Manduca sexta depends on reciprocal interactions between olfactory receptor neuron (ORN) axons growing in from the periphery and centrally-derived glial cells. Early-arriving ORN axons induce a subset of glial cells to proliferate and migrate to form an axon-sorting zone, in which later-arriving ORN axons will change their axonal neighbors and change their direction of outgrowth in order to travel with like axons to their target areas in the olfactory  lobe. These newly fasciculated axon bundles will terminate in protoglomeruli, the formation of which induces other glial cells to migrate to surround them. Glial cells do not migrate unless ORN axons are present, axons fail to fasciculate and target correctly without sufficient glial cells, and protoglomeruli are not maintained without a glial surround. We have shown previously that Epidermal Growth Factor receptors and the IgCAMs Neuroglian and Fasciclin II play a role in the ORN responses to glial cells. In the present work, we present evidence for the importance of glial Fibroblast Growth Factor receptors in glial migration, proliferation, and survival in this developing pathway. We also report changes in growth patterns of ORN axons and of the dendrites of olfactory  neurons following blockade of glial FGFR activation that suggest that glial FGFR activation is important in reciprocal communication between neurons and glial cells.Development of the adult olfactory system of the moth  Manduca sexta, several interactions between neurons and glia have been well characterized The past decade has seen a growing appreciation of the importance of neuron-glia signaling in nervous system development, and glial cells have been shown to play numerous roles affecting axon outgrowth or growth arrest, course changes, fasciculation, and targeting M. sexta. The transmembrane form of M. sexta Fasciclin II  and a homolog of vertebrate NCAM) is found on a subset of ORN axons and the GPI-linked form of M. sexta Fasciclin II (GPI-MFas II) is expressed by antennal nerve (AN) glial cells and in the perineurial sheath In previous studies, we identified several molecular signals that could underlie these neuron-glia interactions in the primary olfactory pathway of cis and in trans) between IgCAMs can lead to activation of both EGFRs and FGFRs with subsequent effects on direction and degree of neuron migration and axon extension In this paper, we pursue evidence that suggests roles for the Fibroblast Growth Factor Receptors (FGFRs), which are present on glial cells during critical stages of development Manduca sexta (Lepidoptera: Sphingidae) were reared from eggs on an artificial diet in a laboratory colony essentially as described by Sanes and Hildebrand 2. The cuticle covering the base of one antenna was removed and the underlying part of the antennal anlage removed with forceps. The opening was then filled with melted wax to prevent ORN axons from surviving distal receptor neurons from extending toward the brain, and the animals were returned to the rearing facility and allowed to develop under standard conditions. Because ORN axons do not project contralaterally, the antennal lobe on the operated side received no input from ORNs. The antenna on the opposite (control) side was not disturbed and therefore received normal afferent input.In some animals, the antennal lobe on one side was deprived of ORN axon input throughout development, using surgical methods described previously Manduca sexta proteins were used. Alternatively, antibodies developed against proteins from vertebrate species were used if the antigenic sequence was a close match to the corresponding amino acid sequence of Manduca or of Bombyx mori, which we have found to exhibit very little sequence difference from Manduca.When possible, antibodies developed against Manduca sexta Fasciclin II (MFas II) Mouse monoclonal antibody P1E1-1C3 (“C3”), developed against the extracellular domain common to all isoforms of Bombyx mori in 8 of 11 amino acids . The antigenic phospho-peptide was used for preadsorption assays. We also used an antibody to the extracellular domain of human FGFR1  and an antibody to heparan sulfate  because heparan sulfate proteoglycans are necessary co-receptors for FGF and are expected to colocalize with the FGFR We used a polyclonal antibody to activated human FGFR1 (phosphorylated on tyrosines 653 and 654) which was developed against a phospho-peptide having amino acid identity between human and Bombyx mori and Manduca sexta. The antibody specificity has been checked with blocking peptides and western blots, and the distribution of activated EGFRs in the moth olfactory pathway during development has been described An antibody to activated human EGFR  was chosen based on sequence homology with the corresponding region of EGFR of Manduca antennal lobes, this antibody recognizes a subset of ORN axons that terminate in a single glomerulus located dorso-posteriorly in the antennal lobe A mouse monoclonal antibody generated against a peptide corresponding to the spectrin-binding domain of human Ankyrin B was purchased from Zymed Laboratories . In Bombyx exhibit 100% amino acid identity.An affinity-purified rabbit polyclonal antibody (#H0412) developed against a phospho-peptide corresponding to amino acids 7–20 (pSer10) of human histone H3 was purchased from Sigma, St Louis, MO. Histone-H3 in humans and 2, 10 mM HEPES, 5 mM glucose, pH 7.0, adjusted to 360 mOsm with mannitol; 2O/glycerol. Glial nuclei were identified by their small size compared to neuronal nuclei, and by their location either in the axonal sorting zone region of the antennal nerve where they are the only cell type present Animals at various stages of metamorphic adult development were anesthetized by cooling on ice. Brains were dissected under insect saline solution  or Lycopersicon esculentum lectin (LEL) .Brains that had been fixed on a shaker ON at 4°C in 4% paraformaldehyde plus 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, were sectioned and incubated ON at 4°C in 0.5 ml lectin buffer  containing 2 µl (10 µg) of fluorescein-labeled DrosophilaManduca sexta, and because our Clustal-W amino acid alignments have shown a high degree of identity between Bombyx and known Manduca proteins, we used the sequence for the published Bombyx mori FGFR Bombyx. An amino acid alignment of human FGFR1 and the Bombyx FGFR showed that the Bombyx sequence matches the human sequence at all the contact sites  or DMSO alone (n = 31) was injected into the headspace at various stages of adult development. The injection sites were sealed with melted dental wax and the animals returned to the rearing room to continue development. Early results suggested that higher single doses sometimes produced less effect than lower single doses, possibly indicating that at high doses the drug, which is dissolved in DMSO, precipitated out as it was injected into the aqueous hemolymph. For this reason, and because we were concerned that newly expressed FGFRs might overwhelm single drug injections, two or three 0.5 mg injections spaced 24 hr apart were used rather than a single, larger injection. We found no difference in phenotype between animals receiving 2 vs 3 injections.The highly selective, cell-permeable FGFR inhibitor PD173074 was the generous gift of Pfizer, Inc. Additional drug was purchased from Sigma (#P2499) and from Tocris Bioscience . It has been used to block activation of FGFRs in vertebrates ct sites . Thus thnd day, and all animals were dissected on the fifth day after injection, so that we could observe animals at two doses and at two developmental stages, mid 5 and late5-early 6. Brains were fixed, sectioned at 100 µm, and processed for phospho-histone H3 immunocytochemistry (a marker of cells undergoing mitosis) as described in Four control (DMSO-injected) and six experimental (0.5 mg PD173074 in DMSO) animals at stage 3 of development were divided into 1× and 2× (24 hr apart) injection groups. The 1× animals were injected with the drug or vehicle only on the 2In Situ Apoptosis Detection Kit , which uses the TUNEL  technique. Brains were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 48 hr at 4°C. Fixed brains were washed in PBS, cryoprotected in 10, 20, and 30% sucrose in 0.1 M phosphate buffer, pH 7.4, at 4°C, flash-frozen in liquid propane, and cryosectioned at 20 µm. Sections were then processed according to the apoptosis detection kit instructions, using propidium iodide to counterstain nuclei.PD173074-treated (n = 9) and control (n = 7) animals were assayed for apoptotic cells using the ApopTag Plus Fluorescein Antennal lobes  of three female animals at stage 7 of adult development were removed and solubilized in Novex lithium dodecyl sulfate sample buffer  containing protease-inhibitor and phosphatase-inhibitor cocktails . Solubilized samples were run on a Novex NuPAGE 4–12% Bis-Tris gel and transferred to a PVDF membrane as described previously An additional blot to compare pFGFR labeling of antennal lobes treated with DMSO or PD173074  was done as described above using lobes and attached nerves from two animals for each treatment. Because immunocytochemistry suggested residual labeling in AL neuron cell bodies following PD173074 treatment, these cell body clusters were removed from the tissue prior to processing in order to assess solely the effect on glia.Sections were viewed on a Nikon PCM 2000 or a Zeiss 510 Meta laser scanning confocal system  using Simple 32 software  or LSM software (Zeiss), respectively. Optical sections were acquired at 1- to 5-µm intervals (depending on the objective used) through the depth of the antennal lobe and saved as three-dimensional stacks. To examine the localization of FGFRs, HSPGs, and Syto dyes to cellular sub-compartments, we used a 40×, oil immersion EC PLAN-NEOFLUAR, N.A. 1.3 lens (Zeiss). Vehicle controls were always imaged along with experimental brains and imaging parameters were always held constant when comparing between controls and experimental brains or across developmental stages. Confocal image stacks were projected and merged in false color using Confocal Assistant  or the Zeiss LSM image browser, and then imported into Corel Photopaint, where image hue, intensity, and contrast were adjusted for maximum clarity. The images were then combined into figures in Corel Draw, where annotations were added.M. sexta consists of a small number of cell types: a) ORNs, whose cell bodies are located distally in the antennae and whose axons extend to form the antennal nerves and antennal lobes, b) antennal-lobe neurons, whose cell bodies are clustered  entirely outside of the antennal lobe neuropil and whose dendrites form synapses with each other and with the terminal arborizations of ORN axons in the glomeruli, c) centrally-derived glial cells which populate the sorting zone (SZ glia) and which surround the glomeruli (NP glia), and d) peripheral glia  glia), which migrate toward the antennal lobes along the antennal nerves and extend processes that surround small groups of ORN axons The primary olfactory pathway of Labeling with an antibody to the activated (phosphorylated on tyrosines 653–654) FGFR reveals that that most, if not all, SZ, NP and antennal nerve (AN) glial cells express activated (phosphorylated) FGFRs throughout adult metamorphic development ,3. Cell As the projections shown in DrosophilaBombyx mori and Spodoptera frugiperdaM. sexta, we performed western blots of antennal lobes plus antennal nerves. The anti-pFGFR antibody, when used on blots of tissue treated with a phosphatase-inhibitor cocktail, produced a strong band at 98 kDa  are known in t 98 kDa , in closSorting zone glial cells respond to the arrival of ORN growth cones by proliferating and migrating to form the sorting zone, and NP glial cells respond to formation of protoglomeruli by ORN axons by extending processes and migrating to form a glial envelope around each protoglomerulus. We asked if glial FGFRs might play a role in these events, which take place predominantly between stages 3 and 8 of adult development. We used the FGFR-specific tyrosine kinase inhibitor, PD173074 EGFR , which marks cells undergoing mitosis at the time the brain was removed and fixed. This is expected to produce a smaller number of labeled cells than was found in our previous studies of NP glial cell division using either BrdU To determine the contribution of apoptosis to the reduction in cell number, TUNEL assays were performed on control and treated animals at various stages of development. At stages mid-5 and early-6, we found evidence of considerable apoptosis of NP (but not SZ or AN) glial cells in treated but not in control animals , Table 3Interestingly, we also found no evidence for apoptosis among SZ glial cells at stages 5–6, even though, like the NP glia, SZ glial cells are centrally–derived. Treated animals allowed to develop to stage 12 displayed the sparse population of NP glia seen previously , and manConsidered together, the results described above indicate that blocking of FGFR activation leads both to glial cell apoptosis and to reduction of glial cell proliferation. The effect on cell number is minimal at early stages (5–6), but becomes significant at later stages.EGFR, as shown earlier  migration of mesodermal cells Drosophila embryos, Heartless has been shown to be necessary for migration of longitudinal glia and for their ability to enwrap longitudinal axon tracts Drosophila visual system, Heartless expressed by CNS glia is activated by glial-cell-derived Pyramus and photoreceptor-axon-produced Thisbe to cause proliferation and migration outward along the optic stalk followed by glial differentiation and wrapping of axons in the optic disc The essential nature of FGFR-mediated signaling for cell differentiation, proliferation, survival, migration, and shape has been well documented in vertebrates and invertebrates M. sexta, glial cells of all types express FGFRs, and that these FGFRs are activated throughout metamorphic adult development  and glia in antennal lobes deprived of ORN innervation do not exhibit functional voltage-gated calcium channels in situ eliminates calcium transients in response to carbamylcholine, an acetylcholine receptor agonist in situ display no calcium influx in response to 200 µM carbamylcholine at stage m5, show maximum influx at stage 6, at the height of glial migration, and then display an influx that declines to about half maximum by stage 9, indicating a strong temporal correlation between acetylcholine-induced glial calcium influx and glial cell migration to surround protoglomeruli As background for assessing the connection between FGFR activation and NP glial cell migration, we know the following: 1) NP glial cells migrate only if a sufficient number of ORN axons have arrived at the antennal lobe Manduca system, similar results have been reported in Drosophila antennal nerves in which glial cells express a dominant-negative form of Heartless In contrast to the effect on NP glial cells, pharmacologic blockade of FGFR activation did not prevent the migration of SZ or AN glial cells , 7B. BloM. sexta, when PD173074-treated animals were allowed to develop to stages later than stage 7, examination of the olfactory pathway revealed an extensive loss of NP, SZ and AN glial cells  is expressed by ORN axons and Neuroglian is expressed by ORN axons and central glia. The GPI-linked isoform of MFas, GPI-Fas II, previously was detected in what appeared to be a subset of AN glia Drosophila embryos, in which glial migration along axons is regulated by cell-surface expression of neuronal TM-Fas II FGFRs and EGFRs in both vertebrates and in Manduca sexta. Our results indicate that these FGFRs are not required for ORN axon targeting, but are essential for 1) the proliferation and survival of glial cells, 2) the ORN-induced migration of NP glial cells to surround glomeruli, 3) the glia-regulated establishment of normal dendritic territory in the glomeruli, and 4) the glia-induced organization of ORN axon fasciculation through the sorting zone. Despite these important effects, ORN axons do manage to target at least one identified glomerulus correctly when FGFR activation is blocked, suggesting a robust targeting system with some degree of redundancy.In summary, we have found evidence for activated FGFRs on central and peripheral glia of the primary olfactory pathway of Figure S1The ATP and PD173074 binding pocket of Bombyx FGFR contains all of the amino acid residues shown to contact PD173074 in the human FGFR1. The relevant regions of the highly conserved tyrosine kinase domains of human and Bombyx FGFRs were aligned using the Clustal-W amino acid alignment program at: http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html(DOC)Click here for additional data file.Figure S2Preadsorption of the anti-pFGFR antibody with its antigenic phospho-peptide eliminates glial labeling.A: Stage-6 brains were labeled with the anti-pFGFR antibody (magenta) using the standard protocol. Syto 13 (green) was used to label nuclei. B: The anti-pFGFR antibody was preadsorbed with the antigenic phospho-peptide. Glial labeling was largely eliminated, as was most of the AL neuron labeling. C: When the anti-pFGFR antibody was omitted from the protocol (No Primary Control), weak labeling of the AL neurons was visible, suggesting that the residual labeling in B was partly due to non-specific labeling by the secondary antibodies. LG, MG = lateral and medial group of AL neuron cell bodies. A′–C′: pFGFR channel alone. Projection depths were 15 µm.(TIF)Click here for additional data file.Figure S3Labeling for FGFRs and HSPG matches that for pFGFRs.A: An antibody to the extracellular domain of human FGFR1 labels glial cells and AL neuron cell bodies as was seen for pFGFRs. A′: An enlarged and deeper region of A reveals labeled glial processes (arrowheads) and colocalization of labels for FGFRs and DNA (arrow). B: An antibody to heparan sulfate proteoglycans, necessary components of the FGF-FGFR-HSPG complexes required for ligand-mediated FGFR activation, labels glial cells and AL neuron cell bodies, but not AL neuron dendrites or ORN axons. Arrowheads point to glial processes surrounding glomeruli (*). C–C″: A higher magnification image of NP glia surrounding a single stage-7 glomerulus. Glial processes were labeled  as was seen for FGFRs (A′). C″: Merged HSPG and Syto 13 images demonstrates colocalization in the nuclei, as was seen for pFGFRs (S3A′). D–D″: AN glia also display labeling of processes (arrowheads) and nuclei. LG, MG = lateral and medial group of AL neuron cell bodies. Projection depths = 5 µm in A, A′. Images were single optical sections in B–D″. 40× objective in C–D″.(TIF)Click here for additional data file.Figure S4Apoptag channel version of.A–F: Control and PD173074-treated animals were allowed to develop to various stages, then dissected and analyzed for apoptotic nuclei using the TUNEL technique. Numerous apoptotic nuclei were seen in treated animals , but few to no apoptotic nuclei were seen in control animals  at all stages examined. Arrowheads in panels B,D indicate apoptotic nuclei among the medial group of antennal lobe neurons. Inset in panel F shows a higher magnification view of a region within the sorting zone of the antennal nerve. Projection depths = 10 µm.(TIF)Click here for additional data file.Figure S5ORNs exhibit no evidence for FGFRs.A: Antennal nerves of untreated stage 7 females were labeled with the pFGFR antibody (magenta). Dark spaces between glia are filled with ORN axons. B: Antennae from the same animals were sectioned in longitudinal section and labeled with the pFGFR antibody. Counterstains (green) were Syto 13 in panel A to show nuclei and LEL-fitc in B to delineate ORN cell bodies and sensilla. Using the collection parameters from panel A we found no labeling of ORN cell bodies or sensillar processes . C: Antennal nerves of untreated stage 7 females were labeled with both the pFGFR  and Fasciclin  antibodies. Syto 13 labeling of nuclei (green) serves to align the images in panels C,C′. We found no evidence of pFGFR labeling in ORN axons. Projection depths were 15 µm in A, 3 µm in C. Image in B was a single optical section (40× objective).(TIF)Click here for additional data file.Figure S6The M. sexta Eph receptor is unlikely to be affected by PD173074. Alignments of a section of the tyrosine kinase domains of the human FGFR1 and the M. sexta Eph receptor shows that many of the amino acids required for binding PD173074 (yellow highlighting) are different in the latter case (gray highlighting), suggesting that, as for vertebrates, PD173074 would not affect M. sexta Eph receptors. Note, too, the large gaps needed to achieve the alignment (compare to (DOC)Click here for additional data file.


Schwann cell (SC) transplantation is currently being discussed as a strategy that may promote functional recovery in patients with multiple sclerosis (MS) and other inflammatory demyelinating diseases of the central nervous system (CNS). However this assumes they will not only survive but also remyelinate demyelinated axons in the chronically inflamed CNS. To address this question we investigated the fate of transplanted SCs in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in the Dark Agouti rat; an animal model that reproduces the complex inflammatory demyelinating immunopathology of MS. We now report that SCs expressing green fluorescent protein (GFP-SCs) allografted after disease onset not only survive but also migrate to remyelinate lesions in the inflamed CNS. GFP-SCs were detected more frequently in the parenchyma after direct injection into the spinal cord, than via intra-thecal delivery into the cerebrospinal fluid. In both cases the transplanted cells intermingled with astrocytes in demyelinated lesions, aligned with axons and by twenty one days post transplantation had formed Pzero protein immunoreactive internodes. Strikingly, GFP-SCs transplantation was associated with marked decrease in clinical disease severity in terms of mortality; all GFP-SCs transplanted animals survived whilst 80% of controls died within 40 days of disease. The development of clinical deficits in multiple sclerosis (MS) is due to repeated episodes of inflammatory demyelination that result in axonal loss and formation of persistently demyelinated plaques of gliotic scar tissue. Enhancing remyelination by transplanting myelin forming cells into the CNS is predicted to provide significant clinical benefits, but as yet there is no consensus as to which cells to use, or the best route of delivery in vitro expansion However, Schwann cells (SCs), the myelinating glia of the peripheral nervous system (PNS) exhibit a range of properties that make them attractive candidates for cell therapy in MShttp://www.myelin.org/0623003.htm).Recent data indicate that exogenous PNS stem/precursor cells are of greater interest for myelin repair than committed SCs due to their greater ability to interface with astrocytes and migrate within the CNS parenchyma We therefore investigated the behaviour and survival of SCs transplanted into the CNS of Dark Agouti (DA) rats with myelin oligodendrocyte glycoprotein (MOG)-induced experimental allergic encephalitis (EAE). This animal model of MS reproduces the histopathology and clinical course of the human disease. In particular animals develop inflammatory demyelinating lesions in the spinal cord that ultimately form confluent plaques of chronically demyelinated gliotic scar tissue, the pathological hallmark of MS In order to trace transplanted SC, we transduced DA rat SCs with a lentiviral vector to induce cytoplasmic expression of GFP .The effect of inflammatory demyelination on SC migration and differentiation was studied by allografting GFP-SCs into the CNS of DA rats with MOG-induced chronic EAE Irrespective of the route of delivery GFP-SCs populated areas of both grey  and whitIn DA rats with MOG-EAE, inflammatory demyelinating lesions develop throughout the CNS including cerebellum and spinal cord + astrocytes in demyelinated lesions both at 7  was compared to that in a group of sham operated controls (n = 5) animals and non-operated animals (n = 9) for 45 days following the first clinical signs of disease. No significant differences were observed in the first 25 days after disease onset, but from day 31 on, the SCs injected group began to show significant improvements relative to the controls . Furtherper se, or an direct or indirect immunomodulatory effect. These results demonstrate that SCs transplantation has a beneficial clinical effect in this model of MS, but also that inflammatory demyelination generates an environment favorable to SCs survival, migration and differentiation.A recent phase I clinical trial of autologous SCs transplantation in MS was discontinued after brain biopsies performed five months after transplantation failed to provide evidence for SCs survival. This is in stark contrast to the situation in experimental models of toxin-mediated demyelination in which the transplanted SCs not only survive but actively participate in lesion repair in the CNS including in conditions of autologous transplantation in large primates. To better understand the behavior of SCs in the inflammatory demyelinating environment of the MS lesion we chose to follow the fate of allogenic SCs transplanted into DA rats with MOG-induced EAE, a chronic relapsing disease model that reproduces many aspects of the complex clinical course and pathology of MS in vitro and in vivoSeveral elements indicate that exogenous SCs behaved differently after transplantation in EAE compared to non-inflammatory models of demyelination. We show that in diffuse EAE, SCs are found along meninges, and associated with perivascular spaces . In addiWe also demonstrated that recruited exogenous SCs differentiated in myelin competent cells as confirmed by their progressive polarization along CNS axons and the detection of Pzero-positive myelin-like internodes. Robust remyelination of central axons by the grafted SCs occurred in the majority of animals, indicating that inflammation does not appear to prevent SCs differentiation. However, myelin repair by exogenous SCs was more frequent after intra-parenchymal than intra-thecal delivery. This could be explained by the fact that transgression of the pia mater may delay their intra-CNS migration and recruitment by the lesion, and consequently decrease their involvement in myelin repair. Thus such as in toxin-induced model of demyelination, the myelination efficiency of exogenous SCs is highly depending upon the rapidity by which the grafted cells arrive at the lesion-site and compete with endogenous cells + oligodendrocytes within the developing lesion clearly provides SCs an opportunity to myelinate axons in the absence of competition from endogenous oligodendrocytes. Whilst remodeling of the lesion environment may reduce expression of factors known to inhibit SCs migration such as Semaphorin 3A (Sema 3A) How inflammatory demyelination supports the survival, recruitment and differentiation of exogenous SCs in this EAE model remains an open question. Immune mediated loss of MOGIn the current study we noticed that clinical amelioration of disease activity was only observed approximately 31 days after disease onset. It was not possible to correlate this effect with the extent of immunoreactivity for P0 protein in these animals suggesting that the reduction in clinical deficits compared to controls is not simply due to remyelination. This indicates that clinical recovery is mediated by other mechanisms. SCs are known to secrete a variety of neurotrophins including nerve growth factor (NGF), neurotrophine-3 (NT3), brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) SCs based therapy is a prominent field of investigation for trauma and demyelinating diseases such as MS. Our findings show that SCs behavior in inflammatory conditions sheds the light on novel SCs function, providing anatomical and clinical recovery in a rodent model of MS without being the target of recurrent immune-mediated demyelination, which characterizes MS pathology.Eight week-old DA female rats (Janvier) were housed in the Pitié-Salpêtrière EOPS animal facility. All animal protocols were performed in accordance with the guidelines published in the National Institute of Health Guide for the Care and Use of Laboratory Animals. The animal studies described in this work were performed under and approved by the French Agricultural Ministry-Animal Welfare license numbers B75-13-08 and A75–585. Chronic demyelination was achieved by injecting sub-cutaneously 100 µl of a solution of MOG (50 µg) in Complete Freund's adjuvant containing Mycobacterium tuberculosis (H37RA) (225 µg) in the rear footpads and flanks (25 µl/site). Clinical scores were evaluated daily using the following scale: 0 =  healthy, 1 =  tail paralysis, 2 =  ataxia or paresis of hind limbs, 3 =  paralysis of hind limbs and/or paresis of forelimbs, 4 =  tetra-paralysis, 5 =  moribund or death. Mann –Whitney rank test was used to test statistical significance between the different groups.www.abdserotec.com) and myelin basic protein  as described previously . Diaminobenzidine (www.sigma.de) was used as chromogenic substrate. Sections were counterstained with hematoxylin, dehydrated and mounted with DePeX (www.serva.de). For immunohistochemical stainings, microwave pre-treatment in 10 mM citric acid buffer, pH 6.0, was performed.Rats were transcardially perfused with 4% PFA, brains and spinal cords dissected, and embedded into paraffin. Two micron-thick sections were cut and deparaffinized. To assess inflammation, demyelination luxol fast blue staining (LFB), was performed. In addition, immunohistochemistry was performed with antibodies against CD68  were transduced with 12 ng p24 of HIV-CMV-GFP, which allowed strong and stable cytoplasmic green fluorescent protein (GFP) expression in SCs (GFP-SCs). Expression of GFP was detected 48–72 h post transduction and was stable over time SCs were purified from adult DA rat sciatic nerves as described previously for adult primate SCs 5cells/µl) were injected. For intra-parenchymal spinal cord delivery, 2 µl of SCs suspension (5.104 cells/µl) or 2 µl of SCs medium were injected in the dorsal spinal cord white matter funiculus. To evaluate the effect of GFP-SCs injection on the evolution of clinical scores, EAE animals were distributed homogeneously according to their clinical score  into 2 groups  before delivery of GFP-SCs or the same volume of medium for controls. Non operated animals induced with EAE were also followed in parallel to the two operated groups.DA rats were anesthetized with ketamine  and xylazine , 2–4 days after the first clinical signs (around 10 days). Allogous GFP-SCs were delivered intra-thecally via the cisterna magna or in the spinal cord parenchyma. For intra-thecal delivery, anesthetized animals were placed in a stereotaxic frame equipped with a Hamilton syringe allowing a direct puncture of the cisterna magna, where 3 µl of SCs suspension  and intra-cardially perfused with 2% paraformaldehyde (PFA). The brain and the spinal cord were post-fixed 1 hour in the same fixative, cryo-protected overnight with 20% sucrose and frozen in isopentane cooled by liquid N2. Immunohistochemistry was performed using MOG antibody to detect the area of demyelination, GFAP  to identify the astrocytes, P0  to identify peripheral myelin and laminin  to localize blood vessels basement membrane. Species-specific secondary fluorescent conjugated antibodies were applied for 1 h in addition to Hoechst 33342 . Coverslips were mounted with fluoromount and analyzed under fluorescent DMC Leica microscope  using Explora Nova Image acquisition software .Figure S1Illustration of GFP SCs migration 7 and 21 days after their delivery in the spinal cord. While at 7 days grafted GFP SCs are detected in the vicinity of the graft (A), 21 days later they spread over a distance of 1.2 mm (B).(TIF)Click here for additional data file.


Increasing preclinical and clinical evidence underscores the strong and rapid antidepressant properties of the glutamate-modulating NMDA receptor antagonist ketamine. Targeting the glutamatergic system might thus provide a novel molecular strategy for antidepressant treatment. Since glutamate is the most abundant and major excitatory neurotransmitter in the brain, pathophysiological changes in glutamatergic signaling are likely to affect neurobehavioral plasticity, information processing and large-scale changes in functional brain connectivity underlying certain symptoms of major depressive disorder. Using resting state functional magnetic resonance imaging (rsfMRI), the „dorsal nexus “(DN) was recently identified as a bilateral dorsal medial prefrontal cortex region showing dramatically increased depression-associated functional connectivity with large portions of a cognitive control network (CCN), the default mode network (DMN), and a rostral affective network (AN). Hence, Sheline and colleagues (2010) proposed that reducing increased connectivity of the DN might play a critical role in reducing depression symptomatology and thus represent a potential therapy target for affective disorders. Here, using a randomized, placebo-controlled, double-blind, crossover rsfMRI challenge in healthy subjects we demonstrate that ketamine decreases functional connectivity of the DMN to the DN and to the pregenual anterior cingulate (PACC) and medioprefrontal cortex (MPFC) via its representative hub, the posterior cingulate cortex (PCC). These findings in healthy subjects may serve as a model to elucidate potential biomechanisms that are addressed by successful treatment of major depression. This notion is further supported by the temporal overlap of our observation of subacute functional network modulation after 24 hours with the peak of efficacy following an intravenous ketamine administration in treatment-resistant depression. Based on the increasing evidence of glutamate-modulating agents having strong and rapid antidepressant properties In general, we hypothesize that the therapeutic potential of ketamine may be explained by reversing disturbances in the glutamatergic system Multimodal imaging studies combining MR spectroscopy and fMRI raised the interest for the investigation of the neurochemical basis of blood oxygen level-dependent (BOLD) signal fluctuations during activity Recent advances in resting state functional connectivity neuroimaging techniques suggest their utility for the investigation of (1) intrinsic brain connections in the healthy human brain, (2) pathophysiological alterations in disease states and (3) changes in neuronal network dynamics following therapeutic interventions Little is known about how ketamine affects large-scale neural network dynamics in the healthy human brain and whether it has the potential to restore the aberrant functional connectivities seen in MDD. Based on previous findings with other antidepressants, we hypothesized that ketamine at subanaesthetic doses will decrease the cortico-limbic resting state connectivity in healthy subjects as a general response pattern. In addition, we aimed to evaluate the pharmacological effects of ketamine on resting state connectivities via the DN The study was approved by the University of Zurich institutional review board, and all subjects gave written informed consent before screening.Healthy subjects  without any psychiatric, neurological, or medical illness were self-referred from online study advertisements. All subjects underwent a psychiatric interview and medical examination. Only medication-free subjects that were healthy according to physical examination, electrocardiogram, blood and urine analyses were included in the study. Exclusion criteria were a history of psychiatric/neurological diseases, drug abuse, concurrent medication, cardiovascular disease, anaemia, thyroid disease, any somatic disease affecting drug metabolism and excretion , MR exclusion criteria, pregnancy, and left handedness.17 out of 19 subjects  completed a total of four rsfMRI sessions in a double-blind, randomized, crossover study design . The ordPsychotomimetic side effects during ketamine infusion were assessed post hoc using the Altered States of Consciousness rating scale ‘5D-ASC’ Measurements were performed on a Philips Achieva TX 3-T whole-body MR unit equipped with an 8-channel head array. The subjects were told to lie still in the scanner with their eyes closed during the acquisition of resting state data. The functional images were collected in 10 min runs (200 volumes) using a sensitivity-encoded single-shot echo-planar sequence  sensitive to BOLD contrast (T2* weighting). Using a midsagittal scout image, 32 contiguous axial slices were placed along the anterior-posterior commissure plane covering the entire brain and acquired with a repetition time of 3000 ms (θ = 82°) in ascending slice order. A 3-dimensional T1-weighted anatomical scan was obtained for structural reference.Data were analyzed using the SPM8  based data processing assistant for resting state fMRI  across whole brain (Gaussian filter width (FWHM) computed from the estimation of spatial smoothness of the residuals using AFNI (http://afni.nimh.nih.gov/afni): sigmax = 2.70, FWHMx = 6.36; sigmay = 2.70, FWHMy = 6.35; sigmaz = 2.78, FWHMz = 6.54; cluster connection radius (rmm) = 5.2 mm, individual voxel threshold probability = 0.001, 1000 iterations). Using the DPARSF toolbox, functional time series were extracted within each of the seed regions and in the target ROI of the dorsal nexus and in the MPFC/PACC voxel cluster, which was determined from the paired t-test significance maps. To quantify changes in functional connectivity of the seed regions to the target voxel clusters, correlation coefficients between the extracted functional time series were computed in Matlab R2009b  for each subject and every session separately. Paired t-tests were applied to compare differences in Fisher z-transformed correlation values between baseline and follow-up sessions. Graphs of the mean changes in z-transformed correlation values were created using SigmaPlot (Systat Software Inc.). Whole brain paired t significance images were thresholded in SPM8 and visualized with the BrainNet Viewer (http://www.nitrc.org/projects/bnv/).Using the DPARSF toolbox, whole brain functional connectivity (FC) maps were obtained from the a priori determined seed regions of interest for each subject and every session separately. Statistical tests on regional functional connectivity maps were computed after application of Fisher's r-to-z transform, which yields variates that are approximately normally distributed. Paired t-test significance maps were computed in SPM8, based on the individual FC maps of the sessions baseline (ketamine) vs. follow-up (ketamine) and baseline (placebo) vs. follow-up (placebo). For the three seed-based comparisons, the correction of the t contrasts was made with a voxelwise threshold of p<0.001 and 15 voxels, achieving a corrected cluster threshold of p<0.05, as determined by the Monte Carlo simulations via AlphaSim , the pregenual anterior cingulate (PACC) and the medioprefrontal cortex (MPFC) following ketamine administration  following ketamine administration compared to placebo  following ketamine administration. Likewise, no significant differences in functional connectivity between amygdala and prefrontal cortical areas could be observed after drug administration. Additional reductions in functional connectivity were found between amygdala and posterior parietal and premotor areas but the pattern was similar for both the drug and placebo condition.No focal changes in functional connectivity with DLPFC were found in the whole brain paired t-test thresholded at p3) based on the overlapping voxels that showed significant changes in functional connectivity to both the posterior and subgenual cingulate cortices after ketamine administration. At the whole brain level (using the same threshold as above), functional connectivity was reduced exclusively to the PCC but nowhere else in the brain.In order to assess the specificity of our findings, we created an additional „dorsal nexus “seed  connectivity via the DN and PACC/MPFC one day after infusion. We could not find any focal change in connectivity to the CCN following ketamine administration and contrary to resting state studies with serotonergic and noradrenergic antidepressants including citalopram and reboxetine In conclusion, we report a reduction of functional connectivity in networks that play a critical role in the pathophysiology of MDD in healthy subjects 24 hours after receiving an antidepressant dose of ketamine. Based on those findings we raise the hypothesis that reducing functional connectivity of the dorsal nexus reflects underlying molecular mechanisms relevant to the antidepressant efficacy of ketamine. Whether this circuit-level glutamatergic effect is likely to be associated with reversing aspects of emotional and behavioral dysregulation has to be further investigated in a clinical study involving MDD patients. This is in further support of the notion of using ketamine as a research tool into the neurobiology of mood disorders and to delineate potential biomarkers and action mechanisms of antidepressant treatment response.


Gemella morbillorum that may result from impaired macrophage function. Finally, we provide evidence that the increased capacity of opportunistic pathogens (e.g. E. coli) to colonize the microbiome is associated with reduced production of antimicrobial peptides.A majority of individuals infected with human immunodeficiency virus (HIV) have inadequate access to antiretroviral therapy and ultimately develop debilitating oral infections that often correlate with disease progression. Due to the impracticalities of conducting host-microbe systems-based studies in HIV infected patients, we have evaluated the potential of simian immunodeficiency virus (SIV) infected rhesus macaques to serve as a non-human primate model for oral manifestations of HIV disease. We present the first description of the rhesus macaque oral microbiota and show that a mixture of human commensal bacteria and “macaque versions” of human commensals colonize the tongue dorsum and dental plaque. Our findings indicate that SIV infection results in chronic activation of antiviral and inflammatory responses in the tongue mucosa that may collectively lead to repression of epithelial development and impact the microbiome. In addition, we show that dysbiosis of the lingual microbiome in SIV infection is characterized by outgrowth of  A majority of HIV-infected individuals world-wide do not have adequate access to antiretroviral therapy and ultimately experience opportunistic oral infections Although studies of oral microbial dynamics can be readily accomplished in HIV patients through non-invasive sampling in vivo gene expression in the tongue epithelium of SIV infected animals with in vitro expression profiles of oral epithelial cells treated with IFNγ provide evidence that chronically activated IFNγ signaling may contribute to dysbiosis by impairing antimicrobial peptide production and compromising barrier development. In addition, we report a potential link between down-modulation of genes that mediate macrophage functions and a dramatic outgrowth of Gemella. morbillorum on the tongue dorsum during SIV infection.In the current study, we seek to initiate development of the rhesus macaque model of oral pathogenesis in SIV infection. We provide the first characterization of the rhesus macaque oral microbiome, and utilize high throughput gene expression profiling to evaluate mechanistic relationships between SIV-induced oral pathogenesis, host immune status, and modulation of the lingual (tongue) microbiota. Our findings indicate that untreated SIV infection results in constitutive induction of antiviral and inflammatory responses in the lingual mucosa, the latter of which is characterized by dysregulated cytokine production that may exclusively promote interferon gamma (IFNγ) driven responses. Comparisons of The study was carried out 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 Institutional Animal Care and Use Committee (IACUC) of the University California, Davis (Protocol #16648). All efforts were made to minimize suffering.A total of 16 male adult rhesus macaques from the California National Primate Research Center (CNPRC) were utilized for the study. Animals were housed in accordance with American Association for Accreditation of Laboratory Animal Care standards, and experiments were designed to adhere to the “Guide for the Care and Use of Laboratory Animals” of the Institute of Laboratory Resources, National Resource Council. Animals used in SIV experiments were housed indoors, carefully paired to facilitate daily social contact, and provided multiple environmental enrichments to promote psychological well-being. Indoor-living animals were fed a commercial monkey chow  supplemented with seasonal fruits and vegetables. Animals used for tongue swab samples and dental plaque scrapings were from naturally formed family groups living outdoors in an enclosed free-range setting with natural vegetation. Outdoor-living animals were fed commercial monkey chow supplemented with fruits and vegetables, occasional peanuts and sunflower seeds, and grass clippings, small branches, insects, and soil that are consumed during their normal daily activities.SIV infected animals were observed on a daily basis by veterinary technicians, and CNPRC veterinarians using established protocols performed all procedures. All possible efforts were made to minimize animal pain and discomfort. Ketamine was given intramuscularly (10 mg/kg) when it was necessary for animals to be immobilized. Consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association, animals in SIV studies were euthanized by overdose of sodium pentobarbital (60 mg/Kg I.V.).TCID50 of SIVmac251 as previously described 16S phylogenetic analyses were performed on lingual epithelial brushings (n = 3) and dental plaque scrapings (n = 5) collected from free-range living animals, and archived lingual tissues collected during previous SIV studies . Archived lingual tissues were also utilized for gene expression analyses and immunohistochemistry. SIV infected animals were inoculated intravenously with 100DNA was extracted from dorsal tongue epithelial brushings, plaque scrapings, and necropsied tongue tissue samples with the Epicentre® MasterPure Complete DNA and RNA Purification Kit (Epicentre) according to previously published protocols Analysis of 16S rRNA encoding sequences was performed as previously described TR146 oral epithelial cells were a generous gift from Cancer Research UK p-value ≤0.05) was used as cut off criteria for generating DEG lists for whole tissue analysis and +/− 1.2 for analysis of epithelial cells isolated by laser capture microdissection. DEG lists were hierarchically clustered and sub-clusters were subjected to pathway analysis using Ingenuity Pathway Analysis (IPA©) and DAVID (http://apps1.niaid.nih.gov/david) web-interfaced software. Microarray data files are deposited at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the following accession number: (GSE51445).Total RNA was extracted from lingual whole tissue samples and TR146 cells utilizing protocols and reagents in the Qiagen RNeasy© RNA isolation kit. Messenger RNA amplification, labeling, hybridization to Rhesus macaque-specific GeneChips© (Affymetrix) staining and scanning were performed as previously described 3 oral epithelial cells from each section were microdissected using an Arcturus XT© LCM system (Arcturus) and total RNA was immediately extracted using a PicoPure© RNA extraction kit (Arcturus) and utilized for transcriptional profiling by microarray.Oral epithelial cells were isolated from tongue tissues of SIV infected and uninfected control animals by laser capture microdissection (LCM). Prior to LCM, fresh frozen tissues embedded in OCT were cut into 5 µm serial sections and briefly stained with hematoxylin and eosin. Approximately 5×10T values was performed based on expression of GAPDH and differential gene expression was determined using previously published protocols T values from SIV infected groups and healthy uninfected controls. Statistical analyses (Student's t-test) were used to determine if differences in the mean values for uninfected and infected groups were significant .Real-time-PCR (RT-PCR) analyses were used to validate microarray findings by comparing mRNA levels of tetherin (BST2), myxovirus protein-1 (MX1), signal transducer and activator of transcription-1 (STAT1), interferon gamma (IFNγ), interferon alpha (IFNα), tumor necrosis factor alpha (TNFα), and interleukin-1 beta (IL1β). RT-PCR was carried out on a Viia 7 Real-Time PCR system  and data was analyzed with the Viia 7 software package. Internal normalization of CMonoclonal antibodies to human psoriasin (Santa Cruz Biotechnology) were utilized to identify and localize macaque psoriasin by fluorescent immunohistochemistry in 5 µm lingual tissue sections from SIV infected macaques and uninfected controls utilizing previously published protocols T values were extrapolated against the standard curve to quantitate viral RNA copies/ml of plasma.SIV RNA copies were detected by quantitative RT-PCR in the blood of infected macaques utilizing previously published protocols and target probes designed to hybridize with the gag region of SIVmac251 CD4+ and CD8+ T cell percentages in the peripheral blood were determined by flow cytometry at the indicated time points. Briefly, Ficol-gradient isolated PBMC were stained with fluorochrome labeled monoclonal antibodies to CD3, CD4, and CD8 (Santa Cruz Biotechnology). Negative controls were stained with isotype control monoclonal antibodies. Fluorescence was measured on a LSRII flow cytometer (Becton Dickinson) and FlowJo (Treestar) was utilized to analyze the data. Student's t-tests were utilized to determine if differences in the mean percentage of T cells in each subset were significant  between SIV infected animals and controls.In implementing a systems biology approach to investigate the relationship between SIV associated oral pathogenesis and host-microbe dysbiosis, we first sought to characterize the types of bacterial species that colonize the rhesus macaque oral microbiome. Lingual mucosal (nylon brush swabs or homogenized tissue) samples and dental plaque scrapings were collected from 11 healthy adult male macaques housed at the California National Primate Research Center (CNPRC). Clonal analysis of 16S rRNA genes revealed that the rhesus macaque oral microbiome harbors many of the same bacterial species that colonize the human oral cavity as well as numerous novel species that appear to be “simian counterparts” of human oral bacteria . ConsistStreptococcus was one of the predominant commensal genera colonizing the lingual epithelium of healthy uninfected macaques, comprising from 15 to 83% of the total community in the animals that were tested  was either the most or the second most abundant genera detected in all but one animal, comprising from 8% to 40% of the total bacterial population. All of the healthy macaques tested also harbored human-colonizing Granulicatella adiacens as 1 to 10% of the total lingual bacterial community. In addition to three core genera, Streptococcus, Gemella, and Granulicatella, 50% of healthy macaques (3 of 6) showed colonization of Lachnospiraceae. Abiotrophia, Haemophilus, and Klebsiella were also detected in 33% (2 of 6) of the samples. Notably, each of the 6 genera identified on the tongue dorsum of at least 2 animals are also found as commensals on the human tongue.As reported in humans e tested . Human-cStreptococcus and Lachnospiraceae, with substantial but comparatively less proportions of Gemella and Abiotrophia  at necropsy . In addition, Gemella spp., consisting primarily of G. morbillorum (human-colonizing), appeared to obtain a significant growth advantage over other all other commensal species  Bacillus cohnii , and a Haemophilus aegyptius-like bacterium . These data indicate that, similar to HIV infected humans In addition to modulations in the proportion of core commensal bacteria, several potentially pathogenic species were detected on the tongue of SIV infected animals that were not found in any controls, including ©) software revealed a statistically significant over-representation of transcripts associated with antiviral response, viral replication, epithelial cell death, epithelial hyperplasia, interferon response, keratinocyte differentiation, and lymphocyte quantity  indicated a total of 905 genes were up-regulated in the SIV infected group and 1329 down-regulated . Biofuncquantity .Closer examination revealed that the antiviral responses  in the tIn light of the increased antiviral expression profile, it was also notable that the lists of up-regulated genes involved in epithelial cell death , keratinThe pathogenic implications of the up-regulated transcriptional profile in the lingual mucosa of SIV infected animals were also supported by functional analysis of genes that were down-regulated. Genes associated with dendritic cell quantity, macrophage function, overall immune system function, and several aspects of epithelial maintenance and regeneration  were all transcriptionally repressed in the tongue . We alsoG. morbillorum on the tongue. Previous reports from bone marrow transplantation studies in mice indicate that macrophages may play a primary role in controlling G. morbillorum colonization Gemella colonization on the tongue of all SIV infected animals and uninfected controls, including, IL-1B ICAM1, CCL2, and CCR6  assay . The dirp-values for the genes assigned to each functional category is presented in Evidence of an association between dysbiosis of the lingual microbiome and alterations in transcriptional programming of epithelial cells in SIV infected animals prompted us to evaluate the role of epithelial deterioration in greater detail. Utilizing laser capture microdissection (LCM), epithelial cells were isolated from lingual tissue sections of a subset of SIV infected animals and healthy controls  and compin vitro transcriptional changes in TR146 cells were then directly compared to the transcriptional profiles of the LCM isolated tongue epithelium to identify in vivo changes in SIV infected macaques that were likely to be driven by chronic induction of IFNγ signaling. This cross comparison revealed that several important factors involved oral epithelial development were similarly repressed in both in vitro and in vivo analyses including ets homologous factor, sciellin, amphiregulin, salvador homolog 1, POU class 3 homeobox 1, runt-related transcription factor 2, and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog , genes encoding antimicrobial peptides  were, in contrast, down-regulated . Furthercontrols . It is a provide support for this conclusion, and further suggest that macrophages may be uniquely responsible for regulating G. morbillorum colonization p-value did not reach our criteria for significance (p = 0.21), it is notable that this transcript was down-regulated approximately 3-fold on average in the tongue mucosa of SIV infected animals, consistent with the down-regulation of other monocyte/macrophage lineage biomarkers . These include down-regulation of genes associated with tight junction integrity Click here for additional data file.


Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.Catalytically defective rare variants of  Siae exhibited enhanced B cell receptor (BCR) signaling, B cell differentiation defects that are consistent with enhanced BCR signaling, circulating anti-chromatin antibodies and immune complex deposits in the kidney Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the 9-OH position of sialic acid, and thus permits α2,6 linked sialic acid on N-glycans on B cell glycoproteins to interact with CD22/Siglec-2, a sialic acid binding lectin that can inhibit B cell antigen receptor signaling siae mutant mice had suggested that this gene functions in a B lymphocyte intrinsic manner We showed in overexpression studies that SIAE can be secreted and decorate the surface of over-expressing transfected non-lymphoid cells presumably by binding to some extracellular component but the physiological relevance of secretion and cell surface expression of this protein was not critically evaluated by in vivo studies SIAE were shown by us to be linked to autoimmune disorders Catalytically defective heterozygous rare variants of SIAE, c.265 A>G (M89V) SIAE is seen in heterozygous form in approximately 10% of control subjects . The examination of this unannotated data indicates that about 0.3% of Caucasians are homozygous for M89V SIAE. Examination of larger sets of subjects by genotyping has revealed that homozygosity of M89V SIAE is frequently observed in controls SIAE was not linked to autoimmunity. Our re-analysis of the data from that study on an allele by allele basis (presented below) indicates that catalytically defective SIAE alleles are indeed enriched in subjects with disease. We also examined the catalytic activity of all common variants of SIAE to determine if any common variant should be considered in terms of disease susceptibility.The most detailed publicly available catalogue of exome sequencing , is the NHLBI Exome Variant Server . The K71R and A467V SIAE variants were re-created on a human SIAE cDNA in a pcDNA3.1 based mammalian expression construct. M89V and the two rare murine Siae variants, C196F and Q335P  were re-created in a murine Siae cDNA. Primers (5′ to 3′) for site-directed mutagenesis were designed based on ENST00000263593 (human) and ENSMUST00000002007 (mouse). The PCR products were digested with Dpn I restriction enzyme (Agilent) and transformed into TOP10 chemically competent E. coli cells (Invitrogen). Clones harboring the desired single nucleotide change were verified by DNA sequencing.Full-length C-terminal Flag-tagged human and murine SIAE variants ) were transfected transiently in HEK 293T cells, in parallel to cDNA’s encoding wild type (WT) and S127A  proteins. Cell lysates were immunoprecipitated with monoclonal anti-Flag antibodies (Sigma) and catalytic activity of immunoprecipitated SIAE was assayed using 4-methylumbelliferyl acetate (USB). Equivalent amounts of cell lysate were immunoprecipitated for the enzyme activity assay, and for quantification of the protein by a quantitative Western blot assay on the LI-COR Odyssey imaging system, using an IR Dye 800 CW labeled Goat anti-mouse IgG as the secondary antibody.Assays for SIAE enzyme activity have been reported by us previously WT, K71R and A467V SIAE were starved for 1 h in methionine/cysteine-free medium and labeled with 0.5 mCi of 35S-methionine for 10 min. Cells were either lysed immediately (10 min) or after 2 and 4 hrs of chase with an excess of complete medium. Lysates were immunoprecipitated with anti-Flag antibodies and protein A sepharose beads (Sigma). Proteins were separated by SDS/PAGE (8%) and revealed by autofluorography.HEK 293T cells transfected with cDNAs encoding SIAE. Cell lysates were immunoprecipitated with anti-SIAE (3 µg), isotype control Mouse IgG (3 µg) (BD Biosciences), or anti-Flag (3 µg) antibodies. Immunoprecipitated samples were analyzed by Western blotting using anti-SIAE (1∶1000) (three hours to overnight) or anti-Flag (1∶1000) antibodies and subsequent incubation with anti-Mouse IgG-HRP  for 1hour. BJAB and Ramos cells were fixed with 2% paraformaldehyde in PBS for 5 minutes at room temperature. The cells were permeabilized with 0.02% Triton-X-100 in 1xPBS-05%BSA (PBS-BSA) for 7 minutes at room temperature. Cells were then incubated with anti-SIAE (3 µg) or isotype control Mouse IgG (3 µg) for 45 minutes at 4°C. The secondary antibody Goat anti-Mouse IgG-FITC (BD Biosciences) was incubated for 40 minutes at 4°C. The cells were washed twice and resuspended with PBS-BSA for flow cytometric analysis.293T human embryonic kidney cells, and the BJAB and Ramos B cells lines (ATCC) were used for characterization of a polyclonal anti-SIAE antibody . 293T cells were transfected with human Flag-tagged Human white blood cells were isolated and examined separately for the presence of surface or intracellular SIAE. Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers using hypotonic water lysis, followed by dilution with 10 X HBSS and two washes with PBS-BSA. For extracellular staining, unfixed or fixed PBMC were incubated with anti-SIAE (3 µg) or Isotype control Mouse IgG (3 µg) followed by a secondary antibody, FITC labeled anti-Mouse IgG. CD8-APC-cy7 (BD Biosciences) was used as a control for both extracellular and intracellular staining. For intracellular staining of PBMC the method was the same as described above for the BJAB and Ramos cell lines.SIAE of which 11 were catalytically defective [4). Rare variants are arbitrarily defined as having a frequency below 1% in the population. In a study on primary biliary cirrhosis, three additional rare variants of SIAE were identified of which two were shown to be defective SIAE. A list of SIAE rare variants that have been identified so far in subjects of European and African- American ancestry is included in SIAE suggests that genotyping studies would be most useful if all possible defective variants are genotyped in order to obtain detailed information regarding susceptibility. Sequencing of all exons of this gene is required in order to accurately identify rare genetic variants. Functional studies on all identified rare variants will most accurately identify those that should be considered relevant to quantify in disease subjects and controls.We originally described 19 rare variants of SIAE were identified in the course of our studies, and all four have also been observed in subsequent exome sequencing approaches  so we determined whether defective human SIAE variants are also catalytically defective when re-created in a murine cDNA. All disease related catalytically defective SIAE variants tested were defective when re-created in the murine cDNA . Both catalytically defective disease-related murine proteins were not secreted in these overexpression studies. However overexpressed murine M89V Siae (unlike human M89V SIAE) was secreted as well as its wild type murine counterpart and like its human counterpart was catalytically normal  and a two tailed p value by Fisher’s exact test of 0.0077 , however these results suggest that it would be premature to assume that catalytically defective SIAE alleles are not associated with disease. Keeping in mind that all the catalytically defective rare variants are present at a very low frequency, the inclusion of even a single high frequency catalytically normal common variant can skew the overall analysis and hinder the detection of associations linked to defective rare variants.Re-analysis of our original sequencing data examining only the frequency of confirmed catalytically defective f 0.0077 . Hunt etanalysis , and somSIAE originally described by us represents a small subset of the defective rare variants in this gene as revealed by our own subsequent sequence analyses and by exome sequencing studies that have been undertaken subsequent to our original publication. The vast majority of the rare genetic variants of SIAE identified by sequencing following our original report represent newly discovered variants, suggesting that the total number of rare variants in this gene will likely be very large. There are currently 67 documented rare variants of SIAE and this number is rapidly growing (The number of catalytically defective rare variants of  growing .A very small fraction of overexpressed wild type SIAE is secreted as revealed in pulse-chase studies, and this minimal secretion observed is an in vitro phenomenon of no proven physiological significance. SIAE is not expressed on the surface of human lymphocytes, and has not been detected in human plasma by sensitive means. Secretion of SIAE per se should not be a criterion for the function of this enzyme. Overexpressed murine M89V SIAE is no different from wild type overexpressed protein, and M89V SIAE in both humans and mice is catalytically normal. On these grounds M89V SIAE should be unequivocally excluded from considerations of disease susceptibility. The failure of secretion of overexpressed mutant catalytically defective SIAE proteins in transfected cells is likely a feature of misfolded mutant proteins that fail to egress the ER – and therefore cannot contribute even to the miniscule in vitro escape from the cell. The likelihood that these disease associated mutant proteins are misfolded is currently being explored using circular dichroism spectroscopy.M89V SIAE from analysis, reveals that defective variants of SIAE remain associated with autoimmunity, with strong Odds Ratios, but our overall numbers are relatively small compared to those used in the genotyping study of Hunt et al. SIAE rare variants that they chose to genotype shows that these rare variants are enriched in the diverse disease cohorts they analyzed (SIAE alleles with disease.Re-analysis of our data analyzed  and thesSIAE locus followed by analyses of catalytic function in order to identify a large proportion of defective rare variants of SIAE that may be relevant to disease. A wide variety of diseases some allergic, others autoimmune, some neither, were grouped together in the study of Hunt et al. Very large genotyping studies even larger than the one conducted by Hunt et al., but including all known catalytically defective alleles may well provide important information when carried out in well-defined disease populations.The analysis of rare genetic variants of any gene will require large scale sequencing analyses followed by functional analyses of novel variants. Large-scale genotyping studies of individual variants may be required in order to attain statistically significant results for any given rare variant allele. Large population studies are currently being carried out with a full sequence analysis of the


A small series of recent studies has examined the possibility of using MRI reporter genes to track the survival of cells injected into the heart following myocardial infarction. In this review, we seek to frame the emerging field of MRI reporter gene based cardiac cell tracking within the larger framework of the needs of cardiac regeneration therapy and the more established field of MRI cell tracking. While initial studies have demonstrated a promising ability to track the viability and proliferation of cells used for cell therapy, the ultimate goal of MR reporter gene imaging in the heart remains the ability to simultaneously correlate cell fate decisions with additional measures of structural and functional recovery.Research into cell therapy based cardiac repair and regeneration has experienced explosive growth over the last decade, however further progress is hindered by an inability to serially and non-invasively image cell survival and fate decisions following implantation. Recent advances in magnetic resonance imaging (MRI) reporter gene techniques have enabled  While early studies sought to replace damaged myocardium with implantation of autologous satellite cells , a vario manner –7.in vivo imaging of cell survival, migration, and differentiation primarily in stationary organs and tumors (for a full review see ref [Magnetic resonance imaging (MRI) has become a reference standard modality for non-invasive imaging of soft tissue with superior contrast. When applied to imaging of the heart, cardiac MRI (CMR) is emerging as a cutting edge modality capable of enabling multi-scale characterization of the heart from the cellular to the whole organ level , 9. Sign see ref ). ApplicIn this review we seek to discuss existing methods for MRI cell tracking and the results of early studies of MR reporter gene cell tracking in the heart. Given the small number of studies performed to date, each study is discussed in detail in order to highlight the important contributions made to the nascent field, and further questions that have arisen as a result of such studies. Finally, we will discuss how important findings from MR reporter gene studies in stationary organs and tumors can potentially lead to more dynamic CMR reporter gene imaging.MRI cell tracking has historically relied upon labeling of cells with chemical agents that generate contrast via modulation of either T1 (bright contrast) or T2 (dark contrast) relaxation times , or throchemical exchange saturation transfer (CEST) [An alternative cell labeling strategy that has recently emerged is to label cells with a class of contrast agents that selectively generate contrast through a process termed r (CEST) –20. CESTr (CEST) . Since tMRI reporter genes can uniquely overcome several of the barriers to conventional cell labeling strategies. First, when initiated with proper genetic reprogramming, MR reporter gene expression will remain constant across successive cell divisions. This eliminates concerns of cell division mediated dilution of contrast agents. Second, since transcription of the reporter gene occurs immediately after division, asymmetric division of an exogenous cell label is removed as a limiting factor to cell tracking. Finally, since production of the MR reporter gene product ceases upon cell death, the likelihood of detecting a false positive signal after cell death is significantly reduced. While such characteristics are highly favorable, it is important to note that many of the first generation of MR reporter genes typically demonstrate lower sensitivity when compared to conventional cell labeling strategies.A large number of MR reporter gene strategies, each employing a different reporter mechanism, have been proposed and tested over the past decade (for a full review see ref ). OverwhMR reporter gene imaging is an exciting new field that is undergoing initial application to cardiac imaging. To date, CMR studies of reporter gene expressing cells have focused on monitoring cell viability and proliferation following engraftment into the rodent heart after surgically induced myocardial infarction. Broadly, these studies have sought to identify reporter gene expressing cells through generation of T2/T2* contrast on gradient echo images of the heart. In one series of studies, contrast was generated through over-expression of iron regulatory elements in order to safely increase intracellular iron content ••, 31. Iin vitro phantom studies revealed a 25 % reduction of T2 relaxation times in ferritin over-expressing C2C12 cells when compared to normal C2C12 cells. In order to examine the impact of exposure to an iron rich environment, as would be the case in vivo, the authors incubated ferritin over-expressing cells in FC supplemented medium for 48 hours prior to phantom imaging. Exposure to FC prior to imaging led to a further 50 % reduction of T2 relaxation times.In an early study by Naumova et al. •• murinein vivo utility of ferritin over-expression for MR cell tracking, the authors transplanted either C2C12 cells or ferritin over-expressing C2C12 cells into the hearts of C3H mice following surgically induced myocardial infarction , normal cardiospheres, or ferritin over-expressing cardio-spheres targeted to border zone tissue. The presence of ferritin heavy chain over-expressing cardiospheres was visualized using standard T2*-weighted imaging as early as 1 week after implantation, and remained constant for 4 weeks after implantation in all rats that received ferritin over-expressing cardiospheres regardless of myocardial infarction status. In animals that underwent myocardial infarction surgery, injection of cardiospheres improved LV ejection fraction at four weeks after MI, with no differences observed between ferritin heavy chain over-expressing and wild type cardiospheres. Prussian blue staining of isolated tissue sections confirmed elevated iron deposits in ferritin heavy chain over-expressing cardiospheres 4 weeks following implantation. Importantly, repeated assessment after in vivo experiments of viability, proliferation, and key cell surface markers demonstrated no difference between ferritin heavy chain over-expressing and wild type cardiospheres.In a similar study, over-expression of human ferritin heavy chain was used as a reporter gene for MR tracking of swine cardiac stem cells following implantation into the infarcted rat heart . Campan These promising early results demonstrate the ability to track the survival and proliferation of cells implanted into the healing infarct using ferritin over-expression as an MRI reporter gene strategy. In both studies, ferritin over-expressing cells appeared as clear regions of signal voids on bright blood T2*-weighted gradient echo images, mimicking the effect of cell labeling with iron oxide nanoparticles. Interestingly, despite relatively similar quantities of injected cells, the signal voids generated by injection of C2C12 cells ••, 31 wein vitro studies demonstrated significant loss of signal in embryonic stem cells expressing HA and myc antigens when co-cultured with targeted SPIO particles that was not observed in non-transduced cells. Later, in vivo studies demonstrated no difference in signal intensity of transplanted cells on T2*-weighted images prior to administration of antibody bound SPIO particles. Studies performed in animals receiving myc and HA positive embryonic stem cells and HA or myc targeted SPIO particles demonstrated reduced signal intensity in areas of injected cells starting at 5 days after cell injection, which was confirmed by whole body bioluminescence imaging. Repeated studies revealed increased binding of targeted SPIO particles between five and ten days after cell implantation as the embryonic stem cells proliferated and began to form teratoma (Fig. In contrast to the previously described studies which sought to generate T2/T2* contrast via increased ferritin expression and subsequent accumulation of intercellular iron, an alternative mechanism has been to adapt immuno-histochemical staining techniques for use with MRI. Recently, Chung et al. •• engineoma Fig. . SimilarAs the field of cardiac MRI reporter gene based cell tracking continues to develop, there are several key objectives that must be part of the field’s evolution. First, the need to genetically alter cells in order to express MR reporter genes remains a barrier to translation beyond pre-clinical models. Although early MR reporter gene studies have demonstrated minimal impact on cell viability, proliferation, function, and differentiation, and newer site specific integration techniques for genetic modification of cells demonstrate superb safety profiles , thorougMR reporter gene imaging is an exciting and burgeoning field that can potentially enable a multi-scale understanding of how cell fate decisions impact subsequent disease and regenerative processes. Application to cardiac imaging is currently in very early stages. However, as this field continues to evolve, the potential to elucidate multiple aspects of cell fate decisions within the framework of cardiac regeneration becomes increasingly possible. With further development, MR reporter gene imaging can someday be used to investigate multiple aspects of cell survival, proliferation, and differentiation in order to gain a deeper understanding of mechanisms by which to promote stem cell survival and regeneration of myocardial tissue.


Graft-versus-host disease (GVHD) is a common complication of allogeneic bone marrow transplantation (BMT). Upregulation of inflammatory cytokines precedes the clinical presentation of GVHD and predicts its severity. In this report, thiol/redox metabolomics was used to identify metabolic perturbations associated with early preclinical (Day+4) and clinical (Day+10) stages of GVHD by comparing effects in Syngeneic  and allogeneic transplant recipients (Allo BMT) in experimental models. While most metabolic changes were similar in both groups, plasma glutathione (GSH) was significantly decreased, and GSH disulfide (GSSG) was increased after allogeneic compared to syngeneic recipient and non-transplant controls. The early oxidation of the plasma GSH/GSSG redox couple was also observed irrespective of radiation conditioning treatment and was accompanied by significant rise in hepatic protein oxidative damage and ROS generation. Despite a significant rise in oxidative stress, compensatory increase in hepatic GSH synthesis was absent following Allo BMT. Early shifts in hepatic oxidative stress and plasma GSH loss preceded a statistically significant rise in TNF-α. To identify metabolomic biomarkers of hepatic GVHD injury, plasma metabolite concentrations analyzed at Day+10 were correlated with hepatic organ injury. GSSG (oxidized GSH) and β-alanine, were positively correlated, and plasma GSH cysteinylglycine, and branched chain amino acids were inversely correlated with hepatic injury. Although changes in plasma concentrations of cysteine, cystathionine (GSH precursors) and cysteinylglycine (a GSH catabolite) were not significant by univariate analysis, principal component analysis (PCA) indicated that accumulation of these metabolites after Allo BMT contributed significantly to early GVHD in contrast to Syn BMT. In conclusion, thiol/redox metabolomic profiling implicates that early dysregulation of host hepatic GSH metabolism and oxidative stress in sub-clinical GVHD before elevated TNF-α levels is associated with GVHD pathogenesis. Future studies will probe the mechanisms for these changes and examine the potential of antioxidant intervention strategies to modulate GVHD. Graft-versus-host disease (GVHD) is an important complication of allogeneic hematopoietic stem cell transplantation (HSCT), and it limits the wider application of this curative treatment option 2-isoprostanes (an in vivo biomarker of lipid oxidation) and activation status of nuclear factor-kappa B (NFkB), a key transcription factor controlling the expression of inflammatory mediators and cytokines Oxidative stress is an unavoidable consequence of HSCT and may be an important exacerbating factor in GVHD. Owing to the contributions of pre-existing disease conditions and the requirement for conditioning regimens that increase cellular reactive oxygen species (ROS), oxidative stress is elevated in all HSCT recipients in vitro, which could explain why GVHD is associated with increased epithelial genomic instability in patients Allogeneic BMT is associated with increased oxidative stress during the active effector phase of GVHD In addition to increased ROS generation, impaired antioxidant defense capacity following Allo BMT could also contribute to oxidative stress. Glutathione (GSH) is an endogenously synthesized sulfur amino acid (SAA)-containing tripeptide, which plays a principal role in cellular redox regulation. GSH synthesis is coordinately regulated through four sequentially interconnected pathways: transmethylation (TM); transsulfuration (TS); glutathione synthesis (GS); and glutathione recycling (GR) The redox state of the GSH/GSSG redox couple is normally tightly regulated (±7%) but it decreases in response to tissue injury, inflammation, and exposures to toxicants Although the importance of GSH in modulating inflammation has been established, it is not clear how GSH metabolism changes early after GVHD initiation and might mediate tissue injury. Clinical studies have shown that plasma and erythrocyte GSH and antioxidant enzymes activities decline after HSCT Given the multidimensional roles of SAA and other amino acid metabolites in processes directly related to GVHD pathology, we hypothesized that comprehensive profiling of these compounds during the development of GVHD would provide new mechanistic insights into its complex pathobiology, particularly in its early phases. In this report, a thiol/redox metabolomics assay Our results demonstrate that oxidation of the host GSH-regulated redox system and failure of compensatory upregulation of GSH-synthesis enzymes occur prior to any evidence of change in amino acids known to be sensitive to inflammation. These results also indicate that this early shift in redox regulation precedes GVHD initiation as established by rise in circulating TNF-α. Lastly, the utility of plasma metabolomics to identify biomarkers of hepatic injury during the early clinical phase of GVHD is demonstrated.Guide and the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. The protocol was approved by the CHORI Institutional Animal Care and Use Committee (IACUC Assurance No: A3631-01). All invasive procedures were performed under isofurane anesthesia, and efforts were made to minimize suffering at all times. Sacrifice procedures were performed in the morning following overnight fasting. Animals were sacrificed in their home cage by CO2 inhalation to effect and followed by exsanguination by cardiac puncture to obtain blood. All other tissues were harvested in sterile hood following cardiac puncture.All procedures were performed in compliance with the recommendations in the b/CD45.2+, Thy1.2), BALB/c (H-2d/CD45.2+), B6D2F1 , and B6.PL-Thy1a  were purchased from The Jackson Laboratories  and/or inbred at the animal facility of Children’s Hospital Oakland Research Institute (CHORI) . All animals were 8–12 weeks of age at the time of transplantation. Following transplantation, animals were fed standard fat-chow and maintained in micro-isolator cages in pathogen free environment. Animals were given Pen/Strep antibiotic water at a dose of 100 units/ml following lethal-radiation.Female mice C57BL/6 (B6: H-26) plus either CD90+ (5×106) or CD4+ (3×106) T cells from respective allogeneic (BALB/c) or syngeneic (B6. Thy1.1) donors were injected intravenously into recipient animals on day 0. To induce GVHD in unirradiated host, 50×106 whole splenocytes from B6 Thy1.1 donor mice were infused into allogeneic (B6D2F1) and syngeneic (B6) mice. Survival was monitored daily, and body weights and GVHD clinical scores of recipients were measured thrice weekly, up to 30 days post-transplantation and then weekly for 6 months. The degree of systemic acute GVHD was assessed by a scoring system that incorporates 5 clinical parameters–weight loss, posture (hunching), activity, fur texture, and skin integrity, as described previously Mice underwent transplantation in accordance with the protocol described previously Representative samples from the liver, gut and lungs were placed in 10% phosphate buffered formalin, embedded in paraffin, cut into 5 µm sections and stained with hematoxylin and eosin for histopathological examination. A pathologist reviewed histopathology sections in a blinded manner to assess for GVHD. A semi-quantitative system of scoring was used as previously described Plasma samples from transplanted animals were aliquoted and batched analysis was performed using FlowCytomix bead based immunoassay  in 96 well microplate format, in accordance with manufacturer’s instructions. The standard curves were created using 5-fold dilution of appropriate standard in culture medium and serum samples were analyzed using mouse Th1/Th2 10 plex kit flowcytomix.Protein carbonyls were measured as a biomarker of hepatic protein oxidation by using the OxiSelect™ Protein Carbonyl ELISA Kit . Briefly, flash-frozen liver tissues (25 mg) were homogenized in 1 ml of phosphate-buffered saline solution by using the Fastprep FP120 centrifuge homogenizer . Homogenized samples were subsequently centrifuged at 20,000 x g to obtain cytosolic protein fraction. Protein carbonyls in hepatic cytosolic fraction (100 µg protein/ml) were quantified by dinitrophenylhydrazine (DNPH) derivatization followed by specific detection by anti-DNP antibody in accordance with manufacturer’s instructions.Hepatocytes were stained with CellROX™ (5 µM) and Thiol Tracker Violet  (Invitrogen) per manufacturers instructions. FACS analysis was performed using LSR Fortessa (BD Biosciences). Data was analyzed using Flowjo software .Plasma and hepatic SAA and other amino acid metabolites were quantified using a liquid chromatography linked tandem mass-spectrometry (LC/MS/MS) assay 2). The standard redox potential used was −264 mV.The plasma redox potential for glutathione (GSH) was calculated using the Nernst equation 5′-TCATGATCGAAGGACACCAA-3′ and for 18S ribosomal RNA were: FP5′-GTAACCCGTTGAACCCCATT-3′ and RP 5′-CCATCCAATCGGTAGTAGCG-3′.A portion of each liver was excised, flash frozen in liquid nitrogen, stored at −80°C and homogenized using a Fast Prep system . Total RNA was isolated from mouse livers using an RNeasy Mini Kit . cDNA was prepared from 1 µg of total RNA per group using QuantiTect Reverse Transcription Kit (Qiagen). Relative transcript amounts of γ-Glutamylcysteine Ligase Catalytic Subunit (GCLC) was quantified using the [delta][delta] Ct method with 18S rRNA as a control. PCR reactions were carried out using the ABI 7900 Real Time qPCR system  under following cycling conditions: 95°C for 10 mins, and 40 cycles at 95°C for 10 mins and 60°C for 1 min. The primers used for GCLC were: FP 5′CCACTGAGCTGGGAAGAGAC-3′ and RP p value <0.05 was considered statistically significant.Survival data in each group were generated using Kaplan–Meier lifetime survival probability methodology and the log-rank (Mantel–Cox) test. GVHD scores and serum cytokine concentrations were analyzed by ANOVA with Tukey’s Honest Significance Test (HSD) post-hoc tests. PRISM software  was used for these tests and a All of the metabolomic statistical tests described below were performed using the Metaboanalyst software platform For univariate analysis of data obtained from baseline controls, Syn, Allo BMT at Day+4 and +10 time points, statistical analysis of microarray (SAM) was performed with Delta values adjusted to minimize false discovery rate below 10%. Tukey’s Honestly Significant (Tukey’s HSD) was used to further quantify the differences between the groups.PCA is an unsupervised classification method that projects high-density data to new coordinated system with much smaller sets of variables , which describe the variability in the data. Each principal component is orthogonal to each other and each PC explains the greatest source of variance remaining after previous PCs have been established. PCA produces two plots; 1) scores plot: projects each sample on a new coordinate system composed of PCs and are used to reveal the intrinsic structure of data set in terms of variance, 2) loading plot: displays variables that contributes to the group separation.PLS-DA is a supervised technique where the discriminant model is built with a prior knowledge of the group membership of set of samples and is created to best explain the group memberships of individual samples. PLS-DA has two outputs: 1) score plot shows the sample distribution in the coordinate system composed to the components selected, 2) Variable importance in Projection (VIP) values which is a computation of influence of every x term in the model on the group classification and larger VIP values indicate a greater influence of x on group discrimination and generally, a VIP value of ≥ 1 are considered significant. Because PLS-DA is prone to over-fitting errors, the significance of group discrimination by multivariate models was cross-validated with the use of “leave-one-out” and permutation testing.Variables that were found to contribute most to the group discrimination were used for hierarchical clustering analysis (HCA) using Pearson’s test for distance measures and Ward’s minimum variance method was used for clustering. Samples are in rows and variables are in columns. The colors vary from deep blue to dark brown to indicate data values change from very low to extremely high.GVHD was induced in the fully MHC-mismatched model consisting of BALB/c → B6 following lethal irradiation .Plasma amino acid metabolomic changes following Syn and Allo BMT at post-transplantation Days +4 and +10 were compared to baseline wild-type (WT) controls. Of the 40 metabolites measured, 16 metabolites changed significantly compared to baseline and are shown in The liver is the major site for GSH synthesis and is also a target organ of GVHD To determine whether the observed decline in GSH is due to GVHD rather than to the conditioning regimen, GVHD was induced in unirradiated recipients using a paternal to F1 hybrid (B6 → B6D2F1) transplantation model  and GSH synthetase. GCL catalyzes the rate-limiting step. It is a heterodimer composed of catalytic (GCLC) and regulatory (GCLM) subunits. GCLC transcription is upregulated in response to increased oxidative stress or xenobiotic exposure. An inadequate GCLC transcriptional induction in response to a cellular ROS burden would result in oxidative stress. At post-transplant Day+4, hepatocytes from Allo BMT mice were freshly isolated and the cellular oxidant burden and relative abundance of GCLC mRNA were estimated by CellROX™ Deep Red reagent (CDRR) flow cytometric and RT-PCR assay, respectively. As shown in Protein carbonyls are formed as a consequence of ROS-dependent protein oxidative modification and are stable biomarkers of oxidative stress. As shown in To establish the temporal relationship between plasma GSH/GSSG redox potential change and inflammatory cytokine upregulation characteristic of GVHD, serum concentrations of IL-2, IFN-γ, and TNF-α were measured at post-transplant Days +4 and +10. 2 = 0.65; p = 0.002). Significant positive correlations were also were observed in the GSH/GSSG redox potential (more positive = more oxidized). Significant inverse correlations were also observed between liver histopathological damage and plasma total cysteinylglycine , GSH, and total GSH (T. GSH), suggesting that concurrent plasma GSH/GSSG oxidation state reflects the extent of GVHD injury in the liver.The correlation between plasma GSH and hepatic GVHD severity was determined using the paternal to F1 hybrid model Figure 4Several metabolites unrelated to GSH metabolism also were significantly correlated with histopathological changes in the liver Figure 4Interestingly, plasma Arg, a requisite precursor for NO synthesis, and Cit, also generated by iNOS enzymes, failed to correlate with liver histopathology. Plasma Trp, which is a substrate of IDO enzymes previously implicated in GVHD, also did not correlate with liver histopathology.Supervised Principle Component Analysis (PCA) and unsupervised (PLS-DA) modeling permit identification of subtle metabolic shifts that may not achieve statistical significance in univariate analyses. These techniques were used to examine the metabolomic separation of the Allo and Syn BMT and baseline control groups. The PCA scores plot Figure5AIn the PC1 versus PC3 loading plot Figure 52 and Q2 are used to test the predictive power of the model. A “Leave-One-Out” cross-validation test using a 3 component model gave an R2(Y) = 0.98 and Q2(cum) = 0.8, indicating that the PLS-DA model explains 98% of response variability. Q2 is an estimate of the predictive ability of the model and a value greater than 0.5 is regarded as good 2 and R2 values than distributions calculated from permuted data.Supervised PLS-DA analysis Figure5CTo investigate additional group segregation, the top variables identified by PLS-DA analysis were subjected to hierarchical cluster analysis (HCA) to generate a heat-map shown in A summary of changes in the GSH metabolic pathway on Day+4 is presented in The major aims of the current work were to identify metabolic pathways and metabolites that are perturbed early in the course of developing GVHD after Allo BMT transplantation. Additionally, we aimed to identify plasma metabolic correlates of hepatic GVHD injury during the early clinical stages of GVHD. Using both conditioning-dependent and independent models of GVHD, rapid loss of plasma GSH and accumulation of its oxidized form occurs in early stages (Day +4) 4. TheseComprehensive analysis of the major plasma amino acids and secondary metabolites of arginine catabolism allowed us to establish the relative sensitivity of plasma and hepatic GSH changes compared to other amino acid catabolic pathways. The panel of 40 analytes quantified included the 20 major amino acids, secondary metabolites produced from Arg catabolism and SAA-derived metabolites. Arg catabolism to citrulline and ornithine is increased during inflammation and we have shown previously in RAW macrophage cells that Arg loss and increased formation of its products are most sensitive biomarkers of macrophage activation To the best of our knowledge, acute changes in plasma and liver GSH metabolism following Allo and Syn BMT and during the early GVHD period have not been systematically examined. Sari and co-workers reported decreases in plasma antioxidant enzyme activities and increases in plasma lipid oxidation in Allo BMT patients 30 days post-HSCT Liver has one of the highest tissue concentrations of GSH. Plasma GSH concentrations are primarily determined by hepatic GSH biosynthesis and efflux Despite an early increase in hepatic oxidative stress, mRNA abundance of GCLC, the rate-limiting enzyme in GSH synthesis, and total GSH were significantly lower in Allo mice in comparison to the Syn group. Total hepatic GSH concentrations in Syn mice were increased by ∼70% relative to baseline Table 4,Key findings in this study are that the GVHD-associated plasma GSH/GSSG ratio declines at Day+4 before TNF-α induction, and that plasma GSH depletion and oxidation at Day+10 correlates with hepatic GVHD severity. Though potential cause-and-effect relationships between early plasma GSH loss and subsequent cytokine elevation or GVHD severity were not addressed in this study, several precedents suggest such a causal relationship is plausible. (a) Host GSH/GSSG redox potential change may impact NFκB-dependent inflammatory signaling These results emphasize the importance of understanding the mechanisms that lead to increased oxidative stress during GVHD pathogenesis, and also point to GSH biosynthesis as a potential target for GVHD prevention. How this might be accomplished depends on which component of the GSH homeostatic system is impaired in the early stages after transplantation. GSH is an endogenously synthesized compound, which is poorly taken up through diet. Clinical approaches to raise GSH have predominantly been focused on N-acetylcysteine (NAC) as a cysteine pro-drug to boost cellular GSH synthesis. However, the metabolic data presented here indicate that loss of GSH during early GVHD is not due to limited cysteine availability, and therefore suggest that such an approach might not be efficacious. Consistent with this idea, a clinical intervention with a moderate dose (100 mg/kg per day) of NAC increased rather than decreased the prevalence of GVHD and veno-occulusive disease of the liver (VOD) in Allo BMT patients This report utilizes a metabolomics approach to demonstrate that oxidative stress driven by impaired hepatic GSH biosynthesis occurs early in GVHD and precedes cytokine upregulation is the hallmark of the onset of GVHD. Results help to clarify the mechanisms resulting in oxidative stress during early GVHD. They also identify hepatic ROS generation and the GSH synthesis pathway as potential targets for early intervention. Plasma GSH and GSSG may also be clinically useful biomarkers for early GVHD prediction. Future work will be directed at investigating upstream events following transplantation that culminate in disruption of the GSH biosynthetic pathway.