Dirk Adriaensen

Cell of Origin of Small Cell Lung Cancer: Inactivation of Trp53 and Rb1 in Distinct Cell Types of Adult Mouse Lung

Small cell lung cancer (SCLC) is one of the most lethal human malignancies. To investigate the cellular origin(s) of this cancer, we assessed the effect of Trp53 and Rb1 inactivation in distinct cell types in the adult lung using adenoviral vectors that target Cre recombinase to Clara, neuroendocrine (NE), and alveolar type 2 (SPC-expressing) cells. Using these cell type-restricted Adeno-Cre viruses, we show that loss of Trp53 and Rb1 can efficiently transform NE and SPC-expressing cells leading to SCLC, albeit SPC-expressing cells at a lesser efficiency. In contrast, Clara cells were largely resistant to transformation. The results indicate that although NE cells serve as the predominant cell of origin of SCLC a subset of SPC-expressing cells are also endowed with this ability.

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract.

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.

Pulmonary intraepithelial vagal nodose afferent nerve terminals are confined to neuroepithelial bodies: an anterograde tracing and confocal microscopy study in adult rats.

Abstract Our present understanding of the morphology of neuroepithelial bodies (NEBs) in mammalian lungs is comprehensive. Several hypotheses have been put forward regarding their function but none has been proven conclusively. Microscopic data on the innervation that appears to affect the reaction of NEBs to stimuli have given rise to conflicting interpretations. The aim of this study has been to check the validity of the hypothesis that pulmonary NEBs receive an extensive vagal sensory innervation. The fluorescent neuronal tracer DiI was injected into the vagal sensory nodose ganglion and NEBs were visualized in toto by using immunocytochemistry and confocal microscopy on 100-µm-thick frozen sections of the lungs of adult rats. The most striking finding was the extensive intraepithelial terminal arborizations of DiI-labelled vagal afferents in intrapulmonary airways, apparently always co-appearing with calcitonin gene-related peptide (CGRP)-immunoreactive NEBs. Not all NEBs received a traced nerve fibre. Intrapulmonary CGRP-containing nerve fibres, including those innervating NEBs, always appeared to belong to a nerve fibre population different from the DiI-traced fibres and hence did not arise from the nodose ganglion. Therefore, at least some of the pulmonary NEBs in adult rats are supplied with sensory nerve fibres that originate from the vagal nodose ganglion and form beaded ramifications between the NEB cells, thus providing support for the hypothesis of a receptor function for NEBs.

Structural organization and neuropeptide distribution in the mammalian enteric nervous system, with special attention to those components involved in mucosal reflexes.

Gastrointestinal events such as peristalsis and secretion/absorption processes are influenced by the enteric nervous system, which is capable of acting largely independently from other parts of the nervous system. Several approaches have been used to further our understanding of the underlying mechanisms of specific enteric microcircuits. Apart from pharmacological and physiological studies, the deciphering of the chemical coding of distinct morphological and functional enteric neuron classes, together with a detailed analysis of their projections by the application of immunocytochemistry, of tracing, and of denervation techniques, have substantially contributed to our knowledge. In view of existing interspecies and regional differences, it is of major importance to expand our knowledge of the enteric nervous system in mammals other than the guinea-pig, the most commonly used experimental animal in this research area. This will increase our chances of finding a valid model, from which well-founded extrapolations can be made regarding the precise function of distinct enteric neuron types regulating motility and ion transport in the human gastrointestinal tract.

Distinct distribution of CGRP-, enkephalin-, galanin-, neuromedin U-, neuropeptide Y-, somatostatin-, substance P-, VIP- and serotonin-containing neurons in the two submucosal ganglionic neural networks of the porcine small intestine

SummaryIn addition to differences between the two submucosal ganglionic neural networks, i.e., the plexus submucosus externus (Schabadasch) and the plexus submucosus internus (Meissner), with respect to the occurrence and distribution of serotonin as neurotransmitter, immunocytochemistry also revealed a distinct distribution for various neuropeptides in these two plexuses. Immunoreactivity for galanin, vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P, neuromedin U, enkephalin, somatostatin and neuropeptide Y was found in varicose and non-varicose nerve fibres of both submucosal ganglionic plexuses, albeit with a distinct distributional pattern. The difference in neurotransmitter and/or neuromodulator content between both neural networks became even more obvious when attention was focussed on the immunoreactivity of the nerve cell bodies for these substances. Indeed, neuropeptide Y, enkephalin-and somatostatin-immunoreactive neuronal perikarya as well as serotonergic neuronal cell bodies appear solely in the plexus submucosus externus. Neuromedin U-immunoreactive perikarya, mostly coexisting with substance P, are observed in large numbers in the plexus submucosus internus, whilst they are rare in the plexus submucosus externus. Double-labelling immunostaining for substance P with CGRP and galanin revealed a different coexistence pattern for the two submucosal ganglionic plexuses. The differing chemical content of the neuronal populations supports the hypothesis that the existence of the two submucosal ganglionic plexuses, present in most large mammals including man, not only reflects a morphological difference but also points to differentiated functions.

Outer submucous plexus: an intrinsic nerve network involved in both secretory and motility processes in the intestine of large mammals and humans.

The architecture of the enteric nerve networks in the gastrointestinal tract appears to be more complex in large mammals, including humans, than in small laboratory animals. At least two distinct ganglionic nerve plexuses could be identified in the submucous layer in the digestive tract of large mammals. While functionally and morphologically similar neuron populations are found in the intestinal wall of both small and large mammals, significant differences in their topographical organization and neurochemical features may be present. This short review clearly illustrates that the close and exclusive association, which has been assumed so far between the efferent pathways of the submucous plexus and regulation of intestinal secretion/absorption on the one hand and between the myenteric plexus and regulation of intestinal motility on the other hand, cannot be interpreted that strictly. An attempt has been made to give a briefoverview of the current status of the identification of distinct functional enteric neuronal classes in the gastrointestinal tract of large mammals using the pig and human intestine as references, and to compare these data with the more extensive information gathered from the guinea‐pig intestine. Anat Rec 262:71–78, 2001.

Mucosal projections of enteric neurons in the porcine small intestine.

In the present study, a combination of immunohistochemistry and retrograde 1,1`‐didodecyl‐3,3,3`,3`‐tetramethylindocarbocyanine perchlorate (DiI) tracing was used to unravel the morphology, distribution, and neurochemical coding of submucous and myenteric neurons with axonal projections to the mucosa of the porcine small intestine. The majority of traced neurons was located in the inner submucous plexus (ISP; 78%), whereas the remaining part was distributed between the outer submucous plexus (OSP; 10%) and myenteric plexus (MP; 12%). Among these traced neurons, some distinct neuronal populations could be distinguished according to their morphologic and neurochemical properties. In the ISP, several types of traced neurons were detected: 1) morphologic type II neurons expressing choline acetyltransferase (ChAT) immunoreactivity, calcitonin gene‐related peptide (CGRP) immunoreactivity, and substance P (SP) immunoreactivity; 2) ChAT/SP‐immunoreactive (‐IR) small neurons; 3) vasoactive intestinal polypeptide (VIP) ‐IR small neurons; and 4) multidendritic ChAT/somatostatin (SOM) ‐IR neurons. The traced neuronal populations of the OSP and MP were similar to each other. In both plexuses, the following DiI‐labelled neurons were found: 1) ChAT/CGRP/(SP)‐IR type II neurons; 2) multidendritic ChAT/SP‐IR neurons; and 3) multidendritic ChAT/SOM‐IR neurons. Comparison of the present findings with previously obtained data concerning the mucosal innervation pattern of the intestine of small mammals, revealed significant species differences with respect to the morphologic and neurochemical features of the involved enteric neuronal classes. Although not identical, a closer resemblance between pig and human enteric nervous system seems to be at hand, as far as the anatomic organization and the presence of neurochemically identified neuronal subtypes within the enteric nervous system are concerned. J. Comp. Neurol. 421:429–436, 2000.

Purinergic Signaling in the Airways

Evidence for a significant role and impact of purinergic signaling in normal and diseased airways is now beyond dispute. The present review intends to provide the current state of knowledge of the involvement of purinergic pathways in the upper and lower airways and lungs, thereby differentiating the involvement of different tissues, such as the epithelial lining, immune cells, airway smooth muscle, vasculature, peripheral and central innervation, and neuroendocrine system. In addition to the vast number of well illustrated functions for purinergic signaling in the healthy respiratory tract, increasing data pointing to enhanced levels of ATP and/or adenosine in airway secretions of patients with airway damage and respiratory diseases corroborates the emerging view that purines act as clinically important mediators resulting in either proinflammatory or protective responses. Purinergic signaling has been implicated in lung injury and in the pathogenesis of a wide range of respiratory disorders and diseases, including asthma, chronic obstructive pulmonary disease, inflammation, cystic fibrosis, lung cancer, and pulmonary hypertension. These ostensibly enigmatic actions are based on widely different mechanisms, which are influenced by the cellular microenvironment, but especially the subtypes of purine receptors involved and the activity of distinct members of the ectonucleotidase family, the latter being potential protein targets for therapeutic implementation.

The bidirectional communication between neurons and mast cells within the gastrointestinal tract

Normal or disordered behaviour of the gastrointestinal tract is determined by a complex interplay between the epithelial barrier, immune cells, blood vessels, smooth muscle and intramurally located nerve elements. Mucosal mast cells (MMCs), which are able to detect noxious and antigenic threats and to generate or amplify signals to the other cells, are assigned a rather central position in this complex network. Signal input from MMCs to intrinsic enteric neurons is particularly crucial, because the enteric nervous system fulfils a pivotal role in the control of gastrointestinal functions. Activated enteric neurons are able to generate an alarm program involving alterations in motility and secretion. MMC signalling to extrinsic nerve fibres takes part in pathways generating visceral pain or extrinsic reflexes contributing to the disturbed motor and secretory function. Morphological and functional studies, especially studies concerning physiological stress, have provided evidence that, apart from the interaction between the enteric nervous system and MMCs, there is also a functional communication between the central nervous system and these mast cells. Psychological factors trigger neuronal pathways, which directly or indirectly affect MMCs. Further basic and clinical research will be needed to clarify in more detail whether basic patterns of this type of interactions are conserved between species including humans.

Triple Immunofluorescence Staining with Antibodies Raised in the Same Species to Study the Complex Innervation Pattern of Intrapulmonary Chemoreceptors

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.