Ronald H. Stead

MyoD or Myf-5 is required for the formation of skeletal muscle

Mice carrying null mutations in the myogenic regulatory factors Myf-5 or MyoD have apparently normal skeletal muscle. To address whether these two factors functionally substitute for one another in myogenesis, mice carrying mutant Myf-5 and MyoD genes were interbred. While mice lacking both MyoD and Myf-5 were born alive, they were immobile and died soon after birth. Northern blot and S1 nuclease analyses indicated that Myf-5(-1-);MyoD(-1-) mice expressed no detectable skeletal muscle-specific mRNAs. Histological examination of these mice revealed a complete absence of skeletal muscle. Immunohistochemical analysis indicated an absence of desmin-expressing myoblast-like cells. These observations suggest that either Myf-5 or MyoD is required for the determination of skeletal myoblasts, their propagation, or both during embryonic development and indicate that these factors play, at least in part, functionally redundant roles in myogenesis.

Dissecting the role of sodium currents in visceral sensory neurons in a model of chronic hyperexcitability using Nav1.8 and Nav1.9 null mice.

Tetrodotoxin‐resistant (TTX‐R) sodium currents have been proposed to underlie sensory neuronal hyperexcitability in acute inflammatory models, but their role in chronic models is unknown. Since no pharmacological tools to separate TTX‐R currents are available, this study employs Nav1.8 and Nav1.9 null mice to evaluate these currents roles in a chronic hyperexcitability model after the resolution of an inflammatory insult. Transient jejunitis was induced by infection with Nippostrongylus brasiliensis (Nb) in Nav1.9 and Nav1.8 null, wild‐type and naïve mice. Retrogradely labelled dorsal root ganglia (DRG) neurons were harvested on day 20–24 post‐infection for patch clamp recording. Rheobase and action potential (AP) parameters were recorded as measures of excitability, and Nav1.9 and Nav1.8 currents were recorded. DRG neuronal excitability was significantly increased in post‐infected mice compared to sham animals, despite the absence of ongoing inflammation (sham = 1.9 ± 0.3, infected = 3.6 ± 0.7 APs at 2× rheobase, P= 0.02). Hyperexcitability was associated with a significantly increased amplitude of TTX‐R currents. Hyperexcitability was maintained in Nav1.9−/− mice, but hyperexcitability was absent and APs were blunted in Nav1.8−/− mice. This study identifies a critical role for Nav1.8 in chronic post‐infectious visceral hyperexcitability, with no contribution from Nav1.9. Nb infection‐induced hyperexcitability is not observed in Nav1.8−/− mice, but is still present in Nav1.9−/− mice. It is not clear whether hyperexcitability is due to a change in the function of Nav1.8 channels or a change in the number of Nav1.8 channels.

Vagal influences over mast cells

The established microanatomical association of rat intestinal mucosal mast cells (IMMC) and mucosal nerves raises the possibility that there is crosstalk between mast cells and extrinsic nerves that connect to the CNS. The idea of mast cell-CNS interactions is supported by the demonstration that rat mast cell protease II (RMCPII), found predominantly in IMMC, can be conditionally released by pairing an audio-visual cue with antigen challenge. That the vagus nerve is involved in the IMMC-nerve axis was further demonstrated in a series of our studies showing that: (a) vagal afferents penetrate the small intestinal mucosa and contact IMMC; (b) vagotomy causes a reduction in IMMC density, suggesting a trophic relationship (typical of nerve-target interactions); and (c) stimulation of the cervical vagus causes an increase in histamine and serotonin in IMMC. To further investigate the IMMC-nerve axis in a model of post-inflammatory bowel disorders, infection with Nippostrongylus brasiliensis (Nb) was used to demonstrate an increase in mast cell numbers in the intestinal mucosa and mucosal nerve remodelling with hyperinnervation. Administration of Nb antigen resulted in dramatic increases in mesenteric afferent nerve firing in Nb infected rats, that was absent in sham animals. Moreover, challenge of post-Nb rats with 2-methyl-5HT caused increased mesenteric afferent firing, indicating that vagal afferent innervation remains intact in the post-infection state. These data suggest a functional connection between mast cells and extrinsic afferent nerves. Nb infection provides a useful model of altered communication between IMMCs, peripheral nerves and the CNS, as may occur in post-inflammatory disease states. Since a close anatomical relationship has also previously been demonstrated between nerves and IMMC in humans, further understanding the mast cell-nerve axis may be of critical importance in the development of treatments for various human disease states, including functional bowel disorders.

Activation of the cannabinoid 2 (CB2) receptor inhibits murine mesenteric afferent nerve activity

Abstract  Cannabinoid 2 (CB2) receptors have both antinociceptive and antihypersensitivity effects, although the precise mechanisms of action are still unclear. In this study, the modulatory role of CB2 receptors on the mesenteric afferent response to the endogenous immunogenic agent bradykinin (BK) was investigated. Mesenteric afferent recordings were obtained from anaesthetized wild‐type and CB2−/− mice using conventional extracellular recording techniques. Control responses to BK were obtained in all experiments prior to administration of either CB2 receptor agonist AM1241, or AM1241 plus the CB2 receptor antagonist AM630. Bradykinin consistently evoked activation of mesenteric afferents (n = 32). AM1241 inhibited the BK response in a dose dependent manner. In the presence of AM630 (10 mg kg−1), however, AM1241 (10 mg kg−1) had no significant effect on the BK response. Moreover, AM1241 had also no significant effect on the BK response in CB2−/− mice. Activation of the CB2 receptor inhibits the BK response in mesenteric afferents, demonstrating that the CB2 receptor is an important regulator of neuroimmune function. This may be a mechanism of action for the antinociceptive and antihypersensitive effects of CB2 receptor agonists.

Impact of 5-HT3 receptor blockade on colonic haemodynamic responses to ischaemia and reperfusion in the rat

Abstract  5‐HT3 receptor antagonists are clinically available for treating patients with irritable bowel syndrome (IBS) but their use is restricted because of a link with some episodes of ischaemic colitis. However, the role of 5‐HT3 receptors in regulating colonic blood flow has not been systematically investigated. Thus, we examined acute and chronic treatment with alosetron, a potent and selective antagonist of the 5‐HT3 receptor, on baseline colonic blood flow and haemodynamic responses during occlusion and reactive hyperaemia in the pentobarbitone‐anaesthetized rat. Colonic haemodynamics were assessed using ultrasonic recordings of superior mesenteric blood flow (MBF) and laser Doppler recordings of colonic vascular perfusion (VP). Blood pressure (BP) was also monitored and in some experiments tissue oxygen was detected polarographically. Alosetron (10, 30, 100 μg kg−1, i.v.) had no effect on baseline haemodynamics nor responses to nitric oxide synthase inhibition with Nω‐nitro‐l‐arginine methyl ester (l‐NAME) (16 mg kg−1). Arterial occlusion (5 min) reduced MBF (−98.6 ± 0.6%) and VP (−70.7 ± 5.4%) followed by a post‐occlusion reactive hyperaemia (MBF = +94.5 ± 19.1%; VP = +60.0 ± 22.3%) the magnitude of which was unchanged following acute (30 μg kg−1) or chronic alosetron administration (0.5 mg kg−1 twice daily, 5 days). Alosetron did not significantly alter baseline colonic blood flow in the anaesthetized rat; nor did it interfere with vascular control mechanisms activated during occlusion and reactive hyperaemia.

Interaction of the Mucosal Immune and Peripheral Nervous Systems

This chapter discusses the interaction of the mucosal immune and peripheral nervous systems. The extensive microanatomical relationships between mast cells and nerves, especially the correlation between the density of these two cell types and the physiological evidence for communication between them, has led to search for growth factors that might sustain both mast cells and nerves. The multitude of different neurotransmitter substances potentially released in mucosal tissues could have a multiplicity of effects on the immune system. The circuitry could be complex, or, perhaps, the delivery of messages from one cell to another could be augmented by local concentration effects such as those seen at the site of release from nerve varicosities. Neuroendocrine complexes have also been described in the lamina propria in the vermiform appendix, and scattered, sparsely granulated neuroendocrine cells have been reported in the gastric mucosa. The complex cellular and molecular circuitry of the in vivo situation might overcome the events observed in vitro . Perhaps changes in vascular permeability and the expression of leukocyte adhesion molecules, which result in the migration of immune cells at a site of injury, may be of considerably more significance than activation of protein synthetic and proliferative pathways, at least in the acute phase.

Comparison of Dako fluorescence in situ hybridization assays (FISH and IQFISH) in the assessment of HER2 amplification in breast cancer

Abstract Determining human epidermal growth factor receptor 2 (HER2) status in breast cancer patients can be attained by fluorescent in situ hybridization (FISH) analysis, which prompts targeted treatment options. This study was performed to compare the staining results of Dako’s manual overnight HER2 FISH pharmDx™ assay with the manual (4 hours) HER2 IQFISH pharmDx™ assay and the Dako Omnis automated (4 hours) HER2 IQFISH assay, for assessment of HER2 amplification. Thirty-four formalin-fixed paraffin-embedded (FFPE) tissue blocks from patients diagnosed with invasive breast cancer were used to compare three FISH assays in three experiments. All assays included Texas Red-labeled HER2 and fluorescein-labeled centromere 17 (CEN-17) probes. First, 20 blocks with known HER2 immunohistochemistry (IHC) and FISH results from a reference laboratory were used in the validation of HER2 FISH pharmDx™ assay. Secondly, 19 of the same blocks were used in a comparison of HER2 FISH pharmDx™ assay and HER2 Instant Quality FISH pharmDx (IQFISH pharmDx™) assays. The third experiment, which included 23 blocks, compared the HER2 FISH pharmDx™ assay with the Dako Omnis IQFISH assay (stained on a Beta version of Dako’s new Omnis platform). Stained slides were counted following American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines (2007 version), with three categories of labeling: <1·8 (negative), 1·8–2·2 (equivocal), and >2·2 (positive) calculated HER2/CEN-17 ratio. The results showed high agreement between all assays employed. Initial validation of HER2 FISH pharmDx™ assay to the results provided by a reference laboratory had a concordance of 95%. The concordance between HER2 FISH pharmDx™ assay and HER2 IQFISH pharmDx™ assay was 100%. There was also 100% concordance between manual and Omnis IQFISH staining. A high level of concordance was obtained in all three experiments, indicating that manual and automated IQFISH are comparable to HER2 FISH pharmDx™. IQFISH has a faster turn-around-time (TAT) and showed comparable results in quality and quantitation.

Neuronal Interaction with Mast Cells

There is significant information available in the literature suggesting that nerves can influence mast cells. Neurotransmitters are able to cause mast cells to secrete mediators in vitro. Stimulation of nerves can result in mast cell degranulation. Stimulation of nerves ex vivo by field stimulation can result in mast cell degranulation with consequent physiological effects. In experimental situations antigen will cause IgE-dependent/mast cell-dependent/nerve-dependent effects both on intestinal and respiratory tract epithelial cell integrity and physiological changes recorded as short circuit current in Ussing chambers. In vitro co-culture of mast cells with nerves grown from superior cervical ganglia show selective association and contact with mast cells, again with physiological consequences. In Pavlovian conditioning experiments of rats, we have been able to show after suitable training, associating antigen injection in sensitized animals with an appropriate stimulus, that on exposure to the stimulus alone, intestinal mast cell degranulation occurred with physiological consequences. Many of these results suggest a bi-directional communication between mast cells and nerves and it is likely that these may have, by extrapolation, significant implications for human physiology and disease.

Nerve growth factor (NGF) receptor expression in human gut associated lymphoid tissues

NGF is a protein required for the survival and differentiation of sympathetic and sensory neurones1. Recently, it has become clear that NGF may have broader biological activity, including effects on immune cells2. NGF has been shown to enhance mitogen stimulated lymphocyte proliferation, when added to rat spleen mononuclear cell cultures3. The cell type mediating this response, presumably through binding to an NGF receptor (NGFR), is not known. In the human, data concerning the effects on NGF on lymphoid cells is not available. However, a recent study by Garin Chesa et al4 demonstrated strong NGFR-immunoreactivity in follicles in lymph nodes and spleen. Because of our interest in mucosal immunity, we decided to see if any cells in the germinal centres of mucosa associated lymphoid tissues (MALT) also expressed the NGFR.