Marc-Henri De Laet

Nitric Oxide Synthase Activity in Infantile Hypertrophic Pyloric Stenosis

BACKGROUND Hypertrophic pyloric stenosis is a common infantile disorder characterized by enlarged pyloric musculature and gastric-outlet obstruction. Its physiopathologic mechanism is not known, but a defect in pyloric relaxation (pylorospasm) has been postulated. Nitric oxide is a mediator of relaxation in the mammalian digestive tract, raising the possibility that pylorospasm could be caused by a defect in nitric oxide production. Since neuronal nitric oxide synthase and NADPH diaphorase are identical, we used the NADPH diaphorase histochemical reaction to study the distribution of nitric oxide synthase in pyloric tissue from patients with infantile hypertrophic pyloric stenosis. METHODS We studied pyloric tissue from nine infants with infantile hypertrophic pyloric stenosis and seven control infants and children. Cryostat sections were processed for NADPH diaphorase histochemical analysis. A polyclonal tau antiserum was used to identify the enteric nervous system by immunohistochemical methods. RESULTS NADPH diaphorase activity was restricted to the enteric nervous system and blood vessels. In the pyloric tissues from the control patients, intense diaphorase activity was present in the nerve fibers of the circular musculature, in the neurons and nerve bundles of the myenteric plexus, and in some nerve fibers of the longitudinal musculature. In the pyloric tissues from patients with infantile hypertrophic pyloric stenosis, the enteric nerve fibers in the hypertrophied circular musculature were enlarged and distorted and did not contain diaphorase activity, whereas the activity in the myenteric plexus and the longitudinal musculature was preserved. CONCLUSIONS We suggest that a lack of nitric oxide synthase in pyloric tissue is responsible for pylorospasm in infantile hypertrophic pyloric stenosis.

Kit-negative fibroblast-like cells expressing SK3, a Ca2+-activated K+ channel, in the gut musculature in health and disease.

Abstract. The apamin-sensitive component of the inhibitory response of the gastrointestinal musculature involves the small conductance Ca2+-activated K+ channel SK3. Kit-immunoreactive (ir) interstitial cells of Cajal appear to be involved in nitrergic inhibition while the role of the recently described CD34-ir fibroblast-like cells adjacent to, but distinct from, the cells of Cajal remains elusive. The distribution of SK3 was studied by immunohistochemistry in the normal human gut, in motility disorders with a lack of cells of Cajal (infantile hypertrophic pyloric stenosis and Hirschsprungs disease) and in mice deficient in cells of Cajal. SK3 immunoreactivity was observed exclusively in Kit-negative interstitial cells adjacent to, but distinct from, the Kit-ir interstitial cells of Cajal in the normal gut. The distribution of SK3-ir cells was not altered in conditions where cells of Cajal were lacking. These cells were CD34-ir fibroblast-like cells in the human gut and in the mouse stomach, while SK3-ir cells in the mouse intestine were CD34 negative. As SK channels are reportedly involved in inhibitory neurotransmission, our morphological observations suggest that SK3-ir interstitial cells, distinct from the Kit-ir interstitial cells of Cajal, may represent a novel cellular component in the control of excitability of the digestive musculature. Further studies will be required to directly address the function of these cells.

CD34 immunoreactivity and interstitial cells of Cajal in the human and mouse gastrointestinal tract.

Abstract. Immunoreactivity for the tyrosine kinase receptor Kit (Kit-ir) is an established marker for the interstitial cells of Cajal (ICC) of the gut. Recently, the presence of CD34 immunoreactivity (CD34-ir) has been reported in Kit-ir ICC around the myenteric plexus in human small intestine. Conversely, we observed that CD34-ir labeled Kit-negative fibroblast-like cells, closely adjacent to, but distinct from, the Kit-ir ICC. The existence of cells expressing both CD34-ir and Kit-ir remains controversial. CD34-ir and Kit-ir were studied by high-resolution confocal microscopy on cryostat sections of human and murine gut as well as murine whole-mounts, using specific antibodies raised to human and murine CD34, respectively. CD34-ir labeled numerous cells in all parts of the gut, in man and in mouse. CD34-ir was consistently observed in Kit-negative cells, distinct from the closely adjacent Kit-ir ICC. Thin processes of both cell types intermingled extensively, often at the limit of resolution for light microscopy. CD34-ir was also observed in Kit-negative mesenchymal cells in the submucosa, in capillaries and in mesothelial cells. CD34-ir is not a marker for Kit-ir ICC in the human and murine gut. No CD34-ir, Kit-ir-expressing cells were encountered. Conversely, CD34-ir cells, closely adjacent to, but distinct from, Kit-ir ICC were consistently identified. The intimate relationship between these cells may offer an alternative explanation for reports of CD34 and Kit co-localization. The ontogeny and function of CD34-ir cells in the gut, as well as the origin of gastrointestinal stromal tumors, remain unclear

The pathology of infantile hypertrophic pyloric stenosis after healing.

INTRODUCTION Infantile hypertrophic pyloric stenosis (IHPS) is a common surgical affection of unknown etiology. The muscular hypertrophy is known to resolve within a few months after pyloromyotomy (PM). The pathology of IHPS has been studied extensively at the time of PM, but the fate of the pylorus after healing remains unknown. MATERIALS AND METHODS We had the rare opportunity to study two pyloric biopsy specimens obtained 4 months and 2 years (respectively) after an uncomplicated PM for IHPS. They were compared with the initial specimen in one case, with 26 other specimens of IHPS, and with five normal controls. Immunohistochemistry using the avidin-biotin complex (ABC) system was performed for S-100 and nerve growth factor receptor, as markers for the enteric nervous system, and for the tyrosine kinase receptor c-kit, as a marker for the interstitial cells of Cajal (pacemaker cells). NADPH-diaphorase histochemistry was performed as a marker for the neuronal enzyme nitric oxide synthase, which produces the inhibitory neurotransmitter nitric oxide. RESULTS In both cases of IHPS, after healing, the circular musculature was not hypertrophic. For all markers studied, the distribution appeared similar to that in the normal pylorus. In contrast, all specimens obtained at the time of PM displayed a severe reduction of the different markers in the hypertrophic musculature. DISCUSSION The pathological features observed in the circular layer in IHPS appear to resolve within a few months after PM. This suggests that the involvement of the enteric nervous system in IHPS might be milder than generally assumed. The etiology remains obscure, but our occasional observations may provide new insight into the pathophysiology of IHPS, and are in agreement with the excellent longterm clinical outcome for IHPS.

Morphological and neurochemical identification of enteric neurones with mucosal projections in the human small intestine.

Data on the axonal projections of enteric neurones in the human intestine are still scarce. The present study aimed to identify the morphology and neurochemical coding of enteric neurones in the human small intestine, which are involved in the innervation of the mucosa. The lipophilic neuronal tracer DiI was applied to one mucosal villus of small intestinal resection specimens. The tissue was kept in organotypic culture and subsequently processed for immunohistochemistry. Neurones labelled from the mucosa were located in all ganglionated nerve networks, including the myenteric plexus. In all plexuses, at least five neurochemical types of neurones could be observed, i.e. SOM‐IR neurones, SP‐IR neurones, SOM/SP‐IR neurones, VIP‐IR neurones and neurones lacking immunoreactivity for any of these markers. Most of the DiI‐labelled neurones were multidendritic; a minority of neurones could be identified as Dogiel type II cells, suggesting the existence of a subgroup of primary afferent neurones in the DiI‐filled cell population. The ratio of labelled multidendritic neurones (assumed to be secretomotor) to labelled Dogiel type II neurones (assumed to be primary afferent) in the myenteric plexus is higher in large mammals (pig and human) than in small mammals (guinea pig). This might point to the existence of a different topographical distribution of subsets of primary afferent neurones and/or topographically distinct intrinsic mucosal reflex circuits in large mammals, including humans.

Distribution of the intermediate filament nestin in the muscularis propria of the human gastrointestinal tract

Abstract. The intermediate filament nestin is expressed in neural stem cells, neuroectodermal tumors and various adult tissues. In the gastrointestinal (GI) tract, nestin has been reported in glial cells. Recently, nestin has been reported in interstitial cells of Cajal (ICC) and in gastrointestinal stromal tumors, thought to derive from ICC. Here we investigated nestin immunoreactivity (-ir) in the normal human GI tract, with emphasis on Kit-ir ICC. Two different antibodies specific for human nestin and multicolor high-resolution confocal microscopy were used on material from our human GI tissue collection. The staining pattern of both nestin antibodies was similar. In labeled cells, nestin-ir appeared filamentous. Most intramuscular ICC in antrum and all myenteric ICC (ICC-MP) in small intestine were nestin-ir, while nestin-ir was not detected in deep muscular plexus ICC. In the colon, some – but not all – ICC-MP and most ICC in the circular musculature were nestin-ir while nestin-ir was not detected in ICC in the longitudinal musculature and in the submuscular plexus. In addition, many Kit-negative cells were nestin-ir in all regions. Neurons and smooth muscle cells were consistently nestin negative, while most S100-ir glial cells were nestin-ir. In addition, nestin-ir was also present in some CD34-ir fibroblast-like cells, in endothelium and in other cell types in the mucosa and serosa. In conclusion, nestin-ir is abundantly present in the normal human GI tract. Among a number of cell types, several, but not all, subpopulations of Kit-ir ICC were nestin-ir. The functional significance of nestin in the GI tract remains obscure.

Pituitary adenylate cyclase activating peptide and its receptors are expressed in human neuroblastomas

Vasoactive intestinal peptide (VIP) has been considered as an autocrine growth factor in neuroblastomas. Pituitary adenylate cyclase activating polypeptides (PA‐CAPs) are newly recognized members of the VIP family of neurohormones. As compared to VIP, PACAP has been reported to be biologically more potent and more efficient in tissues expressing selective PACAP receptors rather than common VIP/PACAP receptors. PACAPs and VIP interact with the same affinity and stimulate adenylate cyclase activity with the same efficacy and potency on the VIP receptors, but PACAPs act also on a more selective PACAP receptor that also recognizes VIP but with a 100‐ to 1,000‐fold lower affinity. Thus, depending on the type of receptors expressed at a cell surface, PACAP may be more potent and efficient than VIP. The capacity of 22 surgical specimens of neuroblastomas and of 5 established cell lines to synthesize PACAP and VIP and to synthesize and express PACAP receptors and VIP receptors was studied. Using the reverse transcriptase‐polymerase chain (RT‐PCR) method with specific primers, we detected the mRNAs coding for PACAP and VIP in 19 and 3 out of 22 samples, respectively. PACAP mRNA was expressed in 3 of the 5 cell lines studied and VIP mRNA in 4. Using the same techniques, PACAP and VIP receptors mRNA were detected in 21, and 13 of the 22 tumor samples and in 5 and 1 of the cell lines studied, respectively. The expression of the PACAP receptor was demonstrated by direct binding studies and/or by the relative potency of PACAPs and VIP to stimulate adenylate cyclase activity in 16 of the 22 tumors and in all the cell lines. In addition, there was no correlation between tumor stage and the expression of mRNA coding for the peptides and the receptors. The present results demonstrated that PACAP could also be a candidate as an autocrine regulator of neuroblastoma which a higher activity than VIP.

Decrease of vasoactive intestinal peptide, methionine-enkephalin, substance P and increase of neuropeptide Y immunoreactive nerve fibres in aganglionic colon of Hirschsprung's disease.

Ganglionic and aganglionic full-thickness samples, at 4 levels of the colon of 26 infants with Hirschsprungs disease, were studied by immunohistochemistry. In the distal part of the aganglionic bowel, we observe a decrease of substance P and vasoactive intestinal peptide, an absence of methionine-enkephalin and an increase in neuropeptide Y nerve fibres. When detected, substance P and vasointestinal peptide are mainly present in abnormal bundle nerve fibres. In the middle part of the aganglionic bowel, a slight increase in the number of normal nerve fibres containing substance P, methionine-enkephalin and vasoactive intestinal peptide is observed. Some vasoactive intestinal peptide abnormal bundle nerve fibres are detected. They are less numerous than in the distal part. In the proximal ganglionic bowel, the number of vasoactive intestinal peptide, substance P and methionine-enkephalin normal nerve fibres is increased compared to the middle aganglionic segment but is slightly lower than in the normal colon. Again vasoactive intestinal peptide abnormal bundle nerve fibres are present at that level and are also detected in more proximal ganglionic bowel up to the hepatic flexure of the colon. Thus, abnormal distribution of neuropeptides is also found in more proximal ganglionic bowel and not only in the aganglionic segment of bowel usually specific of Hirschsprungs disease.

Proenkephalin A associated peptides in the autonomic nervous system of the human infant gastrointestinal tract.

The digestive tract of neonates and infants were examined by immunohistochemistry using specific antisera raised against proenkephalin A related peptides. Proenkephalin A, methionine-enkephalin and leucine-enkephalin are observed in nerve fibres in the smooth muscles in the myenteric and submucosal plexuses or in neuronal cell bodies of the myenteric plexus. In these structures synenkephalin has general distribution as methionine-enkephalin but not the same as leucine-enkephalin. Co-localization of synenkephalin and methionine-enkephalin is found in several neurones. These results suggest that proenkephalin A is the precursor-protein in some enkephalinergic neurones of the human gut. A gradient in the density of immunoreactivity is observed and is maximal in the distal small bowel. This gradient contrasts with observations made in rodents where major enkephalin immunoreactivity is observed in the proximal digestive tract. These findings give evidence that proenkephalin A-derived peptides could have effects in the motility of the human gut.