scholarly journals Untargeted Metabolomic Profiling of Cuprizone-Induced Demyelination in Mouse Corpus Callosum by UPLC-Orbitrap/MS Reveals Potential Metabolic Biomarkers of CNS Demyelination Disorders

2021 ◽  
Vol 2021 ◽  
pp. 1-17
Author(s):  
Zhijie Zhao ◽  
Tongqi Li ◽  
Xiaohua Dong ◽  
Xiaojing Wang ◽  
Zhongxiao Zhang ◽  
...  
Keyword(s):  
Corpus Callosum ◽  
Pathway Analysis ◽  
Metabolic Pathways ◽  
Neurodegenerative Disorder ◽  
Mapk Signaling ◽  
Demyelinating Diseases ◽  
Characteristic Analysis ◽  
Discrimination Ability ◽  
Cns Demyelination ◽  
Orbitrap Ms

Multiple sclerosis (MS) is a neurodegenerative disorder characterized by periodic neuronal demyelination, which leads to a range of symptoms and eventually to disability. The goal of this research was to use UPLC-Orbitrap/MS to identify validated biomarkers and explore the metabolic mechanisms of MS in mice. Thirty-two C57BL/6 male mice were randomized into two groups that were fed either normal food or 0.2% CPZ for 11 weeks. The mouse demyelination model was assessed by LFB and the expression of MBP by immunofluorescence and immunohistochemistry. The metabolites of the corpus callosum were quantified using UPLC-Orbitrap/MS. The mouse pole climbing experiment was used to assess coordination ability. Multivariate statistical analysis was adopted for screening differential metabolites, and the ingenuity pathway analysis (IPA) was used to reveal the metabolite interaction network. We successfully established the demyelination model. The CPZ group slowly lost weight and showed an increased pole climbing time during feeding compared to the CON group. A total of 81 metabolites ( VIP > 1 and P < 0.05 ) were determined to be enriched in 24 metabolic pathways; 41 metabolites were markedly increased, while 40 metabolites were markedly decreased in the CPZ group. The IPA results revealed that these 81 biomarker metabolites were associated with neuregulin signaling, PI3K-AKT signaling, mTOR signaling, and ERK/MAPK signaling. KEGG pathway analysis showed that two significantly different metabolic pathways were enriched, namely, the glycerophospholipid and sphingolipid metabolic pathways, comprising a total of nine biomarkers. Receiver operating characteristic analysis showed that the metabolites (e.g., PE (16 : 0/22 : 6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)), PC (18 : 0/22 : 4(7Z, 10Z, 13Z, 16Z)), cytidine 5 ′ -diphosphocholine, PS (18 : 0/22 : 6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)), glycerol 3-phosphate, SM (d18 : 0/16 : 1(9Z)), Cer (d18:1/18 : 0), galabiosylceramide (d18:1/18 : 0), and GlcCer (d18:1/18 : 0)) have good discrimination ability for the CPZ group. In conclusion, the differential metabolites have great potential to serve as biomarkers of demyelinating diseases. In addition, we identified metabolic pathways associated with CPZ-induced demyelination pathogenesis, which provided a new perspective for understanding the relationship between metabolites and CNS demyelination pathogenesis.

Neurometabolic Diseases in Children: Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy Features

2019 ◽  
Vol 15 (3) ◽  
pp. 255-268
Author(s):  
Direnç Özlem Aksoy ◽  
Alpay Alkan
Keyword(s):  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Metabolic Pathways ◽  
Wide Spectrum ◽  
Demyelinating Diseases ◽  
Resonance Spectroscopy ◽  
Resonance Imaging ◽  
Neurometabolic Disorders ◽  
Brain Involvement ◽  
The Brain

Background: Neurometabolic diseases are a group of diseases secondary to disorders in different metabolic pathways, which lead to white and/or gray matter of the brain involvement. </P><P> Discussion: Neurometabolic disorders are divided in two groups as dysmyelinating and demyelinating diseases. Because of wide spectrum of these disorders, there are many different classifications of neurometabolic diseases. We used the classification according to brain involvement areas. In radiological evaluation, MRI provides useful information for these disseases. Conclusion: Magnetic Resonance Spectroscopy (MRS) provides additional metabolic information for diagnosis and follow ups in childhood with neurometabolic diseases.

scholarly journals Icariin promotes osteoblastic differentiation in OVX mice via MAPK signaling pathway revealed by profiling

2018 ◽  
Vol 01 (01) ◽  
pp. 33-41
Author(s):  
Qin Bian ◽  
Shufen Liu ◽  
Yongjian Zhao ◽  
Jianhua Huang ◽  
Ziyin Shen
Keyword(s):  
Bone Mass ◽  
Signaling Pathway ◽  
Pathway Analysis ◽  
Ex Vivo ◽  
Osteoblastic Differentiation ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
The Self ◽  
Self Renewal ◽  
Colony Forming Unit

Objective: Icariin (ICA), an extract from epimedium, has been reported to be effective in promoting bone formation. The objective of the study is to search for the molecular targets of ICA in bone mesenchymal stem cells (bMSCs) from the mice with ovariectomy (OVX)-induced osteoporosis. Methods: Six-month-old Imprinting Control Region (ICR) mice that underwent OVX were treated with ICA. After three months, bone mass was evaluated by microcomputed tomography, morphometry and immunohistological detection. bMSCs were isolated from the femur and tibia to observe the self-renewal and differentiation capacities using colony-forming unit fibroblastic (CFU-F), colony-forming unit adipocyte (CFU-Adipo) and alkaline phosphatase (ALP) staining. In addition, microarray of bMSCs ex vivo was measured two weeks after ICA treatment and analyzed by heatmap and pathway analysis. The signaling pathway was further explored by western blot assay and inhibitors of p38 and ERK: SB203508 and PD98059. Results: [Formula: see text]CT displayed a decrease in bone mass for three months after OVX. ICA treatment increased the trabecular thickness (Tb.Th), osteoblast number while decreased osteoclast number, elevating osteocalcin (OC) protein levels in vivo and facilitating the self-renewal and osteoblastic differentiation of bMSCs ex vivo. Microarray data indicated ICA rescued several gene expressions that were dysregulated by OVX. Pathway analysis revealed that the core genes acted by ICA were highly involved in MAPK signaling pathway. Further study demonstrated ICA suppressed ERK while stimulated p38 phosphorylation to promote osteoblastic differentiation in vitro. Conclusion: ICA promotes osteoblastic differentiation of bMSCs in OVX mice. MAPK signaling pathway might be involved in the process.

scholarly journals Untargeted metabolomic analysis and pathway discovery in perinatal asphyxia and hypoxic-ischaemic encephalopathy

2017 ◽  
Vol 39 (1) ◽  
pp. 147-162 ◽  
Author(s):  
Niamh M Denihan ◽  
Jennifer A Kirwan ◽  
Brian H Walsh ◽  
Warwick B Dunn ◽  
David I Broadhurst ◽  
...  
Keyword(s):  
Pathway Analysis ◽  
Metabolic Pathways ◽  
Perinatal Asphyxia ◽  
Metabolic Effects ◽  
Metabolomic Analysis ◽  
False Discovery ◽  
Metabolic Feature ◽  
Pathway Discovery ◽  
New Treatment ◽  
Neonatal Population

Elucidating metabolic effects of hypoxic-ischaemic encephalopathy (HIE) may reveal early biomarkers of injury and new treatment targets. This study uses untargeted metabolomics to examine early metabolic alterations in a carefully defined neonatal population. Infants with perinatal asphyxia who were resuscitated at birth and recovered (PA group), those who developed HIE (HIE group) and healthy controls were all recruited at birth. Metabolomic analysis of cord blood was performed using direct infusion FT-ICR mass spectrometry. For each reproducibly detected metabolic feature, mean fold differences were calculated HIE vs. controls (ΔHIE) and PA vs. controls (ΔPA). Putative metabolite annotations were assigned and pathway analysis was performed. Twenty-nine putatively annotated metabolic features were significantly different in ΔPA after false discovery correction ( q < 0.05), with eight of these also significantly altered in ΔHIE. Altered putative metabolites included; melatonin, leucine, kynurenine and 3-hydroxydodecanoic acid which differentiated between infant groups (ΔPA and ΔHIE); and D-erythrose-phosphate, acetone, 3-oxotetradecanoic acid and methylglutarylcarnitine which differentiated across severity grades of HIE. Pathway analysis revealed ΔHIE was associated with a 50% and 75% perturbation of tryptophan and pyrimidine metabolism, respectively. We have identified perturbed metabolic pathways and potential biomarkers specific to PA and HIE, which measured at birth, may help direct treatment.

Characterization of Metabolites of α-mangostin in Bio-samples from SD Rats by UHPLC-Q-Exactive Orbitrap MS

2021 ◽  
Vol 22 ◽  
Author(s):  
Fan Dong ◽  
Shaoping Wang ◽  
Ailin Yang ◽  
Haoran Li ◽  
Pingping Dong ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Garcinia Mangostana ◽  
Sd Rats ◽  
Metabolic Route ◽  
Wide Range ◽  
New Strategy ◽  
Q Exactive ◽  
Orbitrap Ms

Background: α-mangostin, a typical xanthone, often exists in Garcinia mangostana L. (Clusiaceae). α-mangostin was found to have a wide range of pharmacological properties. However, its specific metabolic route in vivo remains unclear, while these metabolites may accumulate to exert pharmacological effects, too. Objective: This study aimed to clarify the metabolic pathways of α-mangostin after oral administration to the rats. Methods: Here, an UHPLC-Q-Exactive Orbitrap MS was used for the detection of potential metabolites formed in vivo. A new strategy for the identification of unknown metabolites based on typical fragmentation routes was implemented. Results: A total of 42 metabolites were detected, and their structures were tentatively identified in this study. The results showed that major in vivo metabolic pathways of α-mangostin in rats included methylation, demethylation, methoxylation, hydrogenation, dehydrogenation, hydroxylation, dehydroxylation, glucuronidation, and sulfation. Conclusions: This study is significant to expand our knowledge of the in vivo metabolism of α-mangostin and to understand the mechanism of action of α-mangostin in rats in vivo.

scholarly journals Impact of a Demyelination-Inducing CNS Virus on Expression of Demyelination Genes in Type 2 Lymphoid Cells (ILC2s)

2020 ◽  
Author(s):  
Satoshi Hirose ◽  
Mihoko Kato ◽  
Kati Tormanen ◽  
Pedram Shafiei Jahani ◽  
Omid Akbari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inflammatory Response ◽  
Expression Profiles ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Demyelinating Diseases ◽  
Viral Gene ◽  
Environmental Cues ◽  
Cns Demyelination

We recently reported the role of innate lymphoid cells type 2 (ILC2s) in CNS demyelination using an HSV-IL-2 model of CNS demyelination. Here we studied how ILC2s respond to HSV-IL-2 at the cellular level using cytokine and gene expression profiling. ILC2s infected with HSV-IL-2 expressed higher levels of GM-CSF, IL-5, IL-6, IL-13, IP-10, MIP-2, and RANTES, which include proinflammatory cytokines, than did those infected with parental control virus. In contrast, TH2 cytokines IL-4 and IL-9, which are typically expressed by ILC2s, were not induced upon HSV-IL-2 infection. RNA-Seq analysis of HSV-IL-2 infected ILC2s showed significant upregulation of over 350 genes and downregulation of 157 genes compared with parental virus-infected ILC2s. GO term analysis indicated that genes related to “mitosis” and “inflammatory response” were among the upregulated genes, suggesting that HSV-IL-2 infection drives the excessive proliferation and atypical inflammatory response of ILC2s. This change in ILC2 activation state could underlie the pathology of demyelinating diseases. IMPORTANCE Innate lymphocytes have plasticity and can change functionality; innate lymphoid cells type 2 (ILC2s) can convert to ILC1 or ILC3 cells, or change their activation state to produce IL-17 or IL-10 depending on environmental cues. In this study, we investigated the gene and cytokine profile of ILC2s, which play a major role in HSV-IL-2-induced CNS demyelination. ILC2s infected with HSV-IL-2 displayed a massive remodeling of cellular state. Additionally, ILC2s infected with HSV-IL-2 differed from those infected with parental HSV in cellular and viral gene expression profiles and in cytokine/chemokine induction, and displayed enhanced activation and proinflammatory responses. These changes in ILC2 activation state could underlie the pathology of demyelinating diseases. These results also highlight the possible importance of pathogens as environmental cues to modify innate lymphocyte functionalities.

scholarly journals Urinary proteome of dogs with kidney injury during babesiosis

2019 ◽  
Author(s):  
Dagmara Winiarczyk ◽  
Katarzyna Michalak ◽  
Łukasz Adaszek ◽  
Mateusz Winiarczyk ◽  
Stanisław Winiarczyk
Keyword(s):  
Metabolic Pathways ◽  
Protein Identification ◽  
Transforming Growth Factor ◽  
Inflammatory Responses ◽  
Urine Samples ◽  
Control Group ◽  
Characteristic Analysis ◽  
Mesenchymal Transition ◽  
Healthy Dogs ◽  
Potential Biomarkers

Abstract This study aimed to identify proteins in the urine of dogs with renal dysfunction during the natural course of babesiosis (n=10) and to compare them with proteins in a control group (n=10) to reveal any potential biomarkers of renal damage. Pooled urine samples from both groups were separated by 2D (two-dimensional) electrophoresis, followed by protein identification using MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometry. In total, 176 proteins were identified in the urine samples from healthy dogs, and 403 proteins were identified in the urine samples from dogs with babesiosis. Of the 176 proteins, 146 were assigned exclusively to healthy dogs, and 373 of the 403 proteins were assigned exclusively to dogs with babesiosis; 30 proteins were common to both groups. Characteristic analysis of the 373 proteins found in dogs with babesiosis led to the isolation of 8 proteins associated with 10 metabolic pathways involved in immune and inflammatory responses. Furthermore, it was hypothesized that epithelial-mesenchymal transition might play an important role in the mechanisms underlying pathological changes in renal tissue during babesiosis, as indicated by a causal relationship network built by combining 5 of the 10 selected metabolic pathways and 4 of the 8 proteins associated with these pathways; this network included cadherins, gonadotropin releasing hormone receptors, inflammatory responses mediated by chemokine and cytokine signalling pathways, integrins, interleukins and TGF-β (transforming growth factor β) pathways. These pathways were linked by interleukin-13, bone morphogenetic protein 7, α2(1) collagen, and FER tyrosine kinase, which are potential biomarkers of damage during babesiosis in dogs that might indicate early renal injury.

scholarly journals Comparative study of the effects of gold and silver nanoparticles on the metabolism of human dermal fibroblasts

2020 ◽  
Vol 7 (2) ◽  
pp. 221-232
Author(s):  
Yan Huang ◽  
Xiaoying Lü ◽  
Rong Chen ◽  
Ye Chen
Keyword(s):  
Silver Nanoparticles ◽  
Pathway Analysis ◽  
Metabolic Pathway ◽  
Metabolic Pathways ◽  
Pattern Analysis ◽  
Dermal Fibroblasts ◽  
Human Dermal Fibroblasts ◽  
Metabolic Pathway Analysis ◽  
Citrate Cycle ◽  
Expression Pattern Analysis

Abstract The purpose of this article was to explore the effects of gold nanoparticles (GNPs) and silver nanoparticles (SNPs) with different cytotoxicities on human dermal fibroblasts (HDFs) at the metabolic level. First, ∼20 nm of GNPs and SNPs were prepared, and their effects on the proliferation of HDFs were evaluated. Then, a metabolomics technique was used to analyse the effects of GNPs and SNPs on the expression profiles of metabolites in HDFs after 4, 8 and 24 h of treatment. Furthermore, the key metabolites and key metabolic pathways involved in the interaction of GNPs and SNPs with HDFs were identified through expression pattern analysis and metabolic pathway analysis of differentially expressed metabolites and were finally verified by experiments. The results of the cytotoxicity experiments showed that there was no cytotoxicity after the treatment of GNPs for 72 h, while the cytotoxicity of the SNPs reached grade 1 after 72 h. By using metabolomics analysis, 29, 30 and 27 metabolites were shown to be differentially expressed in HDFs after GNP treatment, while SNPs induced the differential expression of 13, 33 and 22 metabolites after 4, 8 and 24 h of treatment, respectively. Six and four candidate key metabolites in the GNP and SNP groups were identified by expression pattern analysis and metabolic pathway analysis, respectively. The key metabolic pathways in the GNP and SNP groups were identified as the glutathione metabolic pathway (the key metabolite of which was glutathione) and the citrate cycle pathway (the key metabolite of which was malic acid). Based on the experiments used to verify the key metabolites and key metabolic pathways, it was found that the increase in glutathione after GNP treatment might trigger an oxidative stress protection mechanism and thus avoid cytotoxicity. After exposure to SNPs, the citric acid content was increased, mainly through the citrate cycle pathway, thereby inhibiting the synthesis of malic acid to affect the formation of ATP and finally leading to cytotoxicity.

Comparative Genomic Identification of Unique Signal Transduction Pathways and Targets In Pediatric Burkitt Lymphoma (PBL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 631-631
Author(s):  
Nancy S Day ◽  
Janet Ayello ◽  
Ian Waxman ◽  
Carmella van de Ven ◽  
Megan S. Lim ◽  
...  
Keyword(s):  
Pathway Analysis ◽  
Conflicts Of Interest ◽  
Mapk Signaling ◽  
Negative Control ◽  
Support Vector ◽  
Comparative Genomic ◽  
Toll Like Receptor ◽  
Functional Interpretation ◽  
Significant Difference ◽  
Mock Control

Abstract Abstract 631 Background: The prognosis of PBL has dramatically improved over the past 25 years (> 80% 5 yrs EFS) (Cairo et al BJH 2009). This success, however, has come at the cost of the high toxicity associated with intensive multi-agent chemotherapy. A need to identify less toxic targeted therapy exists. Our recent studies show that while almost 100% of childhood BL (238 children) is characterized by an 8q24 (c-myc) gene rearrangement, 70% of the patients also had a secondary chromosomal aberration (Cairo et al Blood 2007), and those children with BL who had a 13q abnormality had a significantly poorer outcome (20% reduction in EFS; Poirel/Cairo et al Leukemia, 2009). Loss of 13q14.3 was associated with a significant decrease in OS (Nelson/Cairo/Perkins/Sanger et al BJH 2010). DLEU1, proven as a Burkitt specific classifier and a cMYC target gene (Dave/Staudt et al NEJM 2006), is located within the region of 13q14.3. DLEU1 is recognized to interact with c-Myc, TUBB2C, UBR1, and other proteins. When DLEU1 is down-regulated by a DLEU1 siRNA, the spontaneous apoptotic rate was decreased with concomitant significantly reduced levels of UBR1 and TUBB2C gene expression (Day/Cairo et al SIOP 2008). Our hypothesis is that deletion of 13q14.3, which contains DLEU1, in pediatric BL may confer a phenotype of chemoimmunotherapy resistance. Objective: To identify potential drug targets using comparative genomic microarray analysis of specimens in patients with PBL. Methods: PBL data from three different research groups were used; i.e. COG ANHL01P1 by our group (n=11), NCBI's GEO GSE10172 and GSE4475 (Klapper et al) (n=16), and GSE4732 (Dave/Staudt et al) (n=15). For comparison at equal weighted level, ANHL01P1 samples were validated by building a prediction model with Support Vector Machines using Klapper PBL as training database. RNAs were subjected to microarray studies (Affymetrix U133A_2) and analyzed by Agilent GeneSpring or Partek. Functional interpretation of the identified PBL genes was analyzed by Ingenuity Pathways Analysis. One-way ANOVA followed by Tukey test was used. To test our hypothesis, we knocked down DLEU1 and investigated cytoxan (CY) and rituximab effect on apoptotic rate of Ramos. Ramos BL cell lines were transiently transfected (24 hrs) with DLEU1 siRNA (5′-AUACUUGGCAUGAAUGAACUUAUGU-3′ and 3′-UAUGAACCGUACUUACUUGAAUACA-5′) (Day/Cairo SIOP 2008). The siRNA transfected cells were then treated with CY (0, 89.5, 895, 8950 nM) or rituximab (0, 4, 40, 400 mg/mL) for additional 4 hrs. Cells were evaluated for percent apoptosis using Annexin V-FITC and Propidium Iodide followed by FACS using BD LSRII. Results: 1565 genes were identified (p<0.05), among which 376 genes showed no significant difference among three groups. High expression of cMYC (27F) and DLEU1 (9.9F) were detected. Pathway analysis indicates that these genes areinvolved in Toll-like receptor signaling (16 genes; p<0.01), including IRAK1 (22.9F), IFNAR2 (5.4F), NFKBIA (15.3F), and STAT1 (9.2F); JAK-STAT signaling (16 genes; p<0.01), including PTPN11 (25.6F), PTPN6 (8.8F), PIM1 (6.4F), and IL21R (2.9F); and MAPK signaling (19 genes; p<0.01) including MAP2K1 (11.8F), MAP3K7 (6.3F), MAPK9 (6.9F), and RAF1 (9.2F). There was a significant reduction in apoptosis in the CY-treated BL transfected DLEU1 siRNA vs mock control cells (89.5 nM CY: 10.26+0.23% reduction, p<0.05 to negative control; 895 nM CY, 10.86+0.67% reduction, p<0.01; 8950 nM, 9.85+0.32% reduction, p<0.05. There was a similar significant reduction in rituximab-induced apoptosis in the BL transfected DLEU1 siRNA vs mock control cells (4 mg/mL Rituximab: 25.45+2.55% reduction, p<0.01 to negative control; 40 mg/mL Rituximab, 18.31+5.13% reduction, p<0.04; 400 mg/mL rituximab, 32.33+1.77% reduction, p<0.02). Conclusions: Our pathway analysis of the 376 BL signature indicates that Toll-like receptor, JAK-STAT, and MAPK signaling pathways play important roles in gene regulation, anti-apoptosis, and proliferation, respectively, in PBL. The siRNA knock down of DLEU1 resulted in significantly less apoptosis on Ramos with CY or rituximab treatment. These data suggest that DLEU1 may in part play an important role in regulation of programmed cell death in BL and suggest a mechanism whereby BL with a 13q- abnormality may have a more aggressive clinical course. As CY or rituximab does not induce apoptosis in DLEU1 knockdown BL, the absence of DLEU1 may, in part, lead to drug resistance. Disclosures: No relevant conflicts of interest to declare.

The Microarray Signature of Rhesus Macaque CD34+CD123 (IL-3 Receptor)+ Hematopoietic Stem/Progenitor Cells Isolated Following Mobilization with Plerixafor Alone or in Combination with Granulocyte Colony-Stimulating Factor (G-CSF)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1301-1301
Author(s):  
Robert E. Donahue ◽  
Ping Jin ◽  
Johanna I. Klinman ◽  
Aylin C. Bonifacino ◽  
Mark E. Metzger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pathway Analysis ◽  
Rhesus Macaques ◽  
Receptor Activation ◽  
Mapk Signaling ◽  
Data Set ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Custom Made ◽  
Lymphoid Progenitors

Abstract Abstract 1301 CD34+ cells are a heterogeneous population of cells which contain the hematopoietic stem cell (HSC). Previously we determined that the microarray signature of CD34+ cells mobilized by G-CSF differed from those mobilized with either plerixafor (AMD3100) alone or in combination with G-CSF (Donahue RE, et al. Blood 2009; 114:2530–2541). Recently we also determined that a novel subpopulation of CD34+CD123+ cells was mobilized with plerixafor and not G-CSF in rhesus macaques (Uchida N, et al. Exp Hematol 2011; 39:795–805). To explore differences between CD34+CD123+ and CD34+CD123- cells, we evaluated their gene expression signatures. Following mobilization with plerixafor alone or in combination with G-CSF, we collected mononuclear cells by leukapheresis, isolated rhesus CD34+ cells by immunoselection, and further isolated CD34+CD123+ and CD34+CD123- by cell sorting. CD34+CD123+ cells were found not to express CXCR4 on their cell surface. Total RNA was isolated from cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye. Control and experimental labeled cRNA were co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. Gene expression was analyzed by quantifying the fluorescence from the microarray. The raw data set was filtered according to a standard procedure to exclude spots with minimum intensity. The relationship between the cells was analyzed using an unsupervised Eisen's hierarchical clustering method. Statistical analysis was done using Array Class Comparison analysis. Pathway analysis was carried out using Ingenuity Pathway Analysis. Principal Component Analysis demonstrated that CD34+CD123+ and CD34+CD123- cells cluster separately and are not distinguishable on the basis of mobilization regimen. Using Supervised Hierarchical Cluster Analysis for genes which were significantly different (p<0.05) there was further confirmation of clustering of the CD34+CD123+ cells versus the CD34+CD123- cells. Microarray analysis revealed that pathways including retinoic acid receptor activation and TGF-β and MAPK signaling were up-regulated in CD34+CD123+ cells, whereas glucocorticoid receptor and TREM-1 signaling, and genes involved in dendritic cell maturation and production of nitric oxide and reactive oxygen by macrophages were down regulated. The up regulated pathways and the down regulated pathways for the CD34+CD123+ population suggest that CD34+CD123+ cells are lymphoid progenitors. Upon comparing pathways between those previously reported to be unique to CD34+ cells mobilized with plerixafor alone or in combination with G-CSF, the majority are those that have been identified also to be in the CD34+CD123+ subpopulation. It appears as if the CD34+CD123+ population accounts for many of the differences in CD34+ cells mobilized with plerixafor observed in the earlier study. In conclusion, the microarray signature of the CD34+CD123+ cells subpopulation suggests that these cells are lymphoid progenitors which can be effectively mobilized with plerixafor. This may account for the more rapid lymphoid recovery we observed for rhesus macaques transplanted with CD34+ cells mobilized with plerixafor and suggests a therapeutic approach through immunoselection to selectively target CD34+CD123+ progenitor populations. Disclosures: No relevant conflicts of interest to declare.

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