Expression of the sodium channel beta3 subunit in injured human sensory neurons.

Voltage-gated sodium channel alpha-subunits play a key role in pain pathophysiology, and are modulated by beta-subunits. We previously reported that beta1- and beta2-subunits were decreased in human sensory neurons after spinal root avulsion injury. We have now detected, by immunohistochemistry, beta3-subunits in 82% of small/medium and 67% of large diameter sensory neurons in intact human dorsal root ganglia: 54% of beta3 small/medium neurons were NGF receptor trkA negative. Unlike beta1- and beta2, beta3-immunoreactivity did not decrease after avulsion injury, and the beta3:neurofilament ratio was significantly increased in proximal injured human nerves. beta3-subunit expression may thus be regulated differently from beta1, beta2 and Nav1.8. Targeting beta3 interactions with key alpha-subunits, particularly Nav1.3 and Nav1.8, may provide novel selective 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-gamma (IFN-gamma). However, in contrast to previously published reports, the CCR7high CD8 T-cell subpopulation produced both IFN-gamma 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-gamma. 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-gamma-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-gamma.

Decreased potassium channel IK1 and its regulator neurotrophin-3 (NT-3) in inflamed human bowel.

Calcium-activated potassium currents of intermediate conductance (IK1) have been described in the rodent enteric nervous system, where they may regulate afterhyperpolarisation of intrinsic primary afferent neurons. Using specific antibodies for immuno-cytochemistry, we now report IK1-like immunoreactivity for the first time in enteric neurons of human colon, and a significant decrease of IK1-positive cells in myenteric plexus in inflamed colon from patients with Crohn's disease and ulcerative colitis (p = 0.031). Neurotrophin-3 (NT-3), which regulates IK1 expression, was also observed in fewer neurons of the myenteric ganglia in Crohn's bowel (p = 0.048), and in inflamed colonic extracts by Western blotting (p = 0.004); the numbers of neurons expressing the NT-3 high affinity receptor trk C were unchanged. Our findings may explain the diarrhoea and colicky abdominal pain produced by inflammatory bowel disease, and by IK1-blocking pyridine drugs prescribed for neuromuscular disorders.

Resistin is expressed in human macrophages and directly regulated by PPAR gamma 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.

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.

The novel product of a five-exon stargazin-related gene abolishes Ca(V)2.2 calcium channel expression.

We have cloned and characterized a new member of the voltage-dependent Ca(2+) channel gamma subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential gamma subunits identified by their homology to the stargazin gene, CACNG7 is a five-, and not four-exon gene whose mRNA encodes a protein we have designated gamma(7). Expression of human gamma(7) has been localized specifically to brain. N-type current through Ca(V)2.2 channels was almost abolished when co-expressed transiently with gamma(7) in either Xenopus oocytes or COS-7 cells. Furthermore, immunocytochemistry and western blots show that gamma(7) has this effect by causing a large reduction in expression of Ca(V)2.2 rather than by interfering with trafficking or biophysical properties of the channel. No effect of transiently expressed gamma(7) was observed on pre-existing endogenous N-type calcium channels in sympathetic neurones. Low homology to the stargazin-like gamma subunits, different gene structure and the unique functional properties of gamma(7) imply that it represents a distinct subdivision of the family of proteins identified by their structural and sequence homology to stargazin.

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.