Christopher Plumpton

Resistin is expressed in human macrophages and directly regulated by PPARγ activators

Resistin is a cysteine-rich protein postulated to be a molecular link between obesity and type 2 diabetes. The aim of this study was to investigate the role of PPAR gamma in the regulation of resistin expression in human primary macrophages. Fluorescent real-time PCR (Taqman) analysis of resistin expression across a range of human tissues showed that resistin is highly expressed in bone marrow compared to other tissues. Taqman analysis and Western blotting showed that rosiglitazone decreased resistin expression at both the mRNA and protein levels in human primary monocyte-derived macrophages in vitro. Resistin expression was reduced by up to 80% after exposure to 100 nM rosiglitazone for 96 h. Bioinformatics analysis of the genomic sequence upstream of the resistin coding sequence identified several putative PPAR response elements of which one was shown to bind PPAR gamma using electrophoretic mobility shift assays. Our data support a direct role for PPAR gamma in the regulation of resistin expression.

Diversity of Expression of the Sensory Neuron-Specific TTX-Resistant Voltage-Gated Sodium Ion Channels SNS and SNS2

The differential distribution of two tetrodotoxin resistant (TTXr) voltage-gated sodium channels SNS (PN3) and SNS2 (NaN) in rat primary sensory neurons has been investigated. Both channels are sensory neuron specific with SNS2 restricted entirely to those small dorsal root ganglion (DRG) cells with unmyelinated axons (C-fibers). SNS, in contrast, is expressed both in small C-fiber DRG cells and in 10% of cells with myelinated axons (A-fibers). All SNS expressing A-fiber cells are Trk-A positive and many express the vanilloid-like receptor VRL1. About half of C-fiber DRG neurons express either SNS or SNS2, and in most, the channels are colocalized. SNS and SNS2 are found both in NGF-responsive and GDNF-responsive C-fibers and many of these cells also express the capsaicin receptor VR1. A very small proportion of small DRG cells express either only SNS or only SNS2. At least four different classes of A- and C-fiber DRG neurons exist, therefore, with respect to expression of these sodium channels.

Plasticity of TTX-sensitive sodium channels PN1 and brain III in injured human nerves.

Sensory neurones co-express voltage-gated sodium channels that mediate TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) currents, which may contribute to chronic pain after nerve injury. We previously demonstrated that TTX-R channels were decreased acutely in human sensory cell bodies after central axotomy, but accumulated in nerve terminals after peripheral axotomy. We have now studied the TTX-S channels PN1 and Brain III, using specific antibodies for immunohistochemistry, in dorsal root ganglia (DRG) from 10 patients with traumatic central axotomy, nerves from 16 patients with peripheral axotomy, and controls. PN1 showed temporal changes similar to the TTX-R channels in sensory cell bodies of injured DRG. In contrast, Brain III was found only in injured nerves (not control nerves, or control/central axotomy DRG). PN1 and Brain III are distinct targets for novel analgesics.

Sodium channel Nav1.8 immunoreactivity in painful human dental pulp

BackgroundThe tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 (SNS1/PN3) is expressed by nociceptors and may play a role in pain states.MethodsUsing specific antibodies for immunohistochemistry, we studied Nav1.8 – immunoreactivity in human dental pulp in relation to the neuronal marker neurofilament. Human tooth pulp was extracted from teeth harvested from a total of twenty-two patients (fourteen without dental pain, eight patients with dental pain).ResultsFibres immunoreactive for Nav1.8, were significantly increased on image analysis in the painful group: median (range) Nav1.8 to Neurofilament % area ratio, non-painful 0.059 (0.006–0.24), painful 0.265 (0.13–0.5), P = 0.0019.ConclusionNav1.8 sodium channels may thus represent a therapeutic target in trigeminal nerve pain states.

Sodium channel beta1 and beta2 subunits parallel SNS/PN3 alpha-subunit changes in injured human sensory neurons.

Voltage-gated sodium channels consist of a pore-containing alpha-subunit and one or more auxiliary beta-subunits, which may modulate channel function. We previously demonstrated that sodium channel SNS/PN3 alpha-subunits were decreased in human sensory cell bodies after spinal root avulsion injury, and accumulated at injured nerve terminals in pain states. Using specific antibodies for immunohistochemistry, we have now detected sodium channel beta1 and beta2 subunits in sensory cell bodies within control human postmortem sensory ganglia (78% of small/medium (< or = 50 microm) and 68% of large (> or = 50 microm) cells); their changes in cervical sensory ganglia after avulsion injury paralleled those described for SNS/PN3 alpha-subunits. Our results suggest that alpha- and beta-subunits share common regulatory mechanisms, but present distinct targets for novel analgesics.

Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues

Expression of the lymph node homing and CC‐chemokine receptor 7 (CCR7), with L‐selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T‐cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62Lhigh CD44low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7high CD62Lhigh CD44high (central memory) and CCR7low CD62Llow CD44high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short‐term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7high CD4 T cells produced predominantly interleukin (IL)‐2, whereas CCR7low CD4 T cells produced both IL‐2 and interferon‐γ (IFN‐γ). However, in contrast to previously published reports, the CCR7high CD8 T‐cell subpopulation produced both IFN‐γ and IL‐2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down‐regulated CCR7 only after multiple cell divisions, and this coincided with the down‐regulation of CD62L and production of IL‐4 and IFN‐γ. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL‐2‐ and IFN‐γ‐producing cells are CCR7low, while few cytokine‐expressing CCR7high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T‐cell subpopulations, but indicate additional complexity in that CCR7high CD8 T cells also may produce IFN‐γ.

Nerve fibers in lumbar spine structures and injured spinal roots express the sensory neuron-specific sodium channels SNS/PN3 and NaN/SNS2

Study Design. This prospective study examined the innervation of lumbar spine in tissues from patients with lower back pain and spine nerve roots from patients with traumatic brachial plexus injuries. Objectives. To demonstrate the presence of nerve fibers in lumbar spine structures and spine nerve roots, and to determine whether they express the sensory neuron-specific sodium channels SNS/PN3 and NaN/SNS2. Summary of Background Data. The anatomic and molecular basis of low back pain and sciatica is poorly understood. Previous studies have demonstrated sensory nerves in the facet joint capsule and prolapsed intervertebral disc, but not in the ligamentum flavum. The voltage-gated sodium channels SNS/PN3 and NaN/SNS2 are expressed by sensory neurone that mediate pain, but their presence in the lumbar spine is unknown. Methods. Tissue samples of ligamentum flavum (n = 32), facet joint capsule (n = 20), intervertebral disc (n = 15), and spine roots (n = 8) were immunostained with specific antibodies to protein gene product 9.5 (a panneuronal marker), SNS/PN3, and NaN/SNS2. Results. Protein gene product 9.5 immunoreactive nerve fibers were detected in 72% of the ligamentum flavum specimens and 70% of the facet joint capsule specimens, but in only 20% of the intervertebral disc specimens. The study detected SNS/PN3- and NaN/SNS2-positive fibers, respectively, in 28% and 3% of the ligamentum flavum specimens and 25% and 15% of the facet joint capsule specimens. Numerous SNS/PN3- and NaN/SNS2-positive fibers were found in the acutely injured spine roots, and some were still present in the dorsal roots in the chronic state. Conclusions. As the findings showed, SNS/PN3- and NaN/SNS2-immunoreactivity is present in a subset of nerve fibers in lumbar spine structures, including ligamentum flavum, and in injured spine roots. Selective SNS/PN3- and NaN/SNS2-blocking agents may provide new therapy for back pain and sciatica.

LCPTP–MAP kinase interaction: permanent partners or transient associates?

LCPTP (leucocyte-phosphotyrosine phosphatase) is a 42kDa protein tyrosine phosphatase expressed predominantly in haematopoietic cells which has been implicated in the early stages of the T cell receptor signalling pathway. The substrates of LCPTP have been shown to include MAP kinase family members, but it remains unclear whether LCPTP is found in stable constitutive association with these enzymes, or associates transiently during dephosphorylation. Here we report on LCPTP/MAP kinase interactions in CD3-stimulated Jurkat T cells. Pull-downs from Jurkat T cells using a recombinant GST-LCPTP substrate-trap protein, but not wild-type LCPTP show a clear specific association with both ERK1 and ERK2. In Jurkat cells overexpressing LCPTP, a small fraction of cell ERK1 can be immunoprecipitated in stable association with LCPTP. However, in both unstimulated and anti-CD3 antibody stimulated Jurkat T cells, we were unable to demonstrate any constitutive interaction between endogenous LCPTP and any MAP kinase family members. We propose that both ERK1 and ERK2 interact transiently with LCPTP as substrates for the phosphatase rather than as constitutive protein partners.