Three-dimensional visualization of termite (Apicotermitinae) enteric valve using confocal laser scanning microscopy.

Humivorous termites are dominant members of tropical rainforest soil communities. In the soil-feeding subfamily Apicotermitinae (Termitidae), the enteric valve connecting the first section of the hindgut to the paunch often displays a complex sclerotized armature everted towards the lumen of the paunch. This structure is central in termite taxonomy but its function remains hypothetical. Here, we evaluate the potential of confocal laser scanning microscopy to provide detailed imaging of the valve of Anoplotermes parvus, by comparison with bright-field microscopy and scanning electron microscopy. We detected a strong far-red emission of the enteric valve armature that sharply contrasted with the surrounding tissues, providing a convenient method to highlight minute structural elements of the valve and its three-dimensional structure. The method is easy to use and is applicable to standard archival material as demonstrated by images of enteric valves of four other Apicotermitinae species. It may represent a valuable asset for the study of termite enteric valves, for the purpose of taxonomy or functional morphology.

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to ß-cell apoptosis.

Pancreatic ß-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of ß-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in ß-cells following exposure to well-defined ß-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the ß-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to ß-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.

Quantitation of cellular components of the enteric nervous system in the normal human gastrointestinal tract--report on behalf of the Gastro 2009 International Working Group.

BACKGROUND: Patients with gastrointestinal neuromuscular diseases may undergo operative procedures that yield tissue appropriate to diagnosis of underlying neuromuscular pathology. Critical to accurate diagnosis is the determination of limits of normality based on the study of control human tissues. Although robust diagnostic criteria exist for many qualitative alterations in the neuromuscular apparatus, these do not include quantitative values due to lack of adequate control data. PURPOSE: The aim of this report was to summarize all relevant available published quantitative data for elements of the human enteric nervous system (neuronal cell bodies, glial cells, and nerve fibers) from the perspective of the practicing pathologist. Forty studies meeting inclusion criteria were systematically reviewed with data tabulated in detail and discussed in the context of methodological variations and limitations. The results reveal a lack of concordance between observations of different investigators resulting in data insufficient to produce robust normal ranges. This diversity highlights the need to standardize the way pathologists collect, process, and quantitate neuronal and glial elements in enteric neuropathologic samples, as suggested by recent international guidelines on gastrointestinal neuromuscular pathology.

Ulcerative colitis: ultrastructure of interstitial cells in myenteric plexus.

Interstitial cells of Cajal (ICC) are key regulatory cells in the gut. In the colon of patients with severe ulcerative colitis (UC), myenteric ICC had myoid ultrastructural features and were in close contact with nerve terminals. In all patients as opposed to controls, some ICC profiles showed degenerative changes, such as lipid droplets and irregular vacuoles. Nerve terminals often appeared swollen and empty. Glial cells, muscle cells, and fibroblast-like cells (FLC) showed no alterations. FLC enclosed macrophages (MLC), which were in close contact with naked axon terminals. The organization and cytological changes may be of pathophysiological significance in patients with UC.

Interstitial cells of Cajal in the vermiform appendix in childhood.

BACKGROUND AND AIMS: The interstitial cells of Cajal (ICC) have not yet been investigated in the vermiform appendix. They are important for the peristalsis of the gastrointestinal tract and have been found to be altered in various motility disorders. Motor disturbance has been suggested as a possible contributor in the unclear etiology of appendicitis. We wanted to examine the distribution of the ICC in the vermiform appendix. Furthermore we investigated whether ICC are altered in persons with appendicitis. METHODS: We investigated the ICC distribution in 28 appendices of children using immunohistochemistry and anti-c-kit antibodies. Cells and processes were quantified in normal, acute and chronic inflamed appendices. RESULTS: IC(C)-CM and IC(C)-LM were found in the circular and longitudinal muscle layers, respectively. IC(C)-LM, however, were scarce and inhomogeneous in contrast to the IC(C)-CM. The functionally important subgroups of the colon, the IC(C)-SM and IC(C)-MP, however, could not be detected in the appendix with the used antibody. There was no difference in the distribution of detected ICC between normal and inflamed appendices. CONCLUSION: IC(C)-LM are altered and IC(C)-SM and IC(C)-MP are lost in the vermiform appendix with no differences between healthy and inflamed tissue and without a correlation to appendicitis. Thus, other factors must be considered in the etiology of appendicitis.

Differential contribution of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons.

Plasma membrane chloride (Cl(-)) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active Cl(-) uptake mechanism) in Cl(-) handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin-perforated patch-clamp recordings revealed that DRGNs accumulate intracellular Cl(-) through a bumetanide- and Na(+)-sensitive mechanism, indicative of the functional expression of NKCC1. Western blotting confirmed the expression of NKCC1 in both DRGNs and MNs, but immunocytochemistry experiments showed a restricted expression in dendrites of MNs, which contrasts with a homogeneous expression in DRGNs. Both MNs and DRGNs could be readily loaded with or depleted of Cl(-) during GABA(A) receptor activation at depolarizing or hyperpolarizing membrane potentials. After loading, the rate of recovery to the resting Cl(-) concentration (i.e., [Cl(-)](i) decrease) was similar in both cell types and was unaffected by lowering the extracellular Na(+) concentration. In contrast, the recovery on depletion (i.e., [Cl(-)](i) increase) was significantly faster in DRGNs in control conditions but not in low extracellular Na(+). The experimental observations could be reproduced by a mathematical model for intracellular Cl(-) kinetics, in which DRGNs show higher NKCC1 activity and smaller Cl(-)-handling volume than MNs. On the basis of these results, we conclude that embryonic DRGNs show a higher somatic functional expression of NKCC1 than embryonic MNs. The high NKCC1 activity in DRGNs is important for maintaining high [Cl(-)](i), whereas lower NKCC1 activity in MNs allows large [Cl(-)](i) variations during neuronal activity.

Novel smooth muscle markers reveal abnormalities of the intestinal musculature in severe colorectal motility disorders.

Histopathological studies of gastrointestinal motility disorders have mainly focused on enteric nerves and interstitial cells of Cajal, but rarely considered the enteric musculature. Here we used both classical and novel smooth muscle markers and transmission electron microscopy (TEM) to investigate muscular alterations in severe colorectal motility disorders. Full-thickness specimens from Hirschsprung's disease, idiopathic megacolon, slow-transit constipation and controls were stained with haematoxylin/eosin (HE) and Masson's trichrome (MT), incubated with antibodies against smooth muscle alpha-actin (alpha-SMA), smooth muscle myosin heavy chain (SMMHC), smoothelin (SM) and histone deacetylase 8 (HDAC8) and processed for TEM. Control specimens exhibited homogeneous immunoreactivity for all antibodies. Diseased specimens showed normal smooth muscle morphology by HE and MT. While anti-alpha-SMA staining was generally normal, immunoreactivity for SMMHC, HDAC8 and/or SM was either absent or focally lacking in Hirschsprung's disease (80%), idiopathic megacolon (75%) and slow-transit constipation (70%). Ultrastructurally, clusters of myocytes with noticeably decreased myofilaments were observed in all diseases. SMMHC and the novel smooth muscle markers SM and HDAC8 often display striking abnormalities linked to the smooth muscle contractile apparatus unnoticed by both routine stainings and alpha-SMA, suggesting specific defects of smooth muscle cells involved in the pathogenesis of gastrointestinal motility disorders.

Differences in signaling pathways and expression level of the phosphoinositide phosphatase SHIP1 between two oncogenic mutants of the receptor tyrosine kinase KIT.

Oncogenic mutations of the receptor tyrosine kinase KIT are encountered in myeloid leukemia and various solid tumors, including gastrointestinal stromal tumors. We previously identified the human oncogenic germ line mutant KIT(K642E), a substitution in the tyrosine kinase 1 domain (TK1D) in a familial form of gastrointestinal stromal tumors. The effects of oncogenic KIT mutants on cell signaling and regulation are complex. Cellular models are valuable basic tools to tailor novel strategies on specific cellular and molecular bases for tumors expressing KIT oncogenic mutants. Murine KIT(WT) and the murine homologues of human KIT oncogenic mutants, further referred to as KIT(K641E) and KIT(del559), a point deletion in the juxtamembrane domain (JMD), were stably expressed in IL-3-dependent Ba/F3 cells. Major differences in the constitutively activation of Akt/PKB, MAP kinases and STATs pathways were observed between KIT(K641E) and KIT(del559), whereas KIT ligand elicited responses in both mutants. Noteworthy, the protein level of the phosphoinositide phosphatase SHIP1, but not SHIP2 and PTEN, was reduced in KIT(K641E) only while inhibition of KIT phosphorylation reversibly raised SHIP1 level in both JMD and TK1D oncogenic mutants, unraveling the control of SHIP protein level by KIT phosphorylation.

Interstitial cells of Cajal are involved in the afferent limb of the rectoanal inhibitory reflex.

BACKGROUND AND AIMS: Interstitial cells of Cajal (ICC) have been shown to be involved in nitrergic neurotransmission of the lower oesophageal sphincter and pylorus. Here we studied the role of ICC and nitric oxide (NO) in the inhibitory neurotransmission of the murine internal anal sphincter (IAS). METHODS: The rectoanal inhibitory reflex, rectal compliance, and relaxation of the isolated IAS to electrical stimulation were measured in controls, KIT (W)/KIT (Wv) mice, and neuronal NO synthase (nNOS) deficient mice. In addition, we evaluated the effect of blockade of nNOS using N-nitro-L-arginine methyl ester. Distribution of nNOS positive neurones and ICC in the IAS was assessed immunohistochemically. RESULTS: KIT positive ICC were present in a dense network in the IAS of controls but not in KIT (W)/KIT(Wv) mice. Relaxation of IAS muscle strips induced by electrical stimulation was diminished in nNOS-/- mice but not in KIT (W)/KIT (Wv) mice. Blockade of NOS reduced the relaxation of IAS muscle strips in both mice. Relaxation of the IAS to rectal distension was significantly diminished in KIT (W)/KIT (Wv) mice and nNOS deficient mice. In concert, in vivo blockade of NOS attenuated the relaxation of the IAS in controls. No significant difference in compliance was found. CONCLUSION: The inhibitory innervation of the IAS and the rectoanal inhibitory reflex are mediated by NO and the rectoanal inhibitory reflex requires an intact network of ICC in the IAS. Thus both loss of nitrergic innervation and deficiency of ICC lead to impaired anal relaxation and may play an important role in rectal evacuation disorders.

Anatomical and neurochemical features of the extrinsic and intrinsic innervation of the striated muscle in the porcine esophagus: evidence for regional and species differences.

Studies of the intrinsic and extrinsic innervation patterns of esophageal motor endplates (MEPs) are mainly confined to small rodents. Therefore, an immunocytochemical, denervation and tracing study was conducted on the pig, an experimental model in which the distribution of the striated esophageal muscle portion more closely resembles the human situation. The purpose of this study was to analyze the origin and neurochemical content of the nerve fibers participating in the myoneural synapse. Fifteen 6-week-old domestic pigs were studied by immunohistochemistry combined with alpha-bungarotoxin labeling to define the co-innervation patterns of nitrergic and peptidergic nerve terminals in MEPs. Some animals were subjected to unilateral infra- or supranodose vagotomy to determine the origin of the nerve terminals in MEPs. Special attention was paid to the interregional differences in terms of co-innervation rates, and these findings were compared with literature data on small mammals. Double stainings revealed that most of the nNOS-immunoreactive (ir) terminals in MEPs co-stained for VIP, GAL and NPY, but not for PACAP and L-ENK. PACAP- and L-ENK-ir terminals were coarser than nNOS-ir terminals, and largely co-localized VAChT. High percentages of MEPs at the cervical level were contacted by PACAP- (approximately 94%) and L-ENK-ir (approximately 78%) terminals, but the proportion of both decreased in the rostrocaudal direction. Vagotomy significantly reduced their presence in MEPs at the thoracic and abdominal levels, while nNOS-ir terminals observed in approximately 30% of the MEPs were unaffected by vagotomy. Immunostainings on brainstem cryosections after retrograde tracing from the cervical esophagus showed that a large number of FB-positive cells in the nucleus ambiguus were PACAP-ir (approximately 72%). C-kit-positive interstitial cells of Cajal were seen adjacent to the striated muscle fibers, apparently without direct relationship to MEPs. Similar to mouse esophagus, intrinsic nitrergic fibers were found to run close to, or even spiral around, these interstitial cells, an association that might point to a role as specialized spindle proprioceptors. In conclusion, the cholinergic terminals-part of which coexpress PACAP and/or L-ENK-that innervate MEPs in the porcine esophagus have a vagal origin, whereas the nNOS/VIP/GAL/NPY-ir fibers co-innervating these MEPs are intrinsic in nature. The regional differences observed along the esophageal length pertain to the neurochemical content of the vagal motor innervation of the MEPs.

Free cytosolic Ca2+ recordings from myenteric neurones in multilayer intestinal preparations.

The ability to simultaneously monitor different myenteric neurones in a multilayer preparation may enhance our understanding of the enteric nervous system. Longitudinal muscle myenteric plexus preparations were mounted in recording chambers with a coverslip base and loaded with Indo-1-AM. cytosolic Ca2+ concentration ([Ca2+]i); changes were recorded at room temperature with a confocal microscope. In addition to mechanical (pressure-ring) and pharmacological (nifedipine) reduction of muscle contractions, purpose-designed software was developed to reposition regions of interest and avoid artefacts. Confocal scanning permitted optical selection of single cell layers. High K+ depolarization, used to distinguish between excitable and nonexcitable cells, caused a synchronous [Ca2+]i rise in 84.3% of the ganglion cells. Acetylcholine, substance P and serotonin (all at 10(-5) mol L(-1)) induced transient [Ca2+]i changes in subpopulations of myenteric neurones (45.1%, 42.9 and 21.9%, respectively). In addition to immediate responses to agonists, delayed [Ca2+]i changes were also recorded, suggesting the presence of both directly activated and synaptically driven neurones. Functionally identified neurones and other cells in close apposition to the ganglia (interstitial cells of Cajal) could also be studied. This study demonstrates the potential of optical Ca2+ recordings to monitor spread of activity in myenteric neurones and to study their interaction with non-neuronal targets.

Interstitial cells of Cajal in the striated musculature of the mouse esophagus.

Interstitial cells of Cajal (ICC) are important regulatory cells in the smooth muscle coats of the digestive tract. Expression of the Kit receptor tyrosine kinase was used in this study as a marker to study their distribution and development in the striated musculature of the mouse esophagus. Sections and whole-mounts were studied by immunohistochemistry. KitW-lacZ transgenic mice, which carry the lacZ reporter gene inserted in place of the first exon of the Kit gene, were processed for Xgal histochemistry, for quantitative analysis and for ultrastructural studies. Spindle-shaped ICC were scarce in both muscle layers of the thoracic esophagus, while their number increased steeply toward the cardia in the striated portion of the intraabdominal esophagus. They did not form networks and had no relationship with intrinsic myenteric ganglia and motor end-plates. They were often close to nerve fibers immunoreactive for neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) or neuropeptide Y (NPY), but not to fibers immunoreactive for substance P (SP), calcitonin gene related peptide (CGRP), enkephalin, or the capsaicin receptor VRI. They were present in the fetus but absent in adult ICC-deficient KitW-lacZ/KitWv mice. Interstitial cells of Cajal were identified by electron microscopy by their ultrastructure in the striated muscle of the esophagus and exhibited Xgal labeling, while fibroblasts and muscle cells were unlabeled. Interstitial cells of Cajal are scattered between striated muscle cells in the mouse esophagus. They are close to nerves with defined neurochemical coding and could possibly represent specialized esophageal spindle proprioceptors.

Comparative study of hippocampal neuronal loss and in vivo binding of 5-HT1a receptors in the KA model of limbic epilepsy in the rat.

A high density of 5-HT1a receptors is present in pyramidal hippocampal cells. Mapping of these receptors may be performed in vivo using the tracer no-carrier-added 4-(18)F-fluoro-N-2-(1-(2-methoxyphenyl)-1-piperazinyl)ethyl-N-2-pyridinyl-benzamide (MPPF). We tested the hypothesis of a relationship between MPPF binding and post-epileptic neuronal loss in the hippocampus. The model of limbic epilepsy induced by kainic acid (KA) in the rat was used. Rats were sacrificed at various times (1 h-240 days) after systemic injection of 10 mg/kg KA. Determination of MPPF binding in the brain was combined with a quantification of neuronal loss using DNA labeling with propidium iodide and confocal microscopy. Hippocampal MPPF binding varied according to time elapsed from KA injection. An initial decrease from day 1 to day 6 post injection was followed by a relative increase between day 6 and day 30. This effect was observed in rats which showed hippocampal neuronal loss but also in one rat which did not. In KA treated rats, statistically significant relationship between MPPF binding and neuronal count was found during the acute period (rats sacrificed 1 h-day 6 after KA injection) and the chronic phase (rats sacrificed beyond day 60 after KA injection). The late relative increase of MPPF binding suggests an epilepsy-induced increase of 5-HT1a receptors in the hippocampus. This effect needs to be further characterized before considering PET determination of hippocampal MPPF binding as a method of post-epileptic neuronal loss assessment.

The SH2 domain-containing 5-phosphatase SHIP2 is expressed in the germinal layers of embryo and adult mouse brain: increased expression in N-CAM-deficient mice.

The germinative ventricular zone of embryonic brain contains neural lineage progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. The ability to generate neurons persists at adulthood in restricted brain areas. During development, many growth factors exert their effects by interacting with tyrosine kinase receptors and activate the phosphatidylinositol 3-kinase and the Ras/MAP kinase pathways. By its ability to modulate these pathways, the recently identified Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2, SHIP2, has the potential to regulate neuronal development. Using in situ hybridization technique with multiple synthetic oligonucleotides, we demonstrated that SHIP2 mRNA was highly expressed in the ventricular zone at early embryonic stages and subventricular zones at latter stages of brain and spinal cord and in the sympathetic chain. No significant expression was seen in differentiated fields. This restricted expression was maintained from embryonic day 11.5 to birth. In the periphery, large expression was detected in muscle and kidney and moderate expression in thyroid, pituitary gland, digestive system and bone. In the adult brain, SHIP2 was mainly restricted in structures containing neural stem cells such as the anterior subventricular zone, the rostral migratory stream and the olfactory tubercle. SHIP2 was also detected in the choroid plexuses and the granular layer of the cerebellum. The specificity of SHIP2 expression in neural stem cells was further demonstrated by (i) the dramatic increase in SHIP2 mRNA signal in neural cell adhesion molecule (N-CAM)-deficient mice, which present an accumulation of progenitor cells in the anterior subventricular zone and the rostral migratory stream, (ii) the abundant expression of 160-kDa SHIP2 by western blotting in proliferating neurospheres in culture and its downregulation in non-proliferating differentiated neurospheres. In conclusion, the close correlation between the pattern of SHIP2 expression in the brain and the proliferative and early differentiative events suggests that the phosphatase SHIP2 may have important roles in neural development.

The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54.

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.

Palmitoylation of CCR5 is critical for receptor trafficking and efficient activation of intracellular signaling pathways.

CCR5 is a CC chemokine receptor expressed on memory lymphocytes, macrophages, and dendritic cells and also constitutes the main coreceptor for macrophage-tropic (or R5) strains of human immunodeficiency viruses. In the present study, we investigated whether CCR5 was palmitoylated in its carboxyl-terminal domain by generating alanine substitution mutants for the three cysteine residues present in this region, individually or in combination. We found that wild-type CCR5 was palmitoylated, but a mutant lacking all three Cys residues was not. Through the use of green fluorescent fusion proteins and immunofluorescence studies, we found that the absence of receptor palmitoylation resulted in sequestration of CCR5 in intracellular biosynthetic compartments. By using the fluorescence recovery after photobleaching technique, we showed that the non-palmitoylated mutant had impaired diffusion properties within the endoplasmic reticulum. We next studied the ability of the mutants to bind and signal in response to chemokines. Chemokines binding and activation of G(i)-mediated signaling pathways, such as calcium mobilization and inhibition of adenylate cyclase, were not affected. However, the duration of the functional response, as measured by a microphysiometer, and the ability to increase [(35)S]guanosine 5'-3-O-(thio)triphosphate binding to membranes were severely affected for the non-palmitoylated mutant. The ability of RANTES (regulated on activation normal T cell expressed and secreted) and aminooxypentane-RANTES to promote CCR5 endocytosis was not altered by cysteine replacements. Finally, we found that the absence of receptor palmitoylation reduced the human immunodeficiency viruses coreceptor function of CCR5, but this effect was secondary to the reduction in surface expression. In conclusion, we found that palmitoylated cysteines play an important role in the intracellular trafficking of CCR5 and are likely necessary for efficient coupling of the receptor to part of its repertoire of signaling cascades.

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.

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

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.

Distribution and ultrastructure of interstitial cells of Cajal in the mouse colon, using antibodies to Kit and Kit(W-lacZ) mice.

The roles of the interstitial cells of Cajal in the stomach and intestine are becoming increasingly clear. Interstitial cells of Cajal in the colon are less well known, however. We studied the development and distribution of the interstitial cells of Cajal in the mouse colon, using the tyrosine kinase receptor Kit as a marker. Sections and whole mounts were studied by confocal microscopy after double immunofluorescence with specific antibodies. The ultrastructure of Kit-expressing cells was examined by electron microcopy in KitW-lacz/+ transgenic mice, which carry the lacz gene inserted in place of the first exon of the Kit gene. In the subserosa, the interstitial cells of Cajal formed a two-dimensional plexus. In the myenteric area, the interstitial cells of Cajal formed a dense plexus that gradually merged with the interstitial cells of Cajal in the outer half of the circular muscle. The inner half of the circular layer was devoid of interstitial cells of Cajal whereas in the submuscular region the interstitial cells of Cajal formed a two-dimensional plexus. Tertiary nerves with various chemical codings closely followed interstitial cell of Cajal processes. By electron microscopy, Kit-expressing cells in the outer parts of the musculature had scattered caveolae, inconspicuous basal lamina and numerous mitochondria, whereas in the submuscular region they had more pronounced myoid features. Kit-expressing cells in the mouse colon are identifiable as interstitial cells of Cajal by their ultrastructure. The interstitial cells of Cajal in the mouse colon mature postnatally. They are organized into a characteristic plexus, close to the nerves with various chemical codings.