Somatostatin immunoreactivity within the urinary bladder nerve fibers and paracervical ganglion urinary bladder projecting neurons in the female pig.

The study was designed to examine the distribution and chemical coding of somatostatin-immunoreactive (SOM-IR) nerve fibers supplying the urinary bladder wall and to establish the distribution and immunohistochemical characteristics of the subpopulation of paracervical ganglion (PCG) SOM-IR neurons projecting to this organ in female pigs. The PCG-urinary bladder projecting neurons (PCG-UBPN) were visualized with retrograde neuronal tracer Fast Blue (FB). Double-labeling immunohistochemistry performed on cryostat sections from the urinary bladder wall revealed that the greatest density of SOM-IR nerve fibers was found in the muscle layer and around blood vessels, a moderate number of these nerve terminals supplied the submucosa and only single SOM-IR axons were encountered beneath the urothelium. In all the investigated sections the vast majority of SOM-IR nerve fibers were immunopositive to vesicular acetylcholine transporter (VAChT) and many SOM-IR axons contained immunoreactivity to neuropeptide Y (NPY). Approximately 65 % of FB-positive (FB+) PCG-UBPN were immunoreactive to SOM. Moreover, PCG FB+/SOM + nerve cells were simultaneously immunoreactive to choline acetyltransferase (ChAT; 64.6 ± 0.6 %), NPY (59.7 ± 1.2 %), neuronal nitric oxide synthase (nNOS; 46.1 ± 0.7 %), vasoactive intestinal polypeptide (VIP; 29.9 ± 2.2 %), Leu5-enkephalin (L-ENK; 19.5 ± 6.3 %), dopamine ß-hydroxylase (DßH; 14.9 ± 1.9 %) or pituitary adenylate cyclase-activating polypeptide (PACAP; 14.8 ± 2.4 %). The present study reveals the extensive expression of SOM in both the nerve fibres supplying the porcine urinary bladder wall and the PCG neurons projecting to this organ, indicating an important regulatory role of SOM in the control of the urinary bladder function.

Paracervical ganglion in the female pig during prenatal development: Morphology and immunohistochemical characteristics.

The present study investigated the development of the paracervical ganglion in 5-, 7- and 10-week-old porcine foetuses using double labelling immunofluorescence method. In 5-week-old foetuses single PGP-positive perikarya were visible only along the mesonephric ducts. They contained DßH or VAChT, and nerve fibres usually were PGP/VAChT-positive. The perikarya were mainly oval. In 7-week-old foetuses, a compact group of PGP-positive neurons (3144±213) was visible on both sides and externally to the uterovaginal canal mesenchyme of paramesonephric ducts. Nerve cell bodies contained only DßH (36.40±1.63%) or VAChT (17.31±1.13%). In the 10-week-old foetuses, the compact group of PGP-positive neurons divided into several large and many small clusters of nerve cells and also became more expanded along the whole uterovaginal canal mesenchyme reaching the initial part of the uterine canal of the paramesonephric duct. The number of neurons located in these neuronal structures increased to 4121±259. Immunohistochemistry revealed that PGP-positive nerve cell bodies contained DßH (40.26±0,73%) and VAChT (30.73±1.34%) and were also immunoreactive for NPY (33.24±1,27%), SOM (23.6±0,44%) or VIP (22.9±1,13%). Other substances studied (GAL, NOS, CGRP, SP) were not determined at this stage of the development. In this study, for the first time, the morphology of PCG formation in the porcine foetus has been described in three stages of development. Dynamic changes in the number of neurons and their sizes were also noted, as well as the changes in immunochistochemical coding of maturing neurons.

Diagnostic Algorithm in Hirschsprung's Disease: Focus on Immunohistochemistry Markers.

BACKGROUND/AIM: Hirschsprung disease (HD) is caused by the congenital absence of ganglion cells in the distal bowel (aganglionosis). Rectal biopsy is considered important for its diagnosis. The aim of this study was to apply immunohistochemical staining using a minimal set of antibodies and develop an algorithm that will assist in the diagnosis of HD. PATIENTS AND METHODS: Rectal or colonic biopsies were performed in patients with HD (n=26) or patients treated for other bowel diseases (n=34). Immunohistochemical staining was performed for MAP1b, peripherin, S-100, calretinin, NSE, bcl-2 and CD56 proteins. RESULTS: Staining for CD56, S-100, peripherin and calretinin facilitated the identification of ganglion cells. The use of CD56 and S-100 antibodies together resulted in the highest rate of ganglion cell staining intensity (94%). CONCLUSION: We propose a practical diagnostic algorithm with the application of CD56 and S-100 antibodies that can be used in clinical practice in children suspected of Hirschsprung's disease.

Distinct pattern of enteric phospho-alpha-synuclein aggregates and gene expression profiles in patients with Parkinson's disease.

Phosphorylated alpha-synuclein (p-α-syn) containing Lewy bodies (LBs) and Lewy neurites (LNs) are neuropathological hallmarks of Parkinson's disease (PD) in the central nervous system (CNS). Since they have been also demonstrated in the enteric nervous system (ENS) of PD patients, the aim of the study was to analyze enteric p-α-syn positive aggregates and intestinal gene expression. Submucosal rectal biopsies were obtained from patients with PD and controls and processed for dual-label-immunohistochemistry for p-α-syn and PGP 9.5. p-α-syn positive aggregates in nerve fibers and neuronal somata were subjected to a morphometric analysis. mRNA expression of α-syn and dopaminergic, serotonergic, VIP (vaso intestinal peptide) ergic, cholinergic, muscarinergic neurotransmitter systems were investigated using qPCR. Frequency of p-α-syn positive nerve fibers was comparable between PD and controls. Although neuronal p-α-syn positive aggregates were detectable in both groups, total number and area of p-α-syn positive aggregates were increased in PD patients as was the number of small and large sized aggregates. Increased expression of dopamine receptor D1, VIP and serotonin receptor 3A was observed in PD patients, while serotonin receptor 4 and muscarinic receptor 3 (M3R) were downregulated. M3R expression correlated negative with the number of small sized p-α-syn positive aggregates. The findings strengthen the hypothesis that the CNS pathology of increased p-α-syn in PD also applies to the ENS, if elaborated morphometry is applied and give further insights in altered intestinal gene expression in PD. Although the mere presence of p-α-syn positive aggregates in the ENS should not be regarded as a criterion for PD diagnosis, elaborated morphometric analysis of p-α-syn positive aggregates in gastrointestinal biopsies could serve as a suitable tool for in-vivo diagnosis of PD.

Uterine Cervical Neurotransmission and Cervical Remodeling.

Cervical remodeling (CR) is a complex process, which, in part, is believed to be induced by physiological inflammation. Even though the female reproductive tissues are richly innervated by nerves from the parasympathetic pelvic autonomic ganglia, sensory dorsal root and nodose ganglia, their roles (neuronal factors) in this process (CR) has been largely attributed to sex steroid hormones, until recently. Here, we discuss the interaction between neuropeptides derived from peripheral nerves associated with uterine cervix and estrogen, and their likely impact on cervical remodeling. It is likely that these neuronal factors, under the influence of estrogen, could induce physiological inflammation during cervical remodeling by promoting the expression of vascular endothelial growth factor, among other factors.

Functional and molecular changes of the bladder in rats with crushing injury of nerve bundles from major pelvic ganglion to the bladder: role of RhoA/Rho kinase pathway.

Voiding dysfunction is a common complication after radical pelvic surgery. To reduce this complication, nerve-sparing radical pelvic surgery was introduced. However, several patients experienced voiding difficulty despite nerve-sparing radical pelvic surgery. Thus, we investigated the functional and molecular changes of the bladder in rats, which demonstrated voiding dysfunction induced by nerve damage during nerve-sparing radical pelvic surgery. Male rats were used and assigned to normal, sham-operated, and bilateral crushing nerve bundles from major pelvic ganglion (MPG) to bladder group. After one, two, and four-week crushing injury, significantly decreased contractile response and increased connective tissue of the detrusor were observed and these results were reliable findings with voiding difficulty following nerve-sparing radical pelvic surgery. After crushing injury, significantly increased M2 muscarinic receptor expression was observed and this might be regarded as the compensatory response. However, M3 muscarinic receptor expression was not significantly changed. The expression of RhoA, ROCK-α, and ROCK-ß was significantly increased after one, two, and four-week crushing injury. From these results, the down-regulation of RhoA/Rho kinase pathway might lead to the decreased bladder contractility after crushing injury of nerve bundles from MPG to the bladder despite of the compensated up-regulation of M2 muscarinic receptor.

Sildenafil promotes neuroprotection of the pelvic ganglia neurones after bilateral cavernosal nerve resection in the rat.

OBJECTIVE: To determine the gene expression profile of pelvic ganglia neurones after bilateral cavernosal nerve resection (BCNR) and subsequent treatment with sildenafil in relation to neurotrophic-related pathways. MATERIALS AND METHODS: Fisher rats aged 5 months were subjected to BCNR or sham operation and treated with or without sildenafil (20 mg/kg body-weight in drinking water) for 7 days. Total RNA isolated from pelvic ganglia was subjected to reverse transcription and then to quantitative reverse transcriptase-polymerase chain reaction (PCR) with the RAT-neurotrophic array. Results were corroborated by real-time PCR and western blotting. Another set of animals were injected with a fluorescent tracer at the base of the penis, 7 days before BCNR or sham operation, and were sacrificed 7 days after surgery. Sections of pelvic ganglia were used for immunohistochemistry with antibodies against neurturin, neuronal nitric oxide synthase, tyrosine hydroxylase and glial cell line-derived neurotrophic factor receptor α2. RESULTS: A down-regulation of the expression of neuronal nitric oxide synthase accompanied by changes in the level of cholinergic neurotrophic factors, such as neurturin and its receptor glial cell line-derived neurotrophic factor receptor α2, artemin, neurotrophin-4 and cilliary neurotrophic factor, was observed 7 days after BCNR in pelvic ganglia neurones. Treatment with sildenafil, starting immediately after surgery, reversed all these changes at a level similar to that in sham-operated animals. CONCLUSIONS: Sildenafil treatment promotes changes in the neurotrophic phenotype, leading to a regenerative state of pelvic ganglia neurones. The present study provides a justification for the use of phosphodiesterase 5 inhibitors as a neuroprotective agent after BCNR.

Regenerative process evaluation of neuronal subclasses in chagasic patients with megacolon.

Chagas' disease is one of the most serious parasitic diseases of Latin America, with a social and economic impact far outweighing the combined effects of other parasitic diseases such as malaria, leishmaniasis and schistosomiasis. In the chronic phase of this disease, the destruction of enteric nervous system (ENS) components leads to megacolon development. Previous data presented that the regeneration tax in the ENS neurons is augmented in chagasic patients. Although, there are several neuronal types with different functions in the intestine a detailed study about the regeneration of every neuronal type was never performed before. Therefore, the aim of this study was to evaluate the regeneration tax of every neuronal cell type in the ENS from chagasic patients with megacolon and non-infected individuals. A neuronal regeneration marker (GAP-43) was used in combination with a pan-neuronal marker (Peripherin) and several neuropeptides markers (cChat, Substance P, NPY, VIP and NOS), and it was considered as positive just with the combination of these markers. Our results demonstrated that the regeneration levels of cChat, Substance P, and NPY were similar in chagasic patients and non-infected individuals. However, levels of VIP and NOS neuropeptides were increased in chagasic patients when compared with non-infected individuals. We believe that the augment in the regeneration occur due to an increased destruction of selective neuronal types. These results corroborates with previous studies that pointed out to selective destruction of VIP and NOS neurons in chagasic patients.

Localization of the orexin system in the gastrointestinal tract of fallow deer.

The aim of the present study was to investigate by immunohistochemistry the presence and distribution of the orexin system in the stomach and gut of fallow deer. Abundant orexin A-positive cells were localized in the middle and basal portions of the mucosal glands of the cardial and fundic regions of the stomach. In the same gastric areas, orexin B-positive cells were also found, mainly localized in the basal portion of glands. In the intestinal tract, orexin-containing cells were occasionally found in the duodenal epithelium and in the rectal intestinal glands. Immunoreactivity for orexin receptors, type 1 and 2 (OX1R and OX2R), was not detected in the same stomach regions. OX1R-immunopositivity was observed in the enteric neuron ganglia localized in the submucosal and muscular intestinal layers, while OX2R-immunopositivity was found close in contact with the cytoplasmic membrane of epithelial cells in the small intestine.

Developmental determinants of the independence and complexity of the enteric nervous system.

Enteric nervous system (ENS) development is relevant to Hirschsprung's disease (HSCR; congenital aganglionosis of the terminal bowel), which is still imperfectly treated. Mutations in genes encoding the RET receptor tyrosine kinase and endothelin receptor type B (EDNRB) are involved in HSCR pathogenesis; however, also important in ENS development are molecules that mediate events that are more restricted than those of RET and EDNRB, act later in development and which might not be HSCR-associated. Examples are molecules that function in the guidance of enteric neural crest-derived cells (ENCDCs) and vagal axons, and in regulating the terminal differentiation of enteric neurons from ENCDCs. It is probable that highly prevalent disorders of gastrointestinal sensation and motility result from subtle defects in ENS development.

Changes in the number and volume of NPY and VIP neurons from periprostatic accessory vegetative ganglia in pre- and peripubertal rats. A stereological study.

The amount of neurons of periprostatic accessory ganglia in pre- and peripubertal rats was studied to ascertain whether the development of these autonomic ganglia is androgen-dependent. Stereological estimates of the volumes and number of neurons immunoreactive to protein gene product 9.5 (PGP 9.5), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) were carried out. Immunostaining of androgen receptors (AR) in the ganglia was also performed. The ganglionic neurons from the two groups studied were immunoreactive to PGP 9.5, NPY, and VIP. Almost all the neurons were immunostained for AR. The ganglionic volume showed a significant increase in peripubertal prostate in comparison with the prepubertal gland. No significant changes were observed with respect to the absolute number of neurons immunoreactive to all the antigens. The neuronal volume was significantly increased in peripubertal rats in comparison with prepubertal animals. These findings led us to the following conclusions: There is no evidence of neurogenesis during pubertal development in the periprostatic accessory ganglia of the rat. The increase of ganglionic volume in puberty is due to the growth in neuronal volume. There were no differences between the sizes of NPY and VIP neurons in pubertal periprostatic accessory ganglia. The development of periprostatic vegetative neurons is androgen-dependent.

Localization of multiple neurotransmitters in surgically derived specimens of human atrial ganglia.

Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of atrial and ventricular arrhythmias. While this system has been studied extensively in animal models, far less is known about the intrinsic cardiac nervous system of humans. This study was initiated to anatomically identify neurotransmitters associated with the right atrial ganglionated plexus (RAGP) of the human heart. Biopsies of epicardial fat containing a portion of the RAGP were collected from eight patients during cardiothoracic surgery and processed for immunofluorescent detection of specific neuronal markers. Colocalization of markers was evaluated by confocal microscopy. Most intrinsic cardiac neuronal somata displayed immunoreactivity for the cholinergic marker choline acetyltransferase and the nitrergic marker neuronal nitric oxide synthase. A subpopulation of intrinsic cardiac neurons also stained for noradrenergic markers. While most intrinsic cardiac neurons received cholinergic innervation evident as punctate immunostaining for the high affinity choline transporter, some lacked cholinergic inputs. Moreover, peptidergic, nitrergic, and noradrenergic nerves provided substantial innervation of intrinsic cardiac ganglia. These findings demonstrate that the human RAGP has a complex neurochemical anatomy, which includes the presence of a dual cholinergic/nitrergic phenotype for most of its neurons, the presence of noradrenergic markers in a subpopulation of neurons, and innervation by a host of neurochemically distinct nerves. The putative role of multiple neurotransmitters in controlling intrinsic cardiac neurons and mediating efferent signaling to the heart indicates the possibility of novel therapeutic targets for arrhythmia prevention.

Ganglionic acetylcholine receptor autoantibody: oncological, neurological, and serological accompaniments.

OBJECTIVE: To describe the clinical utility of the nicotinic ganglionic acetylcholine receptor (alpha3-AChR) autoantibody as a marker of neurological autoimmunity and cancer. DESIGN: Case-control study. SETTING: Mayo Clinic, Rochester, Minnesota. PATIENTS: A total of 15,000 patients seen at Mayo Clinic (2005-2007) and evaluated on a service basis for paraneoplastic neurological autoimmunity for whom clinical information was obtained retrospectively by medical record review as well as 457 neurologically asymptomatic patients or control subjects of whom 173 were healthy, 245 had lung cancer, and 39 had systemic lupus erythematosus or Sjögren syndrome. OUTCOME MEASURES: Neurological, oncological, and serological associations of alpha3-AChR autoantibody seropositivity. RESULTS: Of 15,000 patients tested on a service basis, 1% were seropositive (median, 0.12 nmol/L; range, 0.03-18.8 nmol/L; normal, < or =0.02 nmol/L), 55% were male, and the median age was 65 years. Cancer was found (new or by history) in 24 of 78 patients evaluated for cancer while at Mayo Clinic (30%); 43% had adenocarcinoma (more patients had breast cancer than prostate, lung, and gastrointestinal cancers; each of the latter groups had about the same number of patients). Of 12 patients with high antibody values (> or =1.00 nmol/L), 83% had pandysautonomia. Of 85 patients with medium antibody values (0.10-0.99 nmol/L), neurological presentations were more diverse and included peripheral neuropathies (36%), dysautonomia (20%, usually limited), and encephalopathy (13%). Of 58 patients with low antibody values (0.03-0.09 nmol/L), 54% had a nonautoimmune neurological disorder or no neurological disorder. Of 245 control patients with lung cancer, 7.8% were seropositive. Only 1 of 212 control patients without cancer (0.5%) was seropositive (P < .001). CONCLUSION: The detection of alpha3-AChR autoantibody aids the diagnosis of neurological autoimmunity and cancer.

Expression of estrogen receptor GPR30 in the rat spinal cord and in autonomic and sensory ganglia.

The G protein-coupled receptor GPR30 has recently been identified as a nonnuclear estrogen receptor. Reverse transcriptase-polymerase chain reaction revealed expression of GPR30 mRNA in varying quantities in the rat spinal cord, dorsal root ganglia, nodose ganglia, trigeminal ganglia, hippocampus, brain stem, and hypothalamus. Immunohistochemical studies that used a rabbit polyclonal antiserum against the human GPR30 C-terminus revealed a fine network of GPR30-immunoreactive (irGPR30) cell processes in the superficial layers of the spinal cord; some of which extended into deeper laminae. A population of neurons in the dorsal horn and ventral horn were irGPR30. Dorsal root, nodose, and trigeminal ganglionic neurons displayed varying intensities of irGPR30. Positively labeled neurons were detected in the major pelvic ganglion, but not in the superior cervical ganglion. A population of chromaffin cells in the adrenal medulla was irGPR30, so were cells of the zona glomerulosa. Double-labeling the adrenal medulla with GPR30 antiserum and tyrosine hydroxylase antibody or phenylethanolamine-N-methyltransferase antiserum revealed that irGPR30 is expressed in the majority of tyrosine hydroxylase-positive chromaffin cells. Last, some of the myenteric ganglion cells were irGPR30. Tissues processed with preimmune serum resulted in no staining. Voltage-sensitive dye imaging studies showed that the selective GPR30 agonist G-1 (1, 10, and 100 nM) depolarized cultured spinal neurons in a concentration-dependent manner. Collectively, our result provides the first evidence that GPR30 is expressed in neurons of the dorsal and ventral horn as well as in sensory and autonomic neurons, and activation of GPR30 by the selective agonist G-1 depolarizes cultured spinal neurons.

Immunohistochemical characterization and quantitative analysis of neurons in the myenteric plexus of the equine intestine.

The present study was performed on whole-mount preparations to investigate the chemical neuroanatomy of the equine myenteric plexus throughout its distribution in the intestinal wall. The objective was to quantify neurons of the myenteric plexus, especially the predominant cholinergic and nitrergic subpopulations. Furthermore, we investigated the distribution of vasoactive intestinal polypeptide and the calcium-binding protein calretinin. Samples from different defined areas of the small intestine and the flexura pelvina were taken from 15 adult horses. After fixation and preparation of the tissue, immunofluorescence labeling was performed on free floating whole-mounts. Additionally, samples used for neuropeptide staining were incubated with colchicine to reveal the neuropeptide distribution within the neuronal soma. The evaluation was routinely accomplished using confocal laser-scanning microscopy. For quantitative and qualitative analysis, the pan-neuronal marker anti-HuC/D was applied in combination with the detection of the marker enzymes for cholinergic neurons and nitrergic nerve cells. Quantitative data revealed that the cholinergic subpopulation is larger than the nitrergic one in several different locations of the small intestine. On the contrary, the nitrergic neurons outnumber the cholinergic neurons in the flexura pelvina of the large colon. Furthermore, ganglia are more numerous in the small intestine compared with the large colon, but ganglion sizes are bigger in the large colon. However, comparison of the entire population of neurons in the different locations of the gut showed no difference. The present study adds further data on the chemoarchitecture of the myenteric plexus which might facilitate the understanding of several gastrointestinal disorders in the horse.

Effects of castration on the immunoreactivity to NGF, BDNF and their receptors in the pelvic ganglia of the male rat.

Nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and are members of the neurotrophin family, a family of neurotrophic factors that also includes neurotrophin (NT) 3 and NT4/5. Neurotrophins have essential roles in the survival, development and differentiation of neurons in the central and peripheral nervous systems. Neurotrophins exert their effects by binding to corresponding receptors which are formed by the tyrosine protein kinases TrkA, TrkB and TrkC, and the low affinity neurotrophic receptor (p75NTR). In the present study, using immunohistochemistry and quantitative analysis, we have investigated immunoreactivity to BDNF, NGF, TrkB, p75NTR and TrkA in the pelvic ganglia of normal and castrated rats. Neurons of the pelvic ganglia expressed both these neurotrophins and their receptors. After castration the immunoreactivity persisted. However, the number of BDNF- and p75NTR-IR cells statistically significant decreased after castration. These results suggest that castration modulates the expression of neurotrophins and their receptors in pelvic autonomic neurons.

Characterization of ganglionic acetylcholine receptor autoantibodies.

In myasthenia gravis (MG), autoantibodies bind to the alpha1 subunit and other subunits of the muscle nicotinic acetylcholine receptor (AChR). Autoimmune autonomic ganglionopathy (AAG) is an antibody-mediated neurological disorder caused by antibodies against neuronal AChRs in autonomic ganglia. Subunits of muscle and neuronal AChR are homologous. We examined the specificity of AChR antibodies in patients with MG and AAG. Ganglionic AChR autoantibodies found in AAG patients are specific for AChRs containing the alpha3 subunit. Muscle and ganglionic AChR antibody specificities are distinct. Antibody crossreactivity between AChRs with different alpha subunits is uncommon but can occur.

Antibodies to muscle and ganglionic acetylcholine receptors (AchR) in celiac disease.

BACKGROUND: About 2.5% of patients with idiopathic peripheral neuropathy or idiopathic dysautonomia have underlying celiac disease (CD). Antibodies to ganglioside have been reported in CD patients with neuropathy. No data are so far available on the presence in CD of acetylcholine receptor (AChR) antibodies. Muscle AChR antibodies are found in patients with myasthenia gravis, and ganglionic AChR antibodies in patients with autoimmune autonomic neuropathy. OBJECTIVE: To determine the frequency of AChR antibodies in CD patients and assess possible correlations with neurological manifestations. METHODS: Seventy CD patients (16 M, 54 F, mean age 36 years) underwent neurological and electrophysiological evaluation. AChR antibodies were detected with radioimmunoprecipitation assay. Sera from 15 age-matched patients with systemic lupus erythematosus (SLE) and 10 with Sjogren syndrome were studied as controls. RESULTS: None of our CD patients complained of autonomic symptoms or fatigable weakness. Borderline titres (0.03-0.05 nmol/l) of ganglionic AChR antibodies were present in 4 patients, one affected with type I diabetes and one with subclinical neuropathy. Three of the 4 patients underwent cardiovascular autonomic function tests, which showed no abnormalities. Low levels of ganglionic AChR antibodies (0.05-0.10 nmol/l) were found in 2 SLE control patients, one of whom had a severe sicca complex. Muscle AChR antibodies (>1.0 nmol/l) were found in two CD patient and one control patient with SLE. Neither had symptoms or signs of myasthenia gravis. DISCUSSION AND CONCLUSIONS: CD is occasionally associated with neurologic disease, and with antibody reactivity to neuronal antigens. None of our CD patients had autonomic failure or significant levels of ganglionic AChR antibodies. Two CD patient and one control with SLE had muscle AChR antibodies without clinical evidence of myasthenia. The presence of antibodies in CD and in SLE patients may reflect a non-specific autoimmune response in these patients or may indicate subclinical autoimmune autonomic and neuromuscular involvement.

Deficiency of purinergic P2Y receptors in aganglionic intestine in Hirschsprung's disease.

Hirschsprung's disease is characterized by the absence of ganglion cells in the distal bowel and extends proximally for varying distances. In recent years, the purinergic P2Y receptors have begun to receive much attention as they have been recognised as major ATP receptors in many regions of the body, including the intestine. ATP has long been established as an inhibitory neurotransmitter in the enteric nervous system. The aim of our study was to analyse the expression of P2Y1 and P2Y2 receptors in the intestine of patients with Hirschsprung's disease. Frozen sections were cut from rectal tissue segments taken from both the aganglionic and ganglionic regions of patients with Hirschsprung's disease, as well as tissue from normal rectal biopsies, which were used as controls. Sections were incubated overnight with P2Y1 and P2Y2 receptor antibodies and results were analysed by light microscopy. Both P2Y1 and P2Y2 immunoreactivity was absent from the submucosal and myenteric plexuses of aganglionic tissue compared to ganglionic tissue and normal controls, in which large number of immunoreactive neurons were evident, arranged in ganglia in both plexuses and positive nerve fibres, in both the smooth and circular muscles. Our results show a lack of expression of P2Y1 and P2Y2 receptors in the aganglionic gut in Hirschsprung's disease. The absence of these receptors suggests the absence of the inhibitory neurotransmitter ATP, which may help to explain the contracted state of the aganglionic gut in Hirschsprung's disease.