Unknown,Transcriptomics,Genomics,Proteomics

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CD34+DNAMbrightCXCR4+ CLP mobilized in chronic inflammation

ABSTRACT: During the course of many chronic inflammatory disorders, a persistent Natural Killer (NK) cell derangement and activation is observed. While increased cell turnover is expected in these cases, little is known about whether and how NK cell homeostatic balance and numbers are maintained by CD34+ progenitor cells. Accordingly, we studied peripheral blood progenitor cells in patients with chronic inflammatory disorders including HIV-1, HCV, TB, COPD and PAPA. Cytometric analysis revealed the presence of a so far unidentified CD34+CD226(DNAM-1)brightCXCR4+ cell population in all these conditions. Further, microarray and PCR analysis showed transcriptional signatures typical of common lymphocyte precursors. Culture of CD34+CD226brightCXCR4+ cells gave rise to NK cell progenies characterized by high expression of activating receptors and mature function. Importantly, in healthy donors CD34+CD226brightCXCR4+ cells reside in bone marrow but do not circulate in peripheral blood and are absent in umbilical cord blood. The proportion of CD34+CD226brightCXCR4+ PBMC were found to correlate with the degree of inflammation, and represent an indicator of lymphoid cell turnover/reconstitution during chronic inflammation. Identification of CD34+CD226brightCXCR4+ circulation offers a novel view of emergency recruitment of cell-precursors upgrade our understanding and monitoring of chronic inflammatory conditions, and provide new insight of intermediate stages of NK cell development. Four samples from CD34+ hematopoietic stem cells of healthy individuals (n=2) and HIV-infected patients (n=2) were analyzed using GeneChip Human Gene 1.0 ST Arrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Maria Ascierto

PROVIDER: E-GEOD-69557 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin- CD34-) hematopoietic stem cells (HSCs) from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR/ABL transcript demonstrated that about one third of CD34- was leukemic. CML CD34- cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures and cytokines induced CD34 expression on some HSCs, cell cycling, acquisition of clonogenic activity and increased expression of BCR/ABL transcript. CML CD34- cells showed an engraftment rate in immunodeficient mice similar to that of CD34+ cells. Gene expression profiling revealed the down-regulation of cell cycle arrest genes together with genes involved in antigen presentation and processing, while the expression of angiogenic factors was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML CD34-cells. Increasing doses of imatinib mesilate (IM) did not affect fusion transcript levels, BCR-ABL kinase activity and the clonogenic efficiency of CML CD34- cells as compared to leukemic CD34+cells. Thus, we identified in CML a novel CD34- leukemic stem cell subset with peculiar molecular and functional characteristics which may be a potential target for CML therapeutics. Leukemic cells were obtained from 12 chronic phase Ph+ CML patients at diagnosis and before treatment. Normal samples were leukapheresis products from 12 healthy stem cell donors receiving recombinant human granulocyte colony-stimulating factor (G-CSF; Lenograstim, Sanofi-Aventis, Milan, Italy). The protocol was approved by the ethical committee of the University Hospital and each patient/donor gave written informed consent. Hemopoietic stem/progenitor cell purification and phenotypic analyses were performed as previously described (Lemoli et al, Br J Haematol, 2003; Lemoli RM et al., Blood, 1997). Aliquots of sorted Lin-CD34-, Lin-CD34+ and Lin+CD34+ were reanalyzed by FacScan (Becton Dickinson, Franklin Lakes, NJ) to assess their purities. Total cellular RNA was extracted from 0.5x105 cells of each sample using RNeasy Micro kit (Qiagen, Valencia, CA) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration and purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. RNAs originating from 12 normal donors or from 12 CML patients were pooled in order to obtain at least 2 mg per sample. One-cycle target labeling assays, as well as the Affymetrix Human HG-U95Av2 GeneChip arrays hybridization, staining, and scanning, were performed, using Affymetrix standard protocols (Affymetrix, Santa Clara,CA).

Project description:CONTEXT Slowly progressive chronic tubulo-interstitial damage jeopardizes long-term renal allograft survival. Both immune and non-immune mechanisms are thought to contribute, but the most promising targets for timely intervention have not been identified. OBJECTIVE In the current study we seek to determine the driving force behind progressive histological damage of renal allografts, without the interference of donor pathology, delayed graft function and acute graft rejection. DESIGN We used microarrays to examine whole genome expression profiles in renal allograft protocol biopsies, and analyzed the correlation between gene expression and the histological appearance over time. The gene expression profiles in these protocol biopsies were then compared with gene expression of biopsies with acute T-cell mediated rejection. PATIENTS Human renal allograft biopsies (N=120) were included: 96 rejection-free protocol biopsies and 24 biopsies with T-cell mediated acute rejection. RESULTS In this highly cross-validated study, we demonstrate the significant association of established, ongoing and future chronic histological damage with regulation of adaptive immune gene expression (T-cell and B-cell transcript sets) and innate immune response gene expression (dendritic cell, NK-cell, mast cell and granulocyte transcripts). We demonstrate the ability of gene expression analysis to perform as a quantitative marker for ongoing inflammation with a wide dynamic range: from subtle subhistological inflammation prior to development of chronic damage, over moderate subclinical inflammation associated with chronic histological damage, to marked inflammation of Banff-grade acute T-cell mediated rejection. CONCLUSION Progressive chronic histological damage after kidney transplantation is associated with significant regulation of both innate and adaptive immune responses, months before the histological lesions appear. This study therefore corroborates the hypothesis that quantitative inflammation below the diagnostic threshold of classic T-cell or antibody-mediated rejection is associated with early subclinical stages of progressive renal allograft damage. We used microarrays to examine whole genome expression profiles in renal allograft protocol biopsies, and analyzed the correlation between gene expression and the histological appearance over time. The gene expression profiles in these protocol biopsies were then compared with gene expression of biopsies with acute T-cell mediated rejection. Human renal allograft biopsies (N=120) were included: 96 rejection-free protocol biopsies and 24 biopsies with T-cell mediated acute rejection.

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CD34+DNAMbrightCXCR4+ CLP mobilized in chronic inflammation

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