyses were performed with SPSS  as previously described , 30. In y et al. .p = 0.005; Fig. Serum samples of critically ill patients were analysed at admission  to the ICU and after 3 days of treatment. As shown in Fig. Serum levels of OPN can be influenced by metabolic disorders such as obesity and diabetes . As thesBased on a recent pilot study suggesting that OPN might represent a novel biomarker for predicting sepsis in critically ill patients , we nextWe next analysed the kinetics of OPN serum concentrations during the first 3 days of ICU treatment. Interestingly, despite remaining elevated compared to healthy controls, longitudinal OPN measurements revealed overall a strong and significant decrease between admission and day 3 Fig. . Of noteWe next compared OPN serum levels at day 3 of ICU treatment between the different predefined subgroups of ICU patients, namely low vs. high APACHE II scores Fig. , pre-exiTo determine factors possibly promoting elevated OPN concentrations in critically ill patients, correlation analyses with extensive sets of laboratory parameters were performed. These analyses revealed that both OPN levels at admission as well as after 3 days of ICU treatment were significantly associated with laboratory markers routinely used in the assessment of organ dysfunction. High OPN levels correlated to decreased renal function assessed by the GFR of cystatin C, elevated creatinine and urea serum concentrations ), hepatic  and renal (creatinine) function at day 3, OPN was identified as an independent significant prognostic parameter to predict ICU survival Table .Table 4MIn our cohort of critically ill patients, 25.8 % died at the ICU, while the overall mortality rate increased to 46.5 % during the  observation period. Patients that died during long-term follow-up showed a trend towards higher initial OPN levels than survivors; strikingly, serum OPN levels at day 3 were significantly higher levels in patients that died during follow-up than for surviving patients Fig. , b. ROC Of note, multivariate Cox regression analyses, including markers of inflammation/infection , hepatic  and renal (creatinine) function at day 3, revealed that OPN represents an independent significant prognostic parameter also for overall survival Table .Table 5MThus, our data indicate that measurement of OPN levels in a medical ICU environment might have a valuable role in the assessment of a critically ill patient’s short-term and long-term prognosis.+ T cells and driving IL-17 expression [In this study, we assessed OPN concentrations upon admission to the medical ICU before specific therapeutic interventions and at day 3 after ICU admission in a well-characterized cohort of critically ill patients , 33, 34.pression . Importapression .+ T helper 1 (Th1) and Th2 cells [Sepsis and the Systemic Inflammatory Response Syndrome (SIRS) represent states of profound dysbalance of the immune system in response to infection and/or organ damage, menacing the prognosis of many patients referred to the ICU . The exah2 cells . Moreoveh2 cells . OPN, thh2 cells  and to dh2 cells . Thus, ih2 cells . On the Of note, Vaschetto et al. even suggested that OPN serum levels may allow differentiating between sepsis and SIRS . In contIn spite of advances in diagnosis and treatment of critically ill patients throughout the recent decades, the triage, diagnostic and therapeutic management of these patients during the first week of ICU treatment still represents a major challenge. The promptness and accuracy of the initial decisions during the initial course of disease are of immense importance for the subsequent outcome of sepsis  or cardiTaken together, our data provide evidence for a role of OPN as a diagnostic tool in the prognostic judgment of critically ill and septic patients during the early phase of their ICU stay. Certainly, these data need to be recapitulated in larger independent cohorts as well as in other ICU settings such as post-surgery care, before implementation into clinical algorithms can be considered. Moreover, although our data imply an important role of OPN in the molecular pathogenesis in critically ill patients, they do not provide a specific molecular mechanism of action. OPN may trigger either pro-inflammatory stimuli like TNF or IL-6 Table  as well Our study identifies osteopontin (OPN) as a stable and robust marker in critically ill patients to assess disease severity and mortality risks. While OPN serum levels are elevated compared to healthy controls both at admission to the ICU and after 3 days of treatment, persistently elevated OPN levels at day 3 of ICU treatment are a strong independent predictor for an unfavourable prognosis. In line with prognostic properties, OPN is closely correlated to established prognosis scores. High OPN levels do not discriminate septic patients from non- septic patients. In contrast, OPN correlates with experimental markers of general inflammation and multi-organ failure.Serum levels of OPN in critically ill patients are elevated compared to healthy controls both at admission to the ICU and after 3 days of treatment.OPN concentrations were related to disease severity and significantly correlated with established prognosis scores and classical as well as experimental markers of inflammation and multi-organ failure.In the total cohort, OPN levels decreased from admission to day 3 of ICU treatment. However, persistently elevated OPN levels at day 3 of ICU treatment were a strong independent predictor for an unfavourable prognosis and indicated short- and long-term mortality with higher diagnostic accuracy than routinely used markers of organ failure and prognostic scoring systems such as SAPS2 or APACHE II score.


We studied three separate animal models of HO including two models of trauma-induced HO and one model of genetic HO, and human HO specimens, to characterize the properties of cells derived from tissue containing pre-and mature ectopic bone in relation to analogous mesenchymal cell populations or osteoblasts obtained from normal muscle tissue. We found that when cultured in vitro, cells isolated from the trauma sites in two distinct models exhibited increased osteogenic differentiation when compared to cells isolated from uninjured controls. Furthermore, osteoblasts isolated from heterotopic bone in a genetic model of HO also exhibited increased osteogenic differentiation when compared with normal osteoblasts. Finally, osteoblasts derived from mature heterotopic bone obtained from human patients exhibited increased osteogenic differentiation when compared with normal bone from the same patients. These findings demonstrate that across models, cells derived from tissues forming heterotopic ossification exhibit increased osteogenic differentiation when compared with either normal tissues or osteoblasts. These cell types can be used in the future for in vitro investigations for drug screening purposes.Heterotopic ossification (HO) is the pathologic formation of bone separate from the normal skeleton. Although several models exist for studying HO, an understanding of the common  ACVR1 causes fibrodysplasia ossificans progressiva (FOP), a debilitating condition in which heterotopic bone forms independent of trauma.[in vivo treatment strategies including mouse and rat trauma models, and transgenic mouse models[in vivo models is the development of representative in vitro cell studies which can be used as a screening tool, especially in the context of studying possible pharmacologic targets. One major barrier to creating representative in vitro studies however, has been the challenge of identifying the tissue origin of the progenitor cell populations which form HO under a variety of trauma-induced conditions. Some studies have utilized muscle resident stromal cells or adipose-derived mesenchymal cells to evaluate in vitro osteogenic differentiation potential[in vitro characteristics of tissues which have either developed HO or will become HO. These models have been selected to represent the clinical scenarios during which heterotopic ossification forms—in situations of high-energy trauma as with our two murine trauma models, and in situations of genetic mutation. The use of human HO samples is intended to represent a more clinically translatable sample, and are derived from patients who developed HO subsequent to trauma. Our characterization of these cell lines will validate further in vitro analyses of potential therapeutics using these cells.Heterotopic ossification (HO), the pathologic formation of ectopic bone in soft tissues, occurs after extremity trauma, burn injury, spinal cord, traumatic brain injuries and orthopaedic procedures.–7 Additif trauma. Due to tse models–15. Critpotential–18. Othepotential. Howeverpotential, 20–23, potential, and humACVR1  with Nfatc1-Cre transgenic mice. F1 offspring mice carrying both transgenes (Nfatc1-cre+/caACVR1fx/wt) were used as experimentals whereas Cre- or Nfatc1-cre+/caACVR1wt/wt littermates were used as controls.[C57BL/6 male mice from Charles River  were used for all experiments with old (16–18 months old) mice between and young (6–8 weeks old) mice. Adult male Sprague Dawley rats (450-600g) were obtained from Taconic Farms . All animals were housed in clean plastic cages and kept on a 12-hour light/dark cycle with unlimited access to food (standard rodent chow) and fresh water ad libitum. Animals were acclimated for at least two weeks prior to experimentation. All animal procedures were carried out in accordance with the guidelines provided in the Guide for the Use and Care of Laboratory Animals from the Institute for Laboratory Animal Research . The study protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Michigan (PRO0001553) and were reviewed and approved by the Walter Reed Army Institute of Research/Naval Medical Research Center Institutional Animal Care and Use Committee (12-OUMD-20S) in compliance with all applicable Federal regulations governing the protection of animals in research. In our genetic model, we crossed mice carrying the conditional constitutively active allele of controls.–2923. Briefly, animals were anesthetized with 3–5% inhaled isoflurane. Hair was closely clipped on the left dorsum to expose the skin. Partial-thickness burn was achieved by placing a metal brand, heated to 60°C in a water bath, against the exposed skin for 18 seconds. Each mouse then received a concurrent Achilles tenotomy with sharp dissection at the midpoint in the left leg. Pain management was achieved with subcutaneous injections of buprenorphine every 12 hours for 3 days. Mice were euthanized using carbon dioxide and cervical dislocation. At indicated time points post injury, tissue surrounding the tendon transection site was collected in addition to tendon from the same region on the uninjured contralateral hindlimb. Mouse mesenchymal cells (mMSCs) were digested and cultured as previously described[All mice used for analysis received a partial thickness burn injury as previously describeddescribed.Rats were anesthetized with isoflurane and received buprenorphine (0.05mg/kg) delivered via intraperitoneal injection and were exposed whole body blast overpressure 120 ± 7 kPa) via a pneumatically driven shock tube. A drop weight apparatus  was used to create a comminuted femur fracture in a similar fashion to that previously described. The wei ± 7 kPa At indicated time points post injury, rats were euthanized with pentobarbital . Tissue surrounding the amputation site was collected from the residual limb in addition to muscle tissue from the same region on the uninjured contralateral hind limb. Rat mesenchymal cells (rMSCs) were digested and cultured as previously described.in vitro culture experiments were run using actively proliferating cells harvested (trypsin-EDTA treatment) from semi-confluent monolayer of adherent cells derived from ectopic heterotopic bone and non-heterotopic skeletal bone tissue.All human subjects and animal research approval was provided by the University of Michigan and Naval Medical Research Center. All participants provided written informed consent to participate in this study. Written consent was approved by the IRB at the University of Michigan. All patients were between the ages of 20 and 38 and none of them had other significant co-morbidities or were on medications related to osteoblast biology. Ectopic heterotopic bone and normal surrounding skeletal bone were excised using normal surgical technique without excising excess tissue. The ectopic bone and skeletal bone tissues were immediately separated and placed in sterile saline. Some of the tissue was processed for histology. The remaining tissues were then vigorously mechanically disrupted with a rongeur and then placed into standard growth medium. Cells were allowed to migrate out of the bone fragments and then subsequently expanded in culture dishes containing Dulbecco’s Modified Eagle Medium  , 10% FBS, 1% Pen/Strep, 2mM L-Glut, and used at passages 1–4. All 5 cells/well and onto a 12-well plate at a density of 3.5 × 104 cells/well, and then cultured at 37°C in 5% CO2 in air as previously described.[Cells (passage 1–4) were resuspended in osteogenic differentiation medium (ODM), seeded in triplicate onto six-well plates at a density of 1× 10escribed. ODM was escribed.. Alizariescribed..RNA was harvested from cells after 7 days in ODM using RNeasy Mini Kit  according to manufacturer’s specifications. Reverse transcription was performed with 1 μg RNA using Taqman Reverse Transcription Reagents . Quantitative real-time PCR was carried out using the Applied Biosystems Prism 7900HT Sequence Detection System and Sybr Green PCR Master Mix (Applied Biosystems) as previously described. SpecifiCultured cells were lysed and protein collected after 7 days differentiation in ODM, separated on polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and assayed with standard immunoblotting technique using the following primary antibodies: anti-phospho-Smad 1/5/8, anti-Smad 5,  as previously described.3 cells per well (n = 3). Cells were grown in standard growth medium. At days 1, 3, 5, and 7 the numbers of viable cells harvested cells following trypsin-EDTA treatment were manually enumerated using Trypan blue stain and a hemocytometer. Additionally, cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation as previously described[Tissue derived cells (P1-4) were seeded in 12-well plates at a density of 5× 10described.Tissue was isolated from the injured mouse specimens, or alternatively cells were isolated from the culture plate. FACS analysis was then performed to interrogate specific subpopulations of cells present in the isolated tissue or from the isolated cells. All collected tissues were mechanically dissociated using sterile scissors. Tissues were digested for 120 minutes in 0.75% Collagenase 2 (Sigma-Aldrich) in Hanks Balanced Salt Solution (HBSS) at 37C under sustained agitation. Samples were filtered using successive 100- and 70-micron sterile strainers and digestion was quenched using equal parts 10% DMEM. Samples were centrifuges at 1,000 rpm for 5 minutes before removing the supernatant and washing in HBSS. This process was repeated in triplicate.® 594). Following incubation, samples were washed three times and filtered through a 45 micron mesh before being run on a FACSAria II (BD Biosciences) Cell Sorter at the University of Michigan Flow Cytometry Core. Greater than 10,000 events were collected for each population. All analyses were carried out in FlowJo.Staining for osteoproliferative cells was performed using previously described markers for osteogenic and proliferative stromal cells in musculoskeletal injury, 36. AntData were analyzed using SPSS software . Means and standard deviations were calculated from numerical data and statistical analysis was performed using an appropriate ANOVA. Equivariance was assessed with Levene’s test and a Welch correction was applied when indicated. Post-hoc analysis was completed with Tukey’s test with equivariant data or Games-Howell if Levene’s test failed. In the figures, the bar graphs represent mean of independent experimental replicates, and error bars represent one standard deviation. For all analyses, significance was defined as a p < 0.05.Nfatc1-cre/caACVR1fl/wt) and human patients. Based on BrDU labeling and manual cell counts, we noted that HO-derived osteoblasts in both settings were significantly more proliferative than their respective normal skeletal osteoblasts , and from the amputation site or contralateral hind limb of rats following blast and polytraumatic extremity injury (rMSCs). Cells derived from the soft tissue of the tenotomy site were significantly more proliferative when compared with those from the contralateral hind limb based on BrDU and manual cell counts . Similarin vitro to differentiate into osteoblasts and produce mineralized matrix. After culture in osteogenic differentiation medium (ODM), mMSCs from the tenotomy site of the burn/tenotomy model had significantly more alkaline phosphatase expression and mineral deposition  when compared with cells derived from the contralateral uninjured hind limb  had a 20.8-fold enrichment (p<0.05) of these osteoproliferative cells when compared with wild type osteoblasts and 43.0-fold enrichment (p<0.05) when compared with normal femur osteoblasts isolated from mutant mice  for CD34+CD29+CD31-CD45-CD11b-, 36 osteant mice .Osteoblasts derived from HO tissue in the transgenic mouse model exhibited increased osteogenic differentiation on the basis of alkaline phosphatase, alizarin red, and gene and protein expression when compared with skeletal osteoblasts . We deteACVR1. Studies have attempted to identify the “putative stem/progenitor cells” responsible for HO, however, the numerous cells identified indicates that differing niches likely plays a role in the cell responsible.[Heterotopic ossification (HO) is a debilitating condition which occurs following trauma and in patients with genetic mutations in the type I bone morphogenetic (BMP) receptor ponsible. ThereforACVR1 is mutated to induce constitutive expression (ACVR1 Q207D) with expression in cells of the Nfatc1-lineage. These mice develop heterotopic bone lesions along tendon and ligamentous structures from birth[in vitro, and increased osteogenic differentiation.Rather than isolate one lineage of cells based on cell surface markers, we have harvested cells from the tissues of two trauma-induced HO models, 20–22, rom birth. Despitein vivo studies. The finding of increased proliferation and osteogenic differentiation also suggests that these cells are particularly aggressive which is consistent with their locally destructive properties observed clinically.In the absence of genetic mutation, human HO-derived osteoblasts exhibit increased proliferation and osteogenic differentiation when compared with normal skeletal osteoblasts. This is similarly true in a transgenic model of HO. This finding suggests that there are intrinsic differences between growing ectopic bone when compared to normal bone, which may be reflective of the aggressive nature of ectopic bone formation. These differences in proliferation may help identify targets for future therapeutic strategies against HO. That HO-derived cells exhibit similar clinicopathologic characteristics further validates their use as a screening tool prior to or in tandem with in vitro setting. These findings are also consistent with those previously reported showing that wounds have osteoproliferative cells with a similar panel to that we have identified (CD34+CD29+CD45-CD31-CD11b-)[in vitro in the absence of injury36. It appears that in both the burn/tenotomy model and the mutant mouse models of HO, an osteoproliferative cell with similar identifying panel exists and may contribute to the overall increased osteogenic differentiation observed in vitro.Furthermore, soft tissue injury portending a high risk of HO development as seen in our burn/tenotomy and blast/polytraumatic extremity injury models yields cells with increased proliferation. Soft tissue-derived cells in these models are derived from two distinct histologic and anatomic locations—in the setting of burn/tenotomy, cells were derived from the Achilles tendon transection site, whereas in the setting of blast/ polytraumatic extremity injury, cells were derived from the primarily muscle components surrounding the amputation stump. Our results demonstrate the cells in both models exhibit higher proliferative capacity than those of the contralateral uninjured hind limb suggests that the trauma site is highly proliferative. This in itself is not surprising; however, we also found that cells from the trauma site of both models were markedly more osteogenic when compared with the contralateral side. The consistency across both models suggests that HO which develops in the setting of tendon injury and muscle injury can be interrogated with similar cellular assays in the 1-CD11b-), 36. AnoOur results suggest that when the cells present from HO producing sites are removed from their environment, they continue to exhibit key factors associated with aggressive bone development as observed in HO including proliferation and osteogenic differentiation. Therefore, ectopic bone formation appears to be mediated, in part, by local factors which can be isolated at the trauma site. Previous studies have identified mesenchymal cells from trauma sites that exhibit increased osteogenic differentiation. It is possible that our soft tissue includes this previously defined population and others with osteogenic potential–40. There.g. myocytes or tenocytes). It is also possible that these cells contribute to osteogenic differentiation either directly, or by eluting cytokines which influence the osteogenic potential of nearby mesenchymal cells. Therefore, use of the aggregate soft tissue may serve as a more representative cell population reflecting the cell types present at the trauma site which develops HO.In addition to mesenchymal cells, the trauma site also includes endothelial cells, inflammatory cells such as T-cells, neutrophils and macrophages, and the native tissue Click here for additional data file.