c-Myc antigens in the mammalian enteric nervous system.

We have investigated the presence of c-Myc-like antigens in the enteric nervous system of the guinea-pig, rat, dog, sheep, monkey and human. c-Myc-like immunoreactivity was demonstrated by immunohistochemistry in the enteric nervous system of all animals tested, with one or more monoclonal antibodies raised against peptide sequences found in the human c-Myc protein. While in most cases the labelling was nuclear, cytoplasmic labelling was also observed. In the guinea-pig enteric nervous system, c-Myc-like immunoreactivity detected by two different antibodies remained detectable for up to 4 h in the presence of cycloheximide. The size and density of labelled nuclei in the ileal submucous plexus were consistent with exclusive neuronal labelling by one antibody and neuronal plus glial labelling by the other. Double-labelling with antiserum directed against vasoactive intestinal peptide revealed a subset of c-Myc-immunoreactive neurons that also contain this neuropeptide. Anti-c-Myc antibodies specifically immunoprecipitated proteins from guinea-pig myenteric plexus-longitudinal muscle preparations whose sizes were consistent with previous observations for c-Myc antigens and whose distribution was consistent with synthesis in the myenteric plexus. We conclude that c-Myc proteins are expressed in mammalian enteric neurons and that they have characteristics similar to those of c-Myc proteins in other nonproliferative cells.

Effect of intracolonic benzalkonium chloride on trinitrobenzene sulphonic acid-induced colitis in the rat.

BACKGROUND: We investigated the effects of benzalkonium chloride (BAC) on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats. METHODS: TNBS was administered intrarectally before and/or after BAC treatment. In the first study, the effects of treatment with BAC 6, 12 or 24 h after TNBS were examined. In the second study, animals were treated with BAC before, after or before and after TNBS, and were examined 7 days later. The severity of colitis was assessed by macroscopic and histological scoring of the colonic damage and by determination of colonic myeloperoxidase (MPO) activity. Macrophages and CD4+ and CD8+ T cells were examined by immunohistochemistry. RESULTS: When BAC was instilled into the colon 6, 12 or 24 h after TNBS, weight loss and macroscopic and histological features of the colon were similar to that of controls (TNBS alone). In contrast, MPO activity was significantly reduced in all three groups post-treated with BAC. In the groups examined 7 days after TNBS treatment, rats post-treated with BAC exhibited increased weight gain and significantly reduced macroscopic damage and MPO activity compared to the TNBS control group. Rats pre-treated with BAC exhibited less macroscopic damage of the colon than rats receiving only TNBS, but histological damage, MPO and weight gain were unchanged from TNBS controls. Immunohistochemistry revealed that BAC pre-treatment increased the numbers of macrophages and T cells in the colon. After TNBS treatment, macrophage accumulation was evident in the colon, but T cells were scarce. However, these cells were preserved or enhanced in the colonic mucosa in TNBS-treated rats that had been pre-treated with BAC. CONCLUSIONS: Treatment with BAC, particularly after induction of colitis, produces a significant reduction in the severity of tissue injury and inflammation through mechanisms that are not fully understood.

c-Fos- and JunB-immunoreactivities in the enteric nervous system of the guinea-pig ileum.

We examined the expression of c-Fos and JunB in four immunohistochemical subtypes of enteric neurones in the guinea-pig ileum. In whole mount preparations of the myenteric and submucous plexuses from isolated segments incubated in normal Krebs' solution, increased numbers of many cells expressed visible c-Fos- and JunB-immunoreactivities. These increases may have been associated with the process of isolation and/or incubation conditions. Depolarizing stimulation by veratridine or 50 mM K+ induced further increases of neuronal c-Fos and JunB expression with no obvious subtype preference. This probably reflected a non-specific activation of most enteric neurones by these stimuli and supports the idea that expression of c-Fos and JunB in most or all enteric neurones may be a useful determinant of activation.

The expression and role of Fas ligand in intestinal inflammation.

Fas ligand (FasL) is involved in the pathogenesis of inflammatory diseases and immune privilege. We examined the expression of FasL in the enteric nervous system (ENS) in murine colitis and guinea-pig ileitis. We studied FasL immunoreactivity, functional integrity of the ENS, severity of colitis, and distribution of neutrophils in wild type and B6/gld mice that lack functional FasL. In ileitis, the distribution of FasL, CD4+ and CD8+ T cells was examined. FasL expression was increased in the ENS of wild type mice with colitis, but decreased labelling of nerve fibres was noted in B6/gld mice. Neutrophils were more abundant and widely distributed in B6/gld mice. Colitis was more severe and persistent in B6/gld mice 7 days after induction. Functional parameters of intestinal secretion and motility in B6/gld mice were the same as controls. In ileitis, FasL expression was increased in the guinea-pig ENS and returned to control levels following the resolution of inflammation. While T cells were not present in the ENS of controls, they were observed during inflammation, but were excluded from ganglia. The number of enteric neurons was unchanged over the course of inflammation. The expression of FasL is altered in intestinal inflammation and contributes to its resolution in experimental colitis.

Hay fever treated with ACTH gel.

ACTH gel was used as a treatment for hay fever during the 1972-1974 hay fever seasons. Patients suffering from hay fever were given ACTH in different dose schedules and other hay fever patients were used as controls. Subjectively, those treated with ACTH gel were pleased with the results and reported more favourably on the injections than those receiving placebo. Objectively, using presence of any symptoms as the index of measurement, those treated with ACTH gel (80 units once weekly or 80 units twice weekly) had a significantly lower incidence of hay fever. Side effects were few.

Multiple mechanisms contribute to myenteric plexus ablation induced by benzalkonium chloride in the guinea-pig ileum.

Ablation of rat myenteric plexus with benzalkonium chloride has provided a model of intestinal aganglionosis, but the degenerative responses are not well understood. We examined the effects of this detergent on neurons and glia, including expression of c-Myc, c-Jun, JunB, and c-Fos, and on immunocytes in the guinea-pig ileum. Benzalkonium chloride (0.1%) or saline was applied to the serosal surface of distal ileum. Tissues were analyzed 2, 3, or 7 days later and compared with cyclosporine-treated and untreated animals. More than 90% of myenteric neurons were destroyed in ileal segments 3-7 days after benzalkonium-chloride treatment. Glia withdrew processes from around neurons after 2 days and were mostly gone after 3 days. Neuronal c-Myc began to disappear while c-Fos, c-Jun, and JunB were evident in some neuronal nuclei after 2 or 3 days. After 3 days, widespread apoptosis was evident in the myenteric plexus. Populations of T cells, B cells, and macrophage-like cells in untreated and saline-treated myenteric plexuses were substantially increased 3 and 7 days after benzalkonium-chloride treatment. Cyclosporine delayed significant neuronal loss. We conclude that a variety of degenerative mechanisms may be active in this model, including an immune response which may actively contribute to tissue destruction.

The use of constitutive nuclear oncoproteins to count neurons in the enteric nervous system of the guinea pig.

Immunohistochemical double labelling of the enteric nervous system of the guinea pig ileum was performed with a monoclonal antibody (anti-MYC 033) directed against a peptide sequence of the human c-Myc protein together with antibodies directed against either the neuron-specific antigens neuron-specific enolase or PGP 9.5 or the glia-specific marker S-100 to demonstrate that anti-MYC 033 labelled the nuclei of all enteric neurons but not glia. This strategy was also employed to demonstrate that another anti-c-Myc monoclonal antibody, anti-MYC 070, labelled the nuclei of all neurons and glia, as well as perhaps all other cells in these preparations. A polyclonal antiserum raised against a peptide sequence of the human c-Fos protein (anti-FOS 4) was shown to label the identical nuclei as anti-MYC 033. The ganglionic density of nuclei labelled by anti-FOS 4 was found to be similar to previous measures of the ganglionic density of neurons. Double labelling with anti-MYC 033 and an antiserum directed against vasoactive intestinal polypeptide was performed to reexamine the ganglionic density of neurons that express this neuropeptide. Our results suggest that the ganglionic density of these neurons might be less than previously determined.

Immunohistochemically-defined subtypes of neurons in the inferior mesenteric ganglion of the guinea-pig.

The distribution of somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), tyrosine hydroxylase (a marker of noradrenergic neurons, NA) and nitric oxide synthase-immunoreactivity (NOS-IR) was examined in the inferior mesenteric ganglion of guinea pigs with double- and triple-labelling immunohistochemistry. About 75% of neurons identified were NA/SOM, almost 20% were NA/NPY and the remainder consisted of small groups of NA/- (1-5%), NA/NPY/SOM (2-5%) and VIP (1-2%) neurons. VIP neurons contained NPY-IR, usually contained SOM-IR and were surrounded by dense pericellular baskets of SP fibres. NOS-IR was found in a small proportion of neurons colocalized with VIP but both NOS- and VIP-IR were also found alone in some neurons. Some NOS reactive varicose fibres throughout the ganglia also contained VIP-IR but much of the NOS- and VIP-IR appeared to be localized in discrete varicosities. SOM-IR was also detectable in TH fibres within myenteric ganglia of the distal colon. We conclude that the subtypes of neurons in the inferior mesenteric ganglion share some properties with other sympathetic and abdominal ganglia but they exist in distinct proportions and may make dissimilar projections along the length of the gut.

Comparison of a low and high dose ACTH gel in the treatment of hay fever.

The present data show that in fourteen patients treated with 200 iu Acthar gel, hay fever symptoms were less during the period of peak pollen challenge during June, than those experienced by an equivalent group treated with 80 iu. Analysis of the data showed that in the seven worst sufferers in both groups the difference between treatments was statistically significant (P=0.033, estimated by analysis of covariance of the symptom scores over the period of peak pollen challenge). No serious side effects were noted in patients from either group.

Effects of inflammation on cell proliferation in the myenteric plexus of the guinea-pig ileum.

This study was undertaken to investigate the occurrence and identity of dividing cells within the myenteric plexus of the guinea-pig ileum and to analyze the effects of inflammation induced by trinitrobenzene sulfonic acid. The incorporation of 5-bromo-2-deoxyuridine, or BrdU, into replicating DNA was used to label proliferative cells, and immunohistochemistry was used to assess the occurrence of BrdU in specific cells of the myenteric plexus. Compared to normal tissue, inflammation is associated with increased BrdU labelling in the crypts and in the substantially thickened muscle layers. In longitudinal muscle/myenteric plexus whole-mounts, there was a significant increase in BrdU labelling of the myenteric plexus after induced inflammation. No BrdU-labelled neurons were detected in tissue double labelled with neuron-specific antibodies. Fluorescein isothiocyanate/dextran-labelled macrophages were rare or absent from the ganglia, and none were double labelled with BrdU in the muscle layers. CD3-immunoreactive T cells were substantially increased in the inflamed longitudinal muscle, but still rare or absent within the enteric ganglia. Some BrdU-labelled T cells were observed in the longitudinal muscle but not in the myenteric ganglia. Lastly, in tissues double labelled with anti-BrdU and anti-S-100, many BrdU-containing cells within the myenteric ganglia were found to be S-100-immunoreactive glial cells. We conclude that inflammation does not stimulate the appearance of new myenteric neurons but does stimulate mitosis in myenteric glia.

Immediate-early gene expression in the inferior mesenteric ganglion and colonic myenteric plexus of the guinea pig.

Activation of neurons in the inferior mesenteric ganglion (IMG) was assessed using c-fos, JunB, and c-Jun expression in the guinea pig IMG and colonic myenteric plexus during mechanosensory stimulation and acute colitis in normal and capsaicin-treated animals. Intracolonic saline or 2% acetic acid was administered, and mechanosensory stimulation was performed by passage of a small (0.5 cm) balloon either 4 or 24 hr later. Lower doses of capsaicin or vehicle were used to activate primary afferent fibers during balloon passage. c-Jun did not respond to any of the stimuli in the study. c-fos and JunB were absent from the IMG and myenteric plexus of untreated and saline-treated animals. Acetic acid induced acute colitis by 4 hr, which persisted for 24 hr, but c-fos was found only in enteric glia in the myenteric plexus and was absent from the IMG. Balloon passage induced c-fos and JunB in only a small subset of IMG neurons and no myenteric neurons. However, balloon passage induced c-fos and JunB in IMG neurons (notably those containing somatostatin) and the myenteric plexus of acetic acid-treated animals. After capsaicin treatment, c-fos and JunB induction by balloon passage was inhibited in the IMG, but there was enhanced c-fos expression in the myenteric plexus. c-fos and JunB induction by balloon stimulation was also mimicked by acute activation of capsaicin-sensitive nerves. These data suggest that colitis enhances reflex activity of the IMG by a mechanism that involves activation of both primary afferent fibers and the myenteric plexus.

The origin and distribution of neurons with projections passing through the inferior mesenteric ganglion of the guinea-pig.

Retrograde tracing with the fluorescent dye, Fast Blue, was used to examine the origin and distribution of neurons whose axons project through the inferior mesenteric ganglion (IMG) of the guinea-pig. These studies were performed by applying the tracer to (a) the rostral cut-end of the hypogastric nerves and (b) the caudal cut-end of the inter-mesenteric nerve (IMN). After application of tracer to the hypogastric nerves retrogradely labelled cell profiles were observed in the IMG and the superior mesenteric ganglion (SMG). The number of labelled cell profiles in the SMG was consistently about 15% of the number in the IMG. In only one of seven animals tested were labelled cells seen in the wall of the colon. Application of tracer to the IMN labelled cells in the IMG and in the wall of the colon. The distribution of the labelled enteric neurons was skewed towards the anal end of the colon. These results confirm that postganglionic sympathetic neurons in the SMG project axons through the guinea-pig IMG and describe the colonic distribution of enteric neurons that project through the IMG and into the IMN.

Intranasal flunisolide, placebo and beclomethasone dipropionate in perennial rhinitis.

Flunisolide nasal spray has been compared with placebo and with beclomethasone dipropionate in the treatment of perennial rhinitis. A double-blind, cross-over study in 26 patients comparing intranasal flunisolide (total dose 300 microgram/day) with placebo showed superiority of the active preparation in the relief of sneezing, stuffiness and runny nose. Physicians and patients significantly preferred the active spray. Side-effects on both sprays were mainly confined to transient nasal irritation. Plasma cortisol levels did not change significantly during the trial. A single-blind, cross-over study in 34 patients comparing flunisolide and beclomethasone dipropionate showed relief of sneezing, stuffiness, runny nose and nose-blowing with both medications. There were no differences between the effects of the two preparations. Physicians and patients favoured the drugs equally. Side-effects were minor.

Immunoreactivity for the Fas ligand in the mammalian enteric nervous system.

The Fas ligand induces apoptosis in activated immunocytes that express the Fas receptor. Fas-ligand transcripts have been found previously in murine intestine but the intestinal tissues that express Fas ligand have not been identified. We used immunohistochemistry to examine the expression of the Fas ligand in the enteric nervous system of rats, mice, guinea-pigs, ferrets and humans. Fas-ligand immunoreactivity was detectable in enteric nerve fibres and neurons in all species tested, representing 25%-50% of the neurons in rats, mice and guinea-pigs. An antigen of approximately 48 kDa was detected by Western blot analysis with Fas-ligand antiserum in the dissected enteric plexuses of duodenum from a C3H/HeJ mouse. In gld mice that harbour a Fas-ligand mutation, Fas-ligand immunoreactivity was slightly more intense in neurons and fibres and was also apparent in submucosal lymphocytes. In the myenteric plexuses of guinea-pig ileum and human colon, Fas-ligand immunoreactivity was not contained in neurons exhibiting nicotinamide-adenine dinucleotide phosphate-diaphorase activity. In the submucosal plexus of guinea-pig ileum, labelled neurons included some neuropeptide-Y-containing neurons but none with vasoactive intestinal polypeptide. We conclude that the Fas ligand is expressed by a large subset of enteric neurons and may provide the basis for cytotoxic neuroimmune interactions in the intestines.