Data from: Extracellular matrix-associated gene expression in adult sensory neurons cultured on laminin substrates

Background: In our previous investigations of the role of the extracellular matrix (ECM) in promoting neurite growth we have observed that a permissive laminin (LN) substrate stimulates differential growth responses in subpopulations of mature dorsal root ganglion (DRG) neurons. DRG neurons expressing Trk and p75 receptors grow neurites on a LN substrate in the absence of neurotrophins, while isolectin B4-binding neurons (IB4+) do not display significant growth under the same conditions. We set out to determine whether there was an expression signature of the LN-induced neurite growth phenotype. Using a lectin binding protocol IB4+ neurons were isolated from dissociated DRG neurons, creating two groups - IB4+ and IB4-. A small-scale microarray approach was employed to screen the expression of a panel of ECM-associated genes following dissociation (t=0) and after 24 hr culture on LN (t=24LN). This was followed by qRT-PCR and immunocytochemistry of selected genes. Results: The microarray screen showed that 36 of the 144 genes on the arrays were consistently expressed by the neurons. The array analyses showed that six genes had lower expression in the IB4+ neurons compared to the IB4- cells at t=0 (CTSH, Icam1, Itgβ1, Lamb1, Plat, Spp1), and one gene was expressed at higher levels in the IB4+ cells (Plaur). qRT-PCR was carried out as an independent assessment of the array results. There were discrepancies between the two methods, with qRT-PCR confirming the differences in Lamb1, Plat and Plaur, and showing decreased expression of AdamTs1, FN, and Icam in the IB4+ cells at t=0. After 24 hr culture on LN, there were no significant differences detected by qRT-PCR between the IB4+ and IB4- cells. However, both groups showed upregulation of Itgβ1 and Plaur after 24 hr on LN, the IB4+ group also had increased Plat, and the IB4- cells showed decreased Lamb1, Icam1 and AdamTs1. Further, the array screen also detected a number of genes (not subjected to qRT-PCR) expressed similarly by both populations in relatively high levels but not detectably influenced by time in culture (Bsg, Cst3, Ctsb, Ctsd, Ctsl, Mmp14, Mmp19, Sparc. We carried out immunohistochemistry to confirm expression of proteins encoded by a number of these genes. Conclusions:Our results show that 1B4+ and IB4- neurons differ in the expression of several genes that are associated with responsiveness to the ECM prior to culturing (AdamTs1, FN, Icam1, Lamb1, Plat, Plaur). The data suggest that the genes expressed at higher levels in the IB4- neurons could contribute to the initial growth response of these cells in a permissive environment and could also represent a common injury response that subsequently promotes axon regeneration. The differential expression of several extracellular matrix molecules (FN, Lamb1, Icam) may suggest that the IB4- neurons are capable of maintaining /secreting their local extracellular environment which could aid in the regenerative process. Overall, these data provide new information on potential targets that could be manipulated to enhance axonal regeneration in the mature nervous system

ECM IB4+ t=24LN expressed genes reps1-6

Data showing genes detected on microarrays for IB4-expressing dorsal root ganglion neurons at t=24LN (sampled after 24 hr culture on laminin-coated substrate). See Read-me file for details. Only genes detected above background are listed; of 144 ECM genes on the arrays, only 36 genes were expressed by both neuron populations

ECM IB4neg t=0 expressed genes reps1-7

Data for non-IB4 expressing DRG neurons at t=0; these were sampled immediately after the selection protocol that removes the IB4-expressing neurons, leaving the IB4-negative neurons behind. Only genes detected as being expressed are presented.Of 144 ECM genes on the arrays, 36 genes were expressed by both neuron populations

ECM IB4neg t=24LN expressed genes reps1-6

Data for non-IB4 expressing DRG neurons at t=24LN; these were sampled after 24 hr culture on laminin-coated substrate. Only genes detected as being expressed are presented.Of 144 ECM genes on the arrays, 36 genes were expressed by both neuron populations

List of genes on microarray ORN-013.2

Complete list of genes on GEArray ORN-013.

ECM IB4+ t=0 expressed genes reps1-7

Data showing genes detected on microarrays for IB4-expressing dorsal root ganglion neurons at t=0 (smapled immediately after selection protocol). See Read-me file for details. Only genes detected above background are listed; of 144 ECM genes on the arrays, only 36 genes were expressed by both neuron populations

List of genes on microarray ORN-013

Complete list of genes on GEArray ORN-01

Cytomegalovirus-Driven Adaption of Natural Killer Cells in NKG2Cnull Human Immunodeficiency Virus-Infected Individuals

Expansion of natural killer (NK) cells expressing NKG2C occurs following human cytomegalovirus (HCMV) infection and is amplified by human immunodeficiency virus (HIV) co-infection. These NKG2C-expressing NK cells demonstrate enhanced CD16-dependent cytokine production and downregulate FcεRIγ and promyelocytic leukemia zinc finger protein (PLZF). Lacking NKG2C diminishes resistance to HIV infection, but whether this affects NK cell acquisition of superior antibody-dependent function is unclear. Therefore, our objective was to investigate whether HCMV-driven NK cell differentiation is impaired in NKG2Cnull HIV-infected individuals. Phenotypic (CD2, CD16, CD57, NKG2A, FcεRIγ, and PLZF expression) and functional (cytokine induction and cytotoxicity) properties were compared between HIV–infected NKG2Cnull and NKG2C-expressing groups. Cytokine production was compared following stimulation through natural cytotoxicity receptors or through CD16. Cytotoxicity was measured by anti-CD16-redirected lysis and by classical antibody-dependent cell-mediated cytotoxicity (ADCC) against anti-class I human leukocyte antigen (HLA) antibody-coated cells. Our data indicate highly similar HCMV-driven NK cell differentiation in HIV infection with or without NKG2C. While the fraction of mature (CD57pos) NK cells expressing CD2 (p = 0.009) or co-expressing CD2 and CD16 (p = 0.03) was significantly higher in NKG2Cnull HIV-infected individuals, there were no significant differences in NKG2A, FcεRIγ, or PLZF expression. The general phenotypic and functional equivalency observed suggests NKG2C-independent routes of HCMV-driven NK cell differentiation, which may involve increased CD2 expression