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ABSTRACTBackgroundDespite achieving endoscopic remission, over 20% of inflammatory bowel disease (IBD) patients experience chronic abdominal pain. Visceral pain and the microbiome exhibit sex-dependent interactions, while visceral pain in IBD shows a sex bias. Our aim was to evaluate whether post-inflammatory microbial perturbations contribute to visceral hypersensitivity in a sex-dependent manner.MethodsMales, cycling females, ovariectomized, and sham-operated females were given dextran sodium sulfate to induce colitis and allowed to recover. Germ-free recipients received sex-appropriate and cross-sex fecal microbial transplants (FMT) from post-inflammatory donor mice. Visceral sensitivity was assessed by recording visceromotor responses to colorectal distention. The composition of the microbiota was evaluated via 16S rRNA gene V4 amplicon sequencing, while the metabolome was assessed using targeted (short chain fatty acids – SCFA) and semi-targeted mass spectrometry.ResultsPost-inflammatory cycling females developed visceral hyperalgesia when compared to males. This effect was reversed by ovariectomy. Both post-inflammatory males and females exhibited increased SCFA-producing species, but only males had elevated fecal SCFA content. FMT from post-inflammatory females transferred visceral hyperalgesia to both males and females, while FMT from post-inflammatory males could only transfer visceral hyperalgesia to males.ConclusionsFemale sex, hormonal status as well as the gut microbiota play a role in pain modulation. Our data highlight the importance of considering biological sex in the evaluation of visceral pain.

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureusand imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.

Specific brain circuits recruited during stress contribute to differential immune responses and affect how the immune system handles viral and autoimmune challenges.

Peripheral inflammatory conditions, including those localized to the gastrointestinal tract, are highly comorbid with psychiatric disorders such as anxiety and depression. These behavioral symptoms are poorly managed by conventional treatments for inflammatory diseases and contribute to quality of life impairments. Peripheral inflammation is associated with sustained elevations in circulating glucocorticoid hormones, which can modulate central processes, including those involved in the regulation of emotional behavior. The endocannabinoid (eCB) system is exquisitely sensitive to these hormonal changes and is a significant regulator of emotional behavior. The impact of peripheral inflammation on central eCB function, and whether this is related to the development of these behavioral comorbidities remains to be determined. To examine this, we employed the trinitrobenzene sulfonic acid-induced model of colonic inflammation (colitis) in adult, male, Sprague Dawley rats to produce sustained peripheral inflammation. Colitis produced increases in behavioral measures of anxiety and elevations in circulating corticosterone. These alterations were accompanied by elevated hydrolytic activity of the enzyme fatty acid amide hydrolase (FAAH), which hydrolyzes the eCB anandamide (AEA), throughout multiple corticolimbic brain regions. This elevation of FAAH activity was associated with broad reductions in the content of AEA, whose decline was driven by central corticotropin releasing factor type 1 receptor signaling. Colitis-induced anxiety was reversed following acute central inhibition of FAAH, suggesting that the reductions in AEA produced by colitis contributed to the generation of anxiety. These data provide a novel perspective for the pharmacological management of psychiatric comorbidities of chronic inflammatory conditions through modulation of eCB signaling.

A recently identified feature of the host response to infection with helminth parasites is suppression of concomitant disease. Dendritic cells (DCs) exposed to antigens from the tapeworm Hymenolepis diminutasignificantly reduce the severity of dinitrobenzene sulfonic acid–induced colitis in mice. Here we elucidate mechanisms underlying this cellular immunotherapy. We show a requirement for Ccr7 expression on transferred H. diminutaantigen–treated (HD)-DCs, suggesting that homing to secondary lymphoid tissues is important for suppression of colitis. Furthermore, sodium metaperiodate–sensitive helminth-derived glycans are required to drive the anti-colitic response in recipient mice. Induction of Th2-type cytokines and Gata-3+Cd4+cells in secondary lymphoid tissues is dependent on major histocompatibility complex class II (MHC II) protein expression on transferred DCs, although remarkably, transfer of MHC II−/−HD-DCs still attenuated dinitrobenzene sulfonic acid–induced colitis in recipient mice. Moreover, transfer of Cd4+splenic T cells retrieved from mice administered MHC II−/−HD-DCs suppressed dinitrobenzene sulfonic acid–induced colitis in recipient mice. Our studies reveal that HD-DCs can suppress colitis via an alternative MHC II–independent pathway that involves, in part, mobilization of T-cell responses. These data support the utility of HD-DCs in blocking colitis, revealing a requirement for Ccr7 and providing for HD-DC autologous immunotherapy for disease in which MHC II expression and/or function is compromised.

Inflammatory bowel diseases (IBD) are chronic, relapsing and remitting gastrointestinal (GI) disorders of unknown etiology. IBD patients commonly exhibit extra-intestinal manifestations and complications of an inflammatory nature, presenting with disorders such as ankylosing spondylitis, uveitis and vasculitis. Although the metabolic syndrome is less prevalent in patients with IBD, they are at an increased risk for atherosclerosis and cardiovascular events. Considerable evidence supports the role of GI microbiota in the development of IBD. Recent studies have also shown a significant interaction between the metabolites of gut microbiota and the development of cardiovascular disease. Here we hypothesize that dysbiosis and/or abnormalities in the function of the intestinal microbiota promote cardiovascular disease in IBD patients, explaining the increased risk of cardiovascular events in these patients.

In November 2004, the very first issue of this journal featured articles on the pathogenesis of ulcerative colitis, mechanisms leading to chronic pancreatitis, and treatment of recurrent Clostridium-difficile-associated diarrhoea. Although those topics might seem familiar, much has changed in the intervening years in our understanding, diagnosis and treatment of many different diseases across the field of gastroenterology and hepatology. Nonetheless, many challenges remain. Here, we have asked five of our Advisory Board members—international experts across different subspecialties in gastroenterology and hepatology—to reflect on the progress and frustrations of the past 10 years. They also comment on where effort and money should be invested now, as well as their predictions for progress in the next 10 years.

Enteric glia are a unique class of peripheral glial cells within the gastrointestinal tract. Major populations of enteric glia are found in enteric ganglia in the myenteric and submucosal plexuses of the enteric nervous system (ENS); these cells are also found outside of the ENS, within the circular muscle and in the lamina propria of the mucosa. These different populations of cells probably represent unique classes of glial cells with differing functions. In the past few years, enteric glia have been found to be involved in almost every gut function including motility, mucosal secretion and host defence. Subepithelial glia seem to have a trophic and supporting relationship with intestinal epithelial cells, but the necessity of these roles in the maintenance of normal epithelial functions remains to be shown. Likewise, glia within enteric ganglia are activated by synaptic stimulation, suggesting an active role in synaptic transmission, but the precise role of glial activation in normal enteric network activity is unclear. Excitingly, enteric glia can also give rise to new neurons, but seemingly only under limited circumstances. In this Review, we discuss the current body of evidence supporting functional roles of enteric glia and identify key gaps in our understanding of the physiology of these unique cells.

Although the cellular prion protein (PrPC) is expressed in the enteric nervous system and lamina propria, its function(s) in the gut is unknown. Because PrPCmay exert a cytoprotective effect in response to various physiologic stressors, we hypothesized that PrPCexpression levels might modulate the severity of experimental colitis. We evaluated the course of dextran sodium sulfate (DSS)–induced colitis in hemizygous Tga20 transgenic mice (approximately sevenfold overexpression of PrPC), Prnp−/−mice, and wild-type mice. On day 7, colon length, disease severity, and histologic activity indices were determined. Unlike DSS-treated wild-type and Prnp−/−animals, PrPCoverexpressing mice were resistant to colitis induction, exhibited much milder histopathologic features, and did not exhibit weight loss or colonic shortening. In keeping with these results, pro-survival molecule expression and/or phosphorylation levels were elevated in DSS-treated Tga20 mice, whereas pro-inflammatory cytokine production and pSTAT3 levels were reduced. In contrast, DSS-treated Prnp−/−mice exhibited increased BAD protein expression and a cytokine expression profile predicted to favor inflammation and differentiation. PrPCexpression from both the endogenous Prnplocus or the Tga20 transgene was increased in the colons of DSS-treated mice. Considered together, these findings demonstrate that PrPChas a previously unrecognized cytoprotective and/or anti-inflammatory function within the murine colon.

The endocannabinoid system is involved in the regulation of gastrointestinal (GI) motility and inflammation. Using the peripherally restricted cannabinoid (CB)(1)/CB(2) receptor agonist naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (SAB378), we investigated the role of peripheral cannabinoid receptors in the regulation of GI motility and the development of colitis in mice. The actions of SAB378 on whole gut transit, upper GI transit, colonic propulsion, and locomotor activity were investigated in C57BL/6N, CB(1) receptor knockout, and CB(2) receptor knockout mice. The potential for SAB378 to modify inflammation was studied by using dextran sulfate sodium (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) models of experimental colitis. SAB378 did not modify locomotor activity. SAB378 slowed all parameters of GI motility, and these effects were significantly reduced by the CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide (AM251), but not by the CB(2) receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H- indol-3-yl](4-methoxyphenyl)methanone (AM630). SAB378 did not inhibit GI transit or colonic propulsion in CB(1) receptor knockout mice, whereas its effects were observed in CB(2) receptor knockout mice. SAB378 did not reduce the degree of colitis induced by DSS or TNBS. The actions of SAB378 on GI motility are mediated by peripherally located CB(1) receptors. SAB378 was not effective against two models of experimental colitis, which may indicate that peripheral cannabinoid receptor stimulation alone may not be sufficient to mediate the anti-inflammatory effects of cannabinoids.

In this review we will consider how central structures involved in the regulation of energy balance gather information from the variety of different peripherally derived signaling molecules that we now believe provide an integrated perspective of energy status of the organism. The existence of the blood brain barrier means that the CNS is theoretically unable to directly monitor many of these circulating signals such as adiponectin, amylin, cholecystokinin (CCK), glucose, ghrelin, leptin, and peptide YY (PYY) which do not freely diffuse across this barrier. Studies have identified five primary mechanisms through which these circulating signals may transmit the afferent information they carry from the periphery to the CNS which we describe below. We will both discuss mechanisms and potential contributions of vagal afferent signaling, peptide transporters, vascular endothelial cell signaling, the arcuate nucleus of the hypothalamus, and brain structures which lack the blood brain barrier known as the sensory circumventricular organs (CVOs), in providing sensory information to the integrative centers of the hypothalamus and medulla which play such essential roles in the regulation of energy balance.

In colitis, chronic and recurrent inflammation is associated with a breakdown in host defence mechanisms that leads to local and systemic infection. Whether this is due to a compromised neuroimmune response has not been studied. Our aim was to determine if colitis altered the host neuroimmune response as reflected in either body temperature rhythm or the febrile responses to lipopolysaccharide (LPS). Body temperature was monitored by telemetry from conscious, unrestrained male rats treated with trinitrobenzene sulphonic acid or saline. Twenty‐six days after initial induction, colitis was reactivated. Animals were given LPS (50 μg kg−1Escherichia coliLPS) during colitis and after reactivation. At the peak of colitis, treated rats showed a disruption of circadian body temperature rhythm, manifested as day‐time fever followed by night‐time hypothermia. In response to LPS, controls displayed a characteristic fever, whereas treated animals had a significantly reduced fever and low plasma levels of interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α). During reactivation of colitis, treated animals did not mount a fever or exhibit increased plasma levels of IL‐6 and TNF‐α after LPS. We conclude that experimental colitis is associated with a compromised neuroimmune status.

The electrical and synaptic properties of myenteric neurones in normal and inflamed guinea‐pig distal colons were evaluated by intracellular microelectrode recording. Chronic inflammation was established 6 days following administration of trinitrobenzene sulfonic acid (TNBS). In S neurones, inflammation only altered synaptic inputs as the amplitude of fast excitatory postsynaptic potentials were significantly larger (31 ± 2 mV compared to 20 ± 1 mV) and they were more likely to receive slow excitatory synaptic input (85% compared to 55%). AH neurones displayed altered electrical properties in colitis compared to control tissues: they generated more action potentials during a maximal depolarising current pulse (7 ± 1 compared to 1.6 ± 0.2); they had a smaller afterhyperpolarisation (9 ± 2 mV s compared to 20 ± 2 mV s); and they were more likely to receive fast excitatory synaptic input (74% compared to 17%), possess spontaneous activity (46% compared to 3%), and generate anodal break action potentials (58% compared to 19%). Although the resting membrane potential, input resistance and action potential characteristics were unaltered in AH neurones from inflamed tissues, they exhibited an enhanced Cs+‐sensitive rectification of the current–voltage relationship. This suggests that the increase in excitability of AH neurones may involve a colitis‐induced augmentation of the hyperpolarisation‐activated cation current (Ih) in these cells. An increased excitability, selectively in AH neurones, suggests that the afferent limb of intrinsic motor reflexes is disrupted in the inflamed colon and this may contribute to dysmotility associated with inflammatory diseases.

The ENS is responsible for the regulation and control of all gastrointestinal functions. Because of this critical role, and probably as a consequence of its remarkable plasticity, the ENS is often relatively well preserved in conditions where the architecture of the intestine is seriously disrupted, such as in IBD. There are structural and functional changes in the enteric innervation in animal models of experimental intestinal inflammation and in IBD. These include both up and down regulation of transmitter expression and the induction of new genes in enteric neurons. Using Fos expression as a surrogate marker of neuronal activation it is now well established that enteric neurons (and also enteric glia) respond to inflammation. Whether this “activation” is limited to a short‐term functional response, such as increased neuronal excitability, or reflects a long‐term change in some aspect of the neuronal phenotype (or both) has yet to be firmly established, but it appears that enteric neurons are highly plastic in their response to inflammation. Anat Rec 262:79–90, 2001. © 2001 Wiley‐Liss, Inc.

The receptor responsible for CGRP‐induced ion transport and permeability was examined in tissues from animals treated 7 days previously with trinitrobenzenesulfonic acid to induce colitis or in controls. CGRP caused a concentration‐dependent increase in short circuit current (Isc, EC5021 nM), which was abolished in chloride‐free buffer but was not blocked by CGRP8–37or tetrodotoxin (TTX). Amylin and adrenomedullin caused only a modest increase in Isc. The responses to the linear CGRP2receptor agonists [Cys(Et)2,7] hCGRPα and [Cys(Acm)2,7] hCGRPα were considerably smaller than the response to CGRP. These responses were abolished in chloride‐free buffer and were TTX sensitive. Atropine, doxantrazole, and indomethacin did not block the effects of CGRP or the CGRP2agonists. The response to [Cys(Et)2,7] hCGRPα was not affected by prior desensitization of the CGRP receptor and vice versa. Inflamed rats had a similar secretory response to CGRP (Isc, EC5015 nM) and [Cys(Et)2,7] hCGRPα as control tissues, while being hyporesponsive to carbachol. CGRP application increased electrical conductance of inflamed preparations. Taken together, these data suggest that CGRP may play an important role in the maintenance of host defense in colitis through an apparently novel CGRP receptor located on the colonic enterocyte.—Esfandyari, T., MacNaughton, W. K., Quirion, R., St. Pierre, S., Junien, J.‐L., Sharkey, K. A. A novel receptor for calcitonin gene‐related peptide (CGRP) mediates secretion in the rat colon: implications for secretory function in colitis. FASEBJ.14, 1439–1446 (2000)

In the present study, we have observed the development of an inflammatory reaction in the rat hindpaw, following the injection of specific agonists of PAR2(two PAR2activating peptides). This inflammation was characterized by oedema and granulocyte infiltration.Two selective PAR2activating peptides, SLGRL‐NH2and trans‐cinnamoyl‐LIGRLO‐NH2induced significant oedema in the rat hindpaw from 1–6 h following subplantar injection. Six hours after the PAR2‐activating peptide injection, the paw tissues showed a complete disruption of tissue architecture along with an inflammatory cell infiltrate.In the inflamed paw, PAR2‐immunoreactivity was expressed on endothelial cells as well as on the infiltrating inflammatory cells.The oedema induced by the injection of the two PAR2activating peptides was slightly reduced in rats pre‐treated with compound 48/80, but was not modified by pre‐treatment of rats with cromolyn, a mast cell stabilizer. Pre‐treatment of rats with a cyclo‐oxygenase inhibitor (indomethacin) or a nitric oxide synthase inhibitor (L‐Nω‐nitro‐L‐arginine methyl ester) had no effect on the oedema induced by the PAR2‐activating peptides.These results demonstrate that the administration of PAR2‐activating peptides into the rat paw induced an acute inflammatory response characterized by a persistent oedema (at least 6 h) and granulocyte infiltration. The PAR2‐induced inflammatory response occurred through a mechanism largely independent of mast cell activation, and of the production of prostanoids and nitric oxide.

Background/Aims:Mechanisms of neuroimmune regulation of intestinal electrolyte transport under pathophysiological conditions are unclear. This study investigated the effect of ionizing radiation on ileal electrolyte transport. Methods:Rats were exposed to 10 Gy γ-radiation and were killed 2, 24, and 48 hours later. Ileal segments were either mounted in Ussing chambers and exposed to electrical field stimulation, prostaglandin E2, leukotriene D4, or theophylline, or they were assayed for biochemical indices of inflammation. Other segments were processed for routine histological screening, mast cell counts, or immunohistochemical analysis of the distribution of vasoactive intestinal polypeptide or substance P immunoreactivity. Results:Basal short-circuit current was unchanged 2, 24, or 48 hours postirradiation. However, there was a reduction of tissue responsiveness to electrical field stimulation, prostaglandin E2, and theophylline but not to leukotriene D4. Decreased responsiveness at 2-hours postirradiation was blocked by pretreatment with the H1antagonist pyrilamine. Tissue myeloperoxidase activity and 5-hydroxytryptamine content were not altered post-irradiation, but tissue histamine and mucosal mast cells were significantly reduced at 24 and 48 hours. There were no significant changes in villus-crypt architecture until 48 hours postirradiation. There was no significant alteration in the distribution of immunoreactive vasoactive intestinal polypeptide or substance P. Conclusions:Ionizing radiation reduced the transport response to neural stimulation. The effect correlated temporally with decreased mast cells and histamine, suggesting a functional role for previously reported mast cell-nerve interactions.

Abstract.: 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.

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.

Nitric oxide (NO) from constitutive NO synthase (NOS) has been postulated to be a homeostatic regulator of leukocyte‐endothelial cell interactions. By contrast, the inducible NO synthase (iNOS) isoform has been invoked as a potential pathogenic enzyme in numerous inflammatory diseases. The objective of this study was to determine whether the iNOS isoform is also capable of functioning as a regulator of leukocyte recruitment. Mice received endotoxin (LPS, 30 μg/kg, i.v.); 2–4 h later, intravital microscopy was used to examine leukocyte rolling and adhesion in postcapillary venules of the cremaster muscle and the sinusoids and postsinusoidal venules of the hepatic microcirculation. Leukocyte recruitment into the lung was also examined. RT‐PCR confirmed that this treatment induced iNOS mRNA expression in wild‐type mice as early as 2 h after LPS treatment. Between 2 and 4 h after LPS administration, the number of rolling and adherent leukocytes in cremasteric postcapillary venules and of adherent cells in liver postsinusoidal venules of iNOS‐deficient mice were significantly higher than in wild‐type mice. Leukocyte accumulation in the lung (measured by myeloperoxidase assay) was also significantly elevated in iNOS‐deficient animals. These effects could not be attributed to differences in systemic blood pressure, shear rates, circulating leukocyte numbers, or baseline levels of rolling and adhesion because these parameters were not different between the two groups. To establish whether the differences in leukocyte recruitment were related to the leukocytes per se, perfusion of iNOS+/+or iNOS−/−septic blood over purified E‐selectin (using parallel plate flow chambers) revealed much larger recruitment of iNOS−/−leukocytes. These results suggest that iNOS induced in response to LPS releases NO that is capable of reducing leukocyte accumulation by affecting leukocytes directly and raises the possibility that induction of iNOS is a homeostatic regulator for leukocyte recruitment.—Hickey, M. J., Sharkey, K. A., Sihota, E. G., Reinhardt, P. H., MacMicking, J. D., Nathan, C., Rubes, P. Inducible nitric oxide synthase‐deficient mice have enhanced leukocyte–endothelium interactions in endotoxemia. FASEB J.11, 955–964 (1997)

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 anti-body, 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.

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.

WE examined the expression of c-Fosand JunBin 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 Kinduced further increases of neuronal c-Fosand JunBexpression 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-Fosand JunBin most or all enteric neurones may be a useful determinant of activation.

We examined and compared a mechanosensitive human sural neuroma and a contralateral sural nerve collected simultaneously from a patient involved in a diabetic neuropathy research protocol. Using indirect immunofluorescence staining, we identified a striking difference in the content within axons of two neuropeptides, substance P (SP) and calcitonin gene‐related peptide (CGRP), between the contralateral nerve and the neuroma. Unlike the contralateral nerve, where immunofluorescence was sparse, a significant number of disorganized axon profiles that stained brightly positive for CGRP or SP were identified in the neuroma. In contrast, staining for tyrosine hydroxylase, a label of sympathetic axons, was largely absent except around one large arteriole. The neuroma specimen also contained large numbers of serotonin‐containing mast cells, only noted occasionally in the contralateral nerve. The peptide staining and mast cell accumulation in the human neuroma closely resembled changes we have previously observed in an animal neuroma model. Local neuropeptides may play a role in the injury response of peripheral nerve, and may be related to mechanosensitivity. © 1997 John Wiley & Sons, Inc. Muscle Nerve20:875–880, 1997.

The presence of immunoreactivity to the neuronal phosphoprotein B-50 and the peptides bombesin, calcitonin gene-related peptide, galanin, neurotensin, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal polypeptide was examined in biopsy specimens from the duodenum and rectum of human immunodeficiency virus (HIV)-seropositive and HIV-seronegative male homosexual patients. The distribution of B-50 and the peptides was correlated with HIV serology, number of CD4+lymphocytes, and the presence of HIV in biopsy culture. There was a very low incidence of enteric pathogens in both groups of patients. It was found that HIV-seropositive patients had a greater incidence of abnormal patterns of immunoreactivity (reduced intensity and/or density of innervation) in enteric nerves and enteroendocrine cells than HIV-seronegative patients. A reduction of substance P immunoreactivity was significantly correlated with reduced CD4+lymphocyte count and HIV status; a similar trend was also seen for somatostatin and vasoactive intestinal polypeptide. Using B-50 as a marker, it was found that both groups of patients had altered patterns of immunoreactivity in rectal nerves. The findings of this study suggest that some of the clinical symptoms associated with HIV infection may be caused by a specific HIV enteropathy that influences enteric nerve and/or enteroendocrine cell function by altering the density of peptide immunoreactivity.

We report the development of novel cannabinergic probes that can stabilize the cannabinoid receptors (CBRs) through tight binding interactions. Ligand design involves the introduction of select groups at a judiciously chosen position within the classical hexahydrocannabinol template (monofunctionalized probes). Such groups include the electrophilic isothiocyanato, the photoactivatable azido, and the polar cyano moieties. These groups can also be combined to produce bifunctionalized probes potentially capable of interacting at two distinct sites within the CBR-binding domains. These novel compounds display remarkably high binding affinities for CBRs and are exceptionally potent agonists. A key ligand (27a, AM11245) exhibits exceptionally high potency in both in vitroand in vivoassays and was designated as “megagonist,” a property attributed to its tight binding profile. By acting both centrally and peripherally, 27adistinguishes itself from our previously reported “megagonist” AM841, whose functions are restricted to the periphery.

Abstract

The aim of these experiments was to determine the contribution of leukocyte‐derived iNOS to total iNOS expression induced by lipopolysaccharide (LPS). By transferring bone marrow between iNOS+/+and iNOS–/–mice, we created chimeric mice in which iNOS expression was limited to either circulating leukocytes (leukocyte‐iNOS mice) or parenchymal cells (parenchyma‐iNOS mice). Analysis of congenic markers demonstrated that >95% of thymocytes in chimeric mice were of donor origin. Also, following LPS treatment, iNOS mRNA was detectable in blood from leukocyte‐iNOS mice but not parenchyma‐iNOS mice. Together these findings indicated that the host marrow had been replaced entirely by donor cells. In the lung, at least 50% of the LPS‐induced iNOS mRNA was derived from leukocytes, and immunohistochemical analysis indicated that leukocytes were the main source of iNOS protein. In contrast in the liver, colon, and muscle, iNOS expression was derived predominantly from parenchymal cells. This divergence is potentially explained by the high level of leukocyte recruitment to the lung, relative to the other tissues. Plasma levels of NOS byproducts indicated that parenchymal iNOS was the dominant source of systemic iNOS activity. These findings indicate that in tissues other than the lung, parenchymal cells are the principal source of iNOS during endotoxemia.

GASTROENTEROLOGY 2002;123:655-656

The gastrointestinal (GI) tract plays a central role in the pathogenesis of transmissible spongiform encephalopathies. These are human and animal diseases that include bovine spongiform encephalopathy, scrapie and Creutzfeldt-Jakob disease. They are uniformly fatal neurological diseases, which are characterized by ataxia and vacuolation in the central nervous system. Alhough they are known to be caused by the conversion of normal cellular prion protein to its infectious conformational isoform (PrPsc) the process by which this isoform is propagated and transported to the brain remains poorly understood. M cells, dendritic cells and possibly enteroendocrine cells are important in the movement of infectious prions across the GI epithelium. From there, PrPsc propagation requires B lymphocytes, dendritic cells and follicular dendritic cells of Peyer’s patches. The early accumulation of the disease-causing agent in the plexuses of the enteric nervous system supports the contention that the autonomic nervous system is important in disease transmission. This is further supported by the presence of PrPsc in the ganglia of the parasympathetic and sympathetic nerves that innervate the GI tract. Additionally, the lymphoreticular system has been implicated as the route of transmission from the gut to the brain. Although normal cellular prion protein is found in the enteric nervous system, its role has not been characterized. Further research is required to understand how the cellular components of the gut wall interact to propagate and transmit infectious prions to develop potential therapies that may prevent the progression of transmissible spongiform encephalopathies.

The Canadian Journal of Gastroenterology continues to grow in strength as a national journal of importance to gastroenterologists and hepatologists. As the official journal of both the Canadian Association of Gastroenterolgy and the Canadian Association for the Study of the Liver, it also serves as an important tool for the dissemination of guidelines and standards for the entire medical community in Canada. We should be proud of these accomplishments. But one also needs to ask, can the Journal be better? Is there something more that can be achieved? Some would say the status quo is fine. But realistically, we regularly look to the journals of our sister associations in the United States and the United Kingdom - Gastroenterology, Hepatology and Gut - for the latest and best in our field. So, I would argue, we can achieve more and make the Journal better.

Since about the 1950s nerves in the wall of the intestine have been postulated to play a role in the pathogenesis of inflammatory bowel disease (IBD). Human and animal studies examining the role of nerves in intestinal inflammation are the focus of this review. Consideration is given to two possible ways that nerves are involved in IBD. First, nerves may play a role in the development or maintenance of inflammation through local release of transmitters. Second, once initiated (by whatever means), the processes of inflammation may disrupt the normal pattern of innervation and the interactions of nerves and their target tissues. Many of the functional disturbances observed in IBD are likely due to an alteration in the enteric nervous system either structurally through disruptions of nerve-target relationships or by modifications of neurotransmitters or their receptors. Finally, it appears that the enteric nervous system may be a potential therapeutic target in IBD and that neuroactive drugs acting locally can represent useful agents in the management of this disease.