Chronic constipation in Parkinson's disease: clinical features and molecular insights on the intestinal epithelial barrier.

Background: Chronic constipation (CC) is a severe symptom in Parkinson's disease (PD), with an unclear pathogenesis. Abnormalities of the enteric nervous system (ENS) and/or intestinal epithelial barrier (IEB) may be pathophysiologically relevant in PD patients with CC. We investigated possible molecular changes of the IEB in PD/CCs compared with CCs and controls. Methods: Twelve PD/CCs (2 female, age range 51-80 years), 20 CCs (15 female, age range 27-78 years), and 23 controls (11 female, age range 32-74 years) were enrolled. Ten PD/CCs and 10 CCs were functionally characterized by anorectal manometry (AM) and transit time (TT). Colon biopsies were obtained and assessed for gene and protein expression, and localization of IEB tight junction markers claudin-4 (CLDN4), occludin-1 (OCCL-1), and zonula occludens-1 (ZO-1) by RT-qPCR, immunoblot and immunofluorescence labeling. Results: PD/CCs were clustered in 2 functional categories: patients with delayed TT and altered AM (60%), and a second group showing only modifications in AM pattern (40%). Gene expression of CLDN4, OCCL-1 and ZO-1 was higher in PD/CCs than controls (P<0.05). Conversely, PD/CCs showed a trend to decrease (P>0.05) in CLDN4 and OCCL-1 protein levels than controls, whereas ZO-1 protein was comparable. In PD/CCs compared with controls, decreasing tendency of vasoactive intestinal polypeptide mRNA, protein and immunoreactive fiber density were observed, although the difference was not statistically significant. Conclusion: Transit and anorectal dysfunctions in PD/CCs are associated with difference in ZO-1, OCCL-1 and CLDN4 expression, thus supporting the role of an altered IEB as a contributory mechanism to possible neuronal abnormalities.

LPAR1 regulates enteric nervous system function through glial signaling and contributes to chronic intestinal pseudo-obstruction.

Gastrointestinal motility disorders involve alterations to the structure and/or function of the enteric nervous system (ENS) but the causal mechanisms remain unresolved in most cases. Homeostasis and disease in the ENS are processes that are regulated by enteric glia. Signaling mediated through type I lysophosphatidic acid receptors (LPAR1) has recently emerged as an important mechanism that contributes to disease, in part, through effects on peripheral glial survival and function. Enteric glia express LPAR1 but its role in ENS function and motility disorders is unknown. We used a combination of genetic, immunohistochemical, calcium imaging, and in vivo pharmacological approaches to investigate the role of LPAR1 in enteric glia. LPAR1 was enriched in enteric glia in mice and humans and LPA stimulated intracellular calcium responses in enteric glia, subsequently recruiting activity in a subpopulation of myenteric neurons. Blocking LPAR1 in vivo with AM966 attenuated gastrointestinal motility in mice and produced marked enteric neuro- and gliopathy. Samples from humans with chronic intestinal pseudo-obstruction (CIPO), a severe motility disorder, showed reduced glial LPAR1 expression in the colon and ileum. These data suggest that enteric glial LPAR1 signaling regulates gastrointestinal motility through enteric glia and could contribute to severe motility disorders in humans such as CIPO.

Ex vivo ultrasonographic and histological morphometry of small intestinal wall layers in horses.

Ultrasonographic morphometry of wall layers is commonly used in veterinary patients with suspected small intestinal disease, however published studies comparing this method with histopathology in horses are limited. This prospective, methods comparison study compared the qualitative and quantitative characteristics of small intestinal wall layers using ex vivo high-frequency ultrasound versus histopathology in a sample of 16 horses. Transverse section images of duodenum, distal jejunum, and ileum were acquired with a high-frequency linear transducer (7-15 MHz). Transverse histological cryosections were obtained at the same level. Appearance and measurements of the intestinal wall layers were assessed on the ultrasonographic and histological images. High-frequency scanning with the probe in close contact with the serosal surface of the equine intestinal wall allowed a clear and detailed definition of wall layers. A hyperechoic line was consistently detected within the tunica muscularis in all the intestinal tracts, corresponding histologically to the interface between its longitudinal and circular muscle layers. The overall trend of the values for wall layers thickness was comparable between ex vivo ultrasonography and histology. However, a poor agreement was found between the two methods for all layers. The ultrasonographic measurements were thicker compared to histological measurements, with the exception of the total wall and the muscular layer thicknesses. These layers were thinner on ultrasonography in the duodenum and in all the intestinal segments, respectively. Findings from the current study can be used as background for future ultrasonographic investigations of small intestinal diseases in horses.

Beyond biologics: advanced therapies in inflammatory bowel diseases.

INTRODUCTION: Inflammatory bowel diseases (IBDs) are conditions affecting the gut at different levels characterized by an abnormal activation of the intestinal immune system. In this narrative review, we will provide the reader with an update on the efficacy and safety of new pharmacological strategies to treat IBD patients. EVIDENCE ACQUISITION: We performed a thorough literature review via PubMed, EMBASE, MEDLINE and Science Direct databases addressing studies reporting on new therapies for IBD management published in the last ten years (January 2010-December 2020). Data from pharmaceutical companies and abstracts of conferences/meetings have also been considered. EVIDENCE SYNTHESIS: The discovery of monoclonal antibodies blocking pro-inflammatory cytokines, e.g., tumor necrosis factor-α (TNF-α) radically changed the management of IBDs. Anti-TNF-α agents represent the prototype molecule of "biologics"/"biologicals." These compounds have significantly improved the therapeutic management of IBDs refractory to standard medications as they provide clinical remission, mucosal healing and prevent extra-intestinal manifestations. However, about 50% of patients treated with biologicals experienced drawbacks, including primary failure or loss of response, requiring new effective treatments. Translational studies have identified new strategies, different from the TNF-α blockade, and new molecules, e.g. sphingosine-1-phosphate agonists and the JAK kinase inhibitors, have been proposed as potential therapeutic options for IBDs. CONCLUSIONS: With the availability of novel approaches reviewed in this article, physicians and especially gastroenterologists will increase the therapeutic options to provide a better management of IBD patients, particularly those poorly responsive to biologicals.

Cellular distribution of cannabinoid-related receptors TRPV1, PPAR-gamma, GPR55 and GPR3 in the equine cervical dorsal root ganglia.

BACKGROUND: The activation of cannabinoid and cannabinoid-related receptors by endogenous, plant-derived or synthetic cannabinoids may exert beneficial effects on pain perception. Of the cannabinoids contained in Cannabis sativa, cannabidiol (CBD) does not produce psychotropic effects and seems to represent a molecule having great therapeutic potential. Cannabidiol acts on a great number of cannabinoid and cannabinoid-related G-protein-coupled receptors and ionotropic receptors which have, to date, been understudied in veterinary medicine particularly in equine medicine. OBJECTIVES: To localise the cellular distribution of four putative cannabinoid-related receptors in the equine cervical dorsal root ganglia (DRG). STUDY DESIGN: A qualitative and quantitative immunohistochemical study. METHODS: The cervical (C6-C8) DRG of six slaughtered horses were obtained from a local slaughterhouse. The tissues were fixed and processed for immunohistochemistry, and the resulting cryosections were used to investigate immunoreactivity for the following putative CBD receptors: Transient receptor potential vanilloid type 1 (TRPV1), nuclear peroxisome proliferator-activated receptor gamma (PPARγ), and G protein-coupled receptors 55 (GPR55) and 3 (GPR3). RESULTS: Large percentages of neuronal cell bodies showed immunoreactivity for TRPV1 (80 ± 20%), PPARγ (100%), GPR55 (64 ± 15%) and GPR3 (63 ± 11%). The satellite glial cells (SGCs) were immunoreactive for TRPV1, PPARγ and GPR55. In addition, GPR55 immunoreactivity was expressed by DRG interneuronal macrophages. In addition, microglia cells were observed surrounding the neuron-SGC complex. MAIN LIMITATIONS: The limited number of horses included in the study. CONCLUSIONS: Cannabinoid-related receptors were distributed in the sensory neurons (TRPV1, PPARγ, GPR55 and GPR3), SGCs (TRPV1, PPARγ and GPR55), macrophages (GPR55) and other interneuronal cells (PPARγ and GPR55) of the equine DRG. Given the key role of DRG cellular elements and cannabinoid receptors in the pathophysiology of pain, the present findings provided an anatomical basis for additional studies aimed at exploring the therapeutic uses of non-psychotropic cannabinoid agonists for the management of pain in horses.

Localisation of Cannabinoid and Cannabinoid-Related Receptors in the Horse Ileum.

Colic is a common digestive disorder in horses and one of the most urgent problems in equine medicine. A growing body of literature has indicated that the activation of cannabinoid receptors could exert beneficial effects on gastrointestinal inflammation and visceral hypersensitivity. The localisation of cannabinoid and cannabinoid-related receptors in the intestine of the horse has not yet been investigated. The purpose of this study was to immunohistochemically localise the cellular distribution of canonical and putative cannabinoid receptors in the ileum of healthy horses. Distal ileum specimens were collected from six horses at the slaughterhouse. The tissues were fixed and processed to obtain cryosections which were used to investigate the immunoreactivity of canonical cannabinoid receptors 1 (CB1R) and 2 (CB2R), and three putative cannabinoid-related receptors: nuclear peroxisome proliferator-activated receptor-alpha (PPARα), transient receptor potential ankyrin 1 and serotonin 5-HT1a receptor (5-HT1aR). Cannabinoid and cannabinoid-related receptors showed a wide distribution in the ileum of the horse. The epithelial cells showed immunoreactivity for CB1R, CB2R and 5-HT1aR. Lamina propria inflammatory cells showed immunoreactivity for CB2R and 5-HT1aR. The enteric neurons showed immunoreactivity for CB1R, transient receptor potential ankyrin 1 and PPARα. The enteric glial cells showed immunoreactivity for CB1R and PPARα. The smooth muscle cells of the tunica muscularis and the blood vessels showed immunoreactivity for PPARα. The present study represents a histological basis which could support additional studies regarding the distribution of cannabinoid receptors during gastrointestinal inflammatory diseases as well as studies assessing the effects of non-psychotic cannabis-derived molecules in horses for the management of intestinal diseases.

Nutritional Treatment in Crohn's Disease.

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) which can affect any part of the whole gastrointestinal tract (from mouth to anus). Malnutrition affects 65-75% of CD patients, and it is now well acknowledged that diet is of paramount importance in the management of the disease. In this review, we would like to highlight the most recent findings in the field of nutrition for the treatment of CD. Our analysis will cover a wide range of topics, from the well-established diets to the new nutritional theories, along with the recent progress in emerging research fields, such as nutrigenomics.

Gastrointestinal Involvement in Anderson-Fabry Disease: A Narrative Review.

Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder leading to a wide array of clinical manifestations. Among these, gastrointestinal (GI) symptoms such as abdominal pain, bloating, and diarrhea affect about half of the FD adults and more than half of FD children. GI symptoms could be the first manifestation of FD; however, being non-specific, they overlap with the clinical picture of other conditions, such as irritable bowel syndrome and inflammatory bowel disease. This common overlap is the main reason why FD patients are often unrecognized and diagnosis is delayed for many years. The present narrative review is aimed to promote awareness of the GI manifestations of FD amongst general practitioners and specialists and highlight the latest findings of this rare condition including diagnostic tools and therapies. Finally, we will discuss some preliminary data on a patient presenting with GI symptoms who turned to be affected by a variant of uncertain significance of alpha-galactosidase (GLA) gene.

Nitrergic and Substance P Immunoreactive Neurons in the Enteric Nervous System of the Bottlenose Dolphin (Tursiops truncatus) Intestine.

Compared with other mammals, the digestive system of cetaceans presents some remarkable anatomical and physiological differences. However, the neurochemical features of the enteric nervous system (ENS) in these animals have only been described in part. The present study gives a description of the nitrergic and selected peptidergic systems in the myenteric plexus (MP) and submucosal plexus (SMP) of the intestine of the bottlenose dolphin (Tursiops truncatus). The distribution and morphology of neurons immunoreactive (IR) for the neuronal nitric oxide synthase (nNOS) and Substance P (SP) were immunohistochemically studied in formalin-fixed specimens from the healthy intestine of three animals, and the data were compared with those described in the literature on other mammals (human and non-human). In bottlenose dolphins, the percentages of nitrergic neurons (expressed as median and interquartile range-IQR) were 28% (IQR = 19-29) in the MP and 1% (IQR = 0-2) in the SMP, while the percentages of SP-IR neurons were 31% (IQR = 22-37) in the MP and 41% (IQR = 24-63) in the SMP. Although morphological features of nNOS- and SP-IR neurons were similar to those reported in other mammals, we found some noticeable differences in the percentages of enteric neurons. In fact, we detected a lower proportion of nNOS-IR neurons in the SMP and a higher proportion of SP-IR neurons in the MP compared to other mammals. To the best of the authors' knowledge, this study represents the first description and quantification of nNOS-IR neurons and the first quantification of SP-IR neurons in the intestine of a cetacean species. As nNOS and SP are important mediators of intestinal functions and the nitrergic population is an important target for many neuroenteropathies, data obtained from a healthy intestine provide a necessary basis to further investigate and understand possible functional differences and motor intestinal dysfunctions/alterations in these special mammals.

Localisation of cannabinoid and cannabinoid-related receptors in the equine dorsal root ganglia.

BACKGROUND: Growing evidence recognises cannabinoid receptors as potential therapeutic targets for pain. Consequently, there is increasing interest in developing cannabinoid receptor agonists for treating pain. As a general rule, to better understand the actions of a drug, it would be of extreme importance to know the cellular distribution of its specific receptors. The localisation of cannabinoid receptors in the dorsal root ganglia of the horse has not yet been investigated. OBJECTIVES: To localise the cellular distribution of canonical and putative cannabinoid receptors in the equine cervical dorsal root ganglia. STUDY DESIGN: Qualitative and quantitative immunohistochemical study. METHODS: Cervical (C6-C8) dorsal root ganglia were collected from six horses (1.5 years of age) at the slaughterhouse. The tissues were fixed and processed to obtain cryosections which were used to investigate the immunoreactivity of canonical cannabinoid receptors 1 (CB1R) and 2 (CB2R), and for three putative cannabinoid-related receptors: nuclear peroxisome proliferator-activated receptor alpha (PPARα), transient receptor potential ankyrin 1 (TRPA1) and serotonin 5-HT1a receptor (5-HT1aR). RESULTS: The neurons showed immunoreactivity for CB1R (100%), CB2R (80% ± 13%), PPARα (100%), TRPA1 (74% ± 10%) and 5-HT1aR (84% ± 6%). The neuronal satellite glial cells showed immunoreactivity for CB2R, PPARα, TRPA1 and 5-HT1aR. MAIN LIMITATIONS: The low number of horses included in the study. CONCLUSIONS: This study highlighted the expression of cannabinoid receptors in the sensory neurons and glial cells of the dorsal root ganglia. These findings could be of particular relevance for future functional studies assessing the effects of cannabinoids in horses to manage pain.

Localization of the Serotonin Transporter in the Dog Intestine and Comparison to the Rat and Human Intestines.

Serotonin is crucial in gastrointestinal functions, including motility, sensitivity, secretion, and the inflammatory response. The serotonin transporter (SERT), responsible for serotonin reuptake and signaling termination, plays a prominent role in gastrointestinal physiology, representing a promising therapeutic target in digestive disorders. Serotonin transporter expression has been poorly investigated in veterinary medicine, under both healthy and pathological conditions, including canine chronic enteropathy, in which the serotonin metabolism seems to be altered. The aim of the present study was to determine the distribution of SERT immunoreactivity (SERT-IR) in the dog intestine and to compare the findings with those obtained in the rat and human intestines. Serotonin transporter-IR was observed in canine enterocytes, enteric neurons, lamina propria cells and the tunica muscularis. Data obtained in dogs were consistent with those obtained in rats and humans. Since the majority of the serotonin produced by the body is synthesized in the gastrointestinal tract, SERT-expressing cells may exert a role in the mechanism of serotonin reuptake.

Expression of cannabinoid and cannabinoid-related receptors in the oral mucosa of healthy cats and cats with chronic gingivostomatitis.

OBJECTIVES: Feline chronic gingivostomatitis (FCGS) is an oral disease. Cats with FCGS experience intense oral pain. Some cats remain refractory to current therapies based on dental extraction and adjuvant medical treatment; it is therefore necessary to investigate alternative therapeutic targets involved in inflammatory mechanisms and pain, namely the endocannabinoid system (ECS). The present study investigated the expression of cannabinoid receptors type 1 (CB1R) and 2 (CB2R), and cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential ankyrin 1 (TRPA1) and serotonin 1a receptor (5-HT1aR), in the oral mucosa of healthy cats to determine whether there was altered expression and distribution in cats with FCGS. METHODS: Samples of caudal oral mucosa were collected from eight control cats (CTRL cats) and from eight cats with FCGS (FCGS cats). Tissue samples were processed using an immunofluorescence assay with cat-specific antibodies, and the immunolabelling of the receptors studied was semiquantitatively evaluated. RESULTS: The mucosal epithelium of the CTRL cats showed CB1R, TRPA1 and 5-HT1aR immunoreactivity (IR), while CB2R and GPR55 IR were generally not expressed. In the CTRL cats, the subepithelial inflammatory cells expressed CB2R, GPR55 and 5-HT1aR IR. In the FCGS cats, all the receptors studied were markedly upregulated in the epithelium and inflammatory infiltrate. CONCLUSIONS AND RELEVANCE: Cannabinoid and cannabinoid-related receptors are widely expressed in the oral mucosa of healthy cats and are upregulated during the course of FCGS. The presence of cannabinoid and cannabinoid-related receptors in healthy tissues suggests the possible role of the ECS in the homeostasis of the feline oral mucosa, while their overexpression in the inflamed tissues of FCGS cats suggests the involvement of the ECS in the pathogenesis of this disease, with a possible role in the related inflammation and pain. Based on the present findings, ECS could be considered a potential therapeutic target for patients with FCGS.

ACE2 Expression in the Cat and the Tiger Gastrointestinal Tracts.

Angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor for Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). It has been identified in the human gastrointestinal tract (GIT), and SARS-CoV-2 has been isolated in human and animal fecal samples. The aim of the present study was to investigate the expression of ACE2 in the gastrointestinal tract of domestic (cat) and wild (tiger) felines. Samples of the pylorus, duodenum, and distal colon were collected from six cats and one tiger. The tissues were processed for immunofluorescence assay with an anti-human ACE2 antibody. Angiotensin-converting enzyme 2 was widely expressed in the gastrointestinal mucosa of the cats and the tiger. In both the species, ACE2-immunoreactivity (ACE2-IR) was expressed by the mucosal epithelial cells of the GIT and by the enteric neurons. In the cats, ACE2-IR was also expressed by the smooth muscle cells of the blood vessels and the tunica muscularis. The expression of the ACE2 receptor in enteric neurons may support the potential neurotropic properties of SARS-CoV-2. Although the evidence of ACE2-IR in the feline GIT does not necessarily indicate the possibility of viral replication and SARS-CoV-2 spread with stool, the findings in the present study could serve as an anatomical basis for additional studies considering the risk of the SARS-CoV-2 fecal-oral transmission between cats/felids, and between cats/felids and humans.

Localization of cannabinoid and cannabinoid related receptors in the cat gastrointestinal tract.

A growing body of literature indicates that activation of cannabinoid receptors may exert beneficial effects on gastrointestinal inflammation and visceral hypersensitivity. The present study aimed to immunohistochemically investigate the distribution of the canonical cannabinoid receptors CB1 (CB1R) and CB2 (CB2R) and the putative cannabinoid receptors G protein-coupled receptor 55 (GPR55), nuclear peroxisome proliferator-activated receptor alpha (PPARα), transient receptor potential ankyrin 1 (TRPA1), and serotonin receptor 5-HT1a 5-HT1aR) in tissue samples of the gastrointestinal tract of the cat. CB1R-immunoreactivity (CB1R-IR) was observed in gastric epithelial cells, intestinal enteroendocrine cells (EECs) and goblet cells, lamina propria mast cells (MCs), and enteric neurons. CB2R-IR was expressed by EECs, enterocytes, and macrophages. GPR55-IR was expressed by EECs, macrophages, immunocytes, and MP neurons. PPARα-IR was expressed by immunocytes, smooth muscle cells, and enteroglial cells. TRPA1-IR was expressed by enteric neurons and intestinal goblet cells. 5-HT1a receptor-IR was expressed by gastrointestinal epithelial cells and gastric smooth muscle cells. Cannabinoid receptors showed a wide distribution in the feline gastrointestinal tract layers. Although not yet confirmed/supported by functional evidences, the present research might represent an anatomical substrate potentially useful to support, in feline species, the therapeutic use of cannabinoids during gastrointestinal inflammatory diseases.

Mast cell-nerve interactions correlate with bloating and abdominal pain severity in patients with non-celiac gluten / wheat sensitivity.

BACKGROUND: Gastrointestinal (GI) and extra-GI symptoms/manifestations represent key clinical features of patients with non-celiac gluten/wheat sensitivity (NCG/WS). This study aimed to investigate neuro-immune (focusing on mast cells, MCs) interactions in the duodenal submucosa of patients with NCG/WS. METHODS: Submucosal whole mounts from duodenal biopsies of 34 patients with self-reported NCG/WS, 28 with celiac disease (CD), 13 with functional dyspepsia (FD), and 24 healthy controls (HC) were analyzed by immunohistochemistry. Quantitative data on neuronal and MCs density and the percentage of MCs in close vicinity to nerves were obtained, and correlations among neurons, MC density and MC-nerve distance (D), and symptoms were assessed in the three groups. KEY RESULTS: The number of submucosal neurons was not different among groups. In NCG/WS, MC density was not different from HC, while it was slightly increased vs. CD (P = .07) and significantly decreased vs. FD (P < .05). The percentage of MCs close to nerves (D < 15 µm) was similarly increased in all three pathological groups vs. HC (P < .001). In NCG/WS, MC infiltration correlated with bloating (P = .001) and abdominal pain severity (P = .03) and the percentage of MCs in proximity to neurons correlated with the number of GI symptoms (D < 5 µm; P = .05), bloating and abdominal pain severity (D < 15um; P = .01). CONCLUSIONS AND INFERENCES: Submucosal MC infiltration and the close (within 15 µm) MC-to-nerve proximity in the duodenum of NCG/WS patients are features providing a histopathological basis to better understand GI symptoms in this condition.

Corrigendum: Cellular Distribution of Canonical and Putative Cannabinoid Receptors in Canine Cervical Dorsal Root Ganglia.

[This corrects the article DOI: 10.3389/fvets.2019.00313.].

Cellular Distribution of Canonical and Putative Cannabinoid Receptors in Canine Cervical Dorsal Root Ganglia.

Growing evidence indicates cannabinoid receptors as potential therapeutic targets for chronic pain. Consequently, there is an increasing interest in developing cannabinoid receptor agonists for treating human and veterinary pain. To better understand the actions of a drug, it is of paramount importance to know the cellular distribution of its specific receptor(s). The distribution of canonical and putative cannabinoid receptors in the peripheral and central nervous system of dogs is still in its infancy. In order to help fill this anatomical gap, the present ex vivo study has been designed to identify the cellular sites of cannabinoid and cannabinoid-related receptors in canine spinal ganglia. In particular, the cellular distribution of the cannabinoid receptors type 1 and 2 (CB1 and CB2) and putative cannabinoid receptors G protein-coupled receptor 55 (GPR55), nuclear peroxisome proliferator-activated receptor alpha (PPARα), and transient receptor potential vanilloid type 1 (TRPV1) have been immunohistochemically investigated in the C6-C8 cervical ganglia of dogs. About 50% of the neuronal population displayed weak to moderate CB1 receptor and TRPV1 immunoreactivity, while all of them were CB2-positive and nearly 40% also expressed GPR55 immunolabeling. Schwann cells, blood vessel smooth muscle cells, and pericyte-like cells all expressed CB2 receptor immunoreactivity, endothelial cell being also PPARα-positive. All the satellite glial cells (SGCs) displayed bright GPR55 receptor immunoreactivity. In half of the study dogs, SGCs were also PPARα-positive, and limited to older dogs displayed TRPV1 immunoreactivity. The present study may represent a morphological substrate to consider in order to develop therapeutic strategies against chronic pain.

Enteric neuron density correlates with clinical features of severe gut dysmotility.

Gastrointestinal (GI) symptoms can originate from severe dysmotility due to enteric neuropathies. Current methods used to demonstrate enteric neuropathies are based mainly on classic qualitative histopathological/immunohistochemical evaluation. This study was designed to identify an objective morphometric method for paraffin-embedded tissue samples to quantify the interganglionic distance between neighboring myenteric ganglia immunoreactive for neuron-specific enolase, as well as the number of myenteric and submucosal neuronal cell bodies/ganglion in jejunal specimens of patients with severe GI dysmotility. Jejunal full-thickness biopsies were collected from 32 patients (22 females; 16-77 yr) with well-characterized severe dysmotility and 8 controls (4 females; 47-73 yr). A symptom questionnaire was filled before surgery. Mann-Whitney U test, Kruskal-Wallis coupled with Dunn's posttest and nonparametric linear regression tests were used for analyzing morphometric data and clinical correlations, respectively. Compared with controls, patients with severe dysmotility exhibited a significant increase in myenteric interganglionic distance (P = 0.0005) along with a decrease in the number of myenteric (P < 0.00001) and submucosal (P < 0.0004) neurons. A 50% reduction in the number of submucosal and myenteric neurons correlated with an increased interganglionic distance and severity of dysmotility. Our study proposes a relatively simple tool that can be applied for quantitative evaluation of paraffin sections from patients with severe dysmotility. The finding of an increased interganglionic distance may aid diagnosis and limit the direct quantitative analysis of neurons per ganglion in patients with an interganglionic distance within the control range.NEW & NOTEWORTHY Enteric neuropathies are challenging conditions characterized by a severe impairment of gut physiology, including motility. An accurate, unambiguous assessment of enteric neurons provided by quantitative analysis of routine paraffin sections may help to define neuropathy-related gut dysmotility. We showed that patients with severe gut dysmotility exhibited an increased interganglionic distance associated with a decreased number of myenteric and submucosal neurons, which correlated with symptoms and clinical manifestations of deranged intestinal motility.

A large deletion in the GP9 gene in Cocker Spaniel dogs with Bernard-Soulier syndrome.

Inherited bleeding disorders including abnormalities of platelet number and function rarely occur in a variety of dog breeds, but are probably underdiagnosed. Genetically characterized canine forms of platelet disorders provide valuable large animal models for understanding similar platelet disorders in people. Breed-specific disease associated genetic variants in only eight different genes are known to cause intrinsic platelet disorders in dogs. However, the causative genetic variant in many dog breeds has until now remained unknown. Four cases of a mild to severe bleeding disorder in Cocker Spaniel dogs are herein presented. The affected dogs showed a platelet adhesion defect characterized by macrothrombocytopenia with variable platelet counts resembling human Bernard-Soulier syndrome (BSS). Furthermore, the lack of functional GPIb-IX-V was demonstrated by immunocytochemistry. Whole genome sequencing of one affected dog and visual inspection of the candidate genes identified a deletion in the glycoprotein IX platelet (GP9) gene. The GP9 gene encodes a subunit of a platelet surface membrane glycoprotein complex; this functions as a receptor for von Willebrand factor, which initiates the maintenance of hemostasis after injury. Variants in human GP9 are associated with Bernard-Soulier syndrome, type C. The deletion spanned 2460 bp, and included a significant part of the single coding exon of the canine GP9 gene on dog chromosome 20. The variant results in a frameshift and premature stop codon which is predicted to truncate almost two-thirds of the encoded protein. PCR-based genotyping confirmed recessive inheritance. The homozygous variant genotype seen in affected dogs did not occur in 98 control Cocker Spaniels. Thus, it was concluded that the structural variant identified in the GP9 gene was most likely causative for the BSS-phenotype in the dogs examined. These findings provide the first large animal GP9 model for this group of inherited platelet disorders and greatly facilitate the diagnosis and identification of affected and/or normal carriers in Cocker Spaniels.

Gut epithelial and vascular barrier abnormalities in patients with chronic intestinal pseudo-obstruction.

BACKGROUND: Chronic intestinal pseudo-obstruction (CIPO) is a rare condition due to severe impairment of gut motility responsible for recurrent subocclusive episodes. Although neuromuscular-glial-ICC abnormalities represent the main pathogenetic mechanism, the pathophysiology of CIPO remains poorly understood. Intestinal epithelial and vascular endothelial barrier (IEVB) abnormalities can contribute to neuroepithelial changes by allowing passage of harmful substances. METHODS: To test retrospectively whether IEVB defects occur in patients with CIPO, we measured the jejunal protein expression of the major tight junction (TJ) components. CIPO patients were subdivided according to gut neuromuscular histopathology: apparently normal (AN); with inflammation (INF); or with degenerative alterations (DEG). The presence of occludin/claudin oligomers (index of TJ assembly), the amount of occludin, claudin-4, and zonula occludens-1 (ZO-1), and the expression of vasoactive intestinal polypeptide (VIP) and glial fibrillary acidic protein (GFAP) immunoreactivities were evaluated on jejunal full-thickness biopsies using Western blot. KEY RESULTS: Oligomers were absent in the 73% of CIPO. Total occludin decreased in CIPO with AN and INF changes. Claudin-4 was upregulated in CIPO with INF and DEG features. ZO-1 and VIP expression decreased selectively in DEG group. GFAP increased in CIPO regardless the histopathological phenotype. CONCLUSIONS & INFERENCES: The absence of oligomers demonstrated in our study suggests that IEBV is altered in CIPO. The mechanism leading to oligomerization is occludin-dependent in AN and INF, whereas is ZO-1-dependent in DEG. Our study provides support to IEVB abnormalities contributing to CIPO clinical and histopathological features.