Expression of Calretinin in the Cecal Muscularis Interna: Observation and Hypothetical Relevance to Appendicitis.

BACKGROUND: The unexpected observation of calretinin immunoreactivity in smooth muscle cells in the muscularis propria of the cecum led to a more detailed examination of calretinin expression and its possible relationship to propulsive contractile activity around the vermiform appendix. METHODS: Immunohistochemistry and RNA in situ hybridization were performed to analyze calretinin expression in intestinal samples from 33 patients at ages ranging from mid-gestation fetuses to adults, as well as in some potentially relevant animal models. Dual immunolabeling was done to compare calretinin localization with markers of smooth muscle and interstitial cells of Cajal. RESULTS: Calretinin expression was observed consistently in the innermost smooth muscle layers of the muscularis interna in the human cecum, appendiceal base, and proximal ascending colon, but not elsewhere in the intestinal tract. Calretinin-positive smooth muscle cells did not co-express markers located in adjacent interstitial cells of Cajal. Muscular calretinin immunoreactivity was not detected in the ceca of mice or macaques, species which lack appendices, nor in the rabbit cecum or appendix. CONCLUSIONS: Localized expression of calretinin in cecal smooth muscle cells may reduce the likelihood of retrograde, calcium-mediated propulsive contractions from the proximal colon and suppress pro-inflammatory fecal stasis in the appendix.

Next generation sequencing targeted detection of somatic mutations in patients with mucinous adenocarcinoma of the appendix.

The aim of this study was to investigate the mutations in mucinous adenocarcinoma of the appendix (MAA). SNV was detected in 15 patients with MAA, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and reactome pathway analyses were performed. Tumor mutational burden (TMB), mutant-allele tumor heterogeneity (MATH), microsatellite instability (MSI) was analysis. Finally, the human leukocyte antigen (HLA) typing of the samples was detected. The results showed that TP53 (27 %) and KRAS (20 %) were the highest mutation frequency in the sample, mainly occur in p53 pathway and RTK-RAS pathway. GO analysis reveals mutated genes are closely related to the regulation of GTPase activity, regulation of small GTPase mediated signal transduction and other BP, related to the CC and MF. Analysis of KEGG pathways indicated that the top canonical pathways associated with SNV was Wnt signaling pathway. Reactome pathway analysis further revealed that the mutant genes were closely related to muscle contraction. Only one patient had moderate TMB level and one patient with high MSI. In conclusion, the most common mutated genes and the signaling pathways closely related to MAA development were detected in this study, which will contribute to the development of immunotherapy for patients with MAA.

The First Evaluation of Proteinase K-Resistant Prion Protein (PrPSc) in Korean Appendix Specimens.

Background and Objectives: Prion diseases are fatal neurodegenerative disorders caused by the abnormal proteinase K-resistant prion protein (PrPSc). Since variant Creutzfeldt-Jakob disease (CJD) was first reported in the United Kingdom (UK) in 1996, the occurrence of variant CJD has been reported in over 10 countries. To date, variant CJD has not been reported in Korea. However, the E211K somatic mutation in the prion protein gene (PRNP), which is related to bovine spongiform encephalopathy (BSE), was reported in Korean Holstein cattle, and atypical BSE, which is supposed to be sporadic BSE, has been occurring in many countries, including Japan and the USA. These results suggest that BSE may occur naturally in Korea. Thus, we performed a preemptive PrPSc test in appendix specimens to diagnose variant CJD in a Korean population. Materials and Methods: In the present study, we investigated CJD-related mutations and polymorphisms of the PRNP gene and carried out an examination on PrPSc in appendix specimens of Korean patients after appendectomy. Results: In all Korean appendix specimens tested, PrPSc bands were not detected. Conclusion: To the best of our knowledge, this was the first evaluation of PrPSc in Korean appendix specimens.

Angiotensin-Converting Enzyme 2 and Furin Expression in the Appendix of Children with COVID-19.

Background: The sudden outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in more than 261 million infections and an estimated 5.1 million deaths. Some vital organs such as the kidneys, heart, intestines, and lungs could be damaged by SARS-CoV-2. Questions remain regarding the effect of SARS-CoV-2 on the mucous membrane of the appendix in children. The aim of this study was to evaluate the knowledge of cytologic processes in appendix tissue in children with coronavirus disease 2019 (COVID-19). Patients and Methods: Fragments of the appendices of children with confirmed COVID-19 (n = 42) were studied by real-time polymerase chain reaction (PCR) to determine the expression of SARS-CoV-2 RNA and genes encoding protein complexes: ACE-2 and Furin. Results: We found traces of coronavirus genetic material in the appendices of children with COVID-19. We also found increased expression of ACE-2 and Furin in the tissue, which indicates favorable conditions for coronavirus infection. Conclusions: Viral load in the appendices of children can be reported based on the results of real-time PCR for SARS-CoV-2 and the expression of ACE-2 and Furin.

Epigenetic inactivation of the autophagy-lysosomal system in appendix in Parkinson's disease.

The gastrointestinal tract may be a site of origin for α-synuclein pathology in idiopathic Parkinson's disease (PD). Disruption of the autophagy-lysosome pathway (ALP) may contribute to α-synuclein aggregation. Here we examined epigenetic alterations in the ALP in the appendix by deep sequencing DNA methylation at 521 ALP genes. We identified aberrant methylation at 928 cytosines affecting 326 ALP genes in the appendix of individuals with PD and widespread hypermethylation that is also seen in the brain of individuals with PD. In mice, we find that DNA methylation changes at ALP genes induced by chronic gut inflammation are greatly exacerbated by α-synuclein pathology. DNA methylation changes at ALP genes induced by synucleinopathy are associated with the ALP abnormalities observed in the appendix of individuals with PD specifically involving lysosomal genes. Our work identifies epigenetic dysregulation of the ALP which may suggest a potential mechanism for accumulation of α-synuclein pathology in idiopathic PD.

What is New in the 2019 World Health Organization (WHO) Classification of Tumors of the Digestive System: Review of Selected Updates on Neuroendocrine Neoplasms, Appendiceal Tumors, and Molecular Testing.

CONTEXT.­: The 5th edition of the World Health Organization classification of digestive system tumors discusses several advancements and developments in understanding the etiology, pathogenesis, and diagnosis of several digestive tract tumors. OBJECTIVE.­: To provide a summary of the updates with a focus on neuroendocrine neoplasms, appendiceal tumors, and the molecular advances in tumors of the digestive system. DATA SOURCES.­: English literature and personal experiences. CONCLUSIONS.­: Some of the particularly important updates in the 5th edition are the alterations made in the classification of neuroendocrine neoplasms, understanding of pathogenesis of appendiceal tumors and their precursor lesions, and the expanded role of molecular pathology in establishing an accurate diagnosis or predicting prognosis and response to treatment.

sB7H3 in Children with Acute Appendicitis: Its Diagnostic Value and Association with Histological Findings.

BACKGROUND: Several efforts have been made to find out a valuable marker to assist the diagnosis and differentiation of gangrenous/perforated appendicitis. We aimed to determine the diagnostic capacity of soluble B7H3 (sB7H3) in acute appendicitis (AA) and its accuracy as a predictor of the severity of appendicitis. METHODS: 182 children were allocated into four groups as follows: control group (CG, 90), simple appendicitis (SA, 12), purulent appendicitis (PA, 49), and gangrenous appendicitis (GA, 31). Prior to appendectomy, blood was collected and sent for analysis of routine examination and cytokines (sB7H3 and TNF-α). We compared values of all measured parameters according to histological findings. Furthermore, we assigned AA patients into the nonperforated appendicitis group and the perforated appendicitis group. The diagnostic effects of significant markers were assessed by ROC curves. RESULTS: Only the levels of CRP, FIB, and sB7H3 had a remarkable rising trend in AA-based groups, while differences in the levels of CRP and FIB between simple appendicitis and purulent appendicitis were not statistically significant. In addition, sB7H3 was found as the only marker in children with AA, which was markedly associated with the degree of histological findings of the appendix. Furthermore, sB7H3 had a high diagnostic value in predicting AA and complex appendicitis (PA+GA) in children. However, the diagnostic performance of sB7H3 for distinguishing PA from GA was not remarkable. Additionally, only the levels of CRP and sB7H3 were statistically different between the nonperforated appendicitis group and the perforated appendicitis group. The diagnostic performance of CRP and sB7H3 could not merely predict perforation of AA in children; however, the diagnostic performance was improved after combination. CONCLUSIONS: sB7H3 could be used as a valuable marker to predict the presence of AA and complex AA in children. However, the diagnostic value of sB7H3 to predict gangrenous/perforated appendicitis was not found to be remarkable. The combination of sB7H3 and CRP might improve the prediction of perforated appendicitis.

Prevalence in Britain of abnormal prion protein in human appendices before and after exposure to the cattle BSE epizootic.

Widespread dietary exposure of the population of Britain to bovine spongiform encephalopathy (BSE) prions in the 1980s and 1990s led to the emergence of variant Creutzfeldt-Jakob Disease (vCJD) in humans. Two previous appendectomy sample surveys (Appendix-1 and -2) estimated the prevalence of abnormal prion protein (PrP) in the British population exposed to BSE to be 237 per million and 493 per million, respectively. The Appendix-3 survey was recommended to measure the prevalence of abnormal PrP in population groups thought to have been unexposed to BSE. Immunohistochemistry for abnormal PrP was performed on 29,516 samples from appendices removed between 1962 and 1979 from persons born between 1891 through 1965, and from those born after 1996 that had been operated on from 2000 through 2014. Seven appendices were positive for abnormal PrP, of which two were from the pre-BSE-exposure era and five from the post BSE-exposure period. None of the seven positive samples were from appendices removed before 1977, or in patients born after 2000 and none came from individuals diagnosed with vCJD. There was no statistical difference in the prevalence of abnormal PrP across birth and exposure cohorts. Two interpretations are possible. Either there is a low background prevalence of abnormal PrP in human lymphoid tissues that may not progress to vCJD. Alternatively, all positive specimens are attributable to BSE exposure, a finding that would necessitate human exposure having begun in the late 1970s and continuing through the late 1990s.

Appendix Tumor Microenvironment.

The pathological features of the appendix tumors fundamentally recall those of the more frequent colorectal neoplasms, although with a higher relative incidence of carcinoids, due to the abundant presence of enteroendocrine cells in the appendix wall. Moreover, different types of lymphomas, Hodgkin and non-Hodgkin, arising from the extra-nodal mucosal-associated lymphatic tissue, can be encountered. The appendix tumor microenvironment (TME) consists of a cellular component and of a noncellular component: the former includes the immunocompetent cells, while the latter represents the support stroma. Particularly in carcinoids, the immune cell reaction can be explicated by tumor-infiltrating lymphocytes, which, in some circumstances, may arrange around and inside the tumor in a brisk fashion influencing favorably the prognosis. This active reaction has to be distinguished from any preexisting inflammatory condition of the appendix and from superimposed tumor complications, such as infection or ischemia. In practice, we consider the appendix TME a complex framework with immunological, mechanic, and metabolic functions, all supported by a marked neo-lymphoangiogenesis.

Comparing the rates of methane production in patients with and without appendectomy: results from a large-scale cohort.

There is no clear study identifying the microbiome of the appendix. However, in other diverticular conditions, such as diverticulosis, methanogens appear important. We investigated whether patients who had undergone appendectomies had decreased levels of exhaled methane (CH4). Consecutive patients who underwent breath testing (BT) from November 2005 to October 2013 were deterministically linked to electronic health records. The numbers of patients with CH4 ≥ 1 ppm (detectable) and ≥ 3 and ≥ 10 ppm (excess) were compared between patients who did and did not undergo appendectomy using a multivariable model adjusted for age and sex. Of the 4977 included patients (48.0 ± 18.4 years, 30.1% male), 1303 (26.2%) had CH4 ≥ 10 ppm, and 193 (3.9%) had undergone appendectomy. Appendectomy was associated with decreased odds of CH4 ≥ 1, ≥ 3, and ≥ 10 ppm (ORs (95% CI) = 0.67 (0.47-0.93), p = 0.02; 0.65 (0.46-0.92), p = 0.01; and 0.66 (0.46-0.93), p = 0.02, respectively). Additionally, the percentage of CH4 producers increased 4-fold from the first to ninth decade of life. This is the first study to report that appendectomy is associated with decreased exhaled CH4. The appendix may play an active physiologic role as a reservoir of methanogens.

The Appendix in Parkinson's Disease: From Vestigial Remnant to Vital Organ?

Parkinson's disease (PD) has long been considered a brain disease, but studies now point to the gastrointestinal (GI) tract as a potential starting point for PD. In particular, the human vermiform appendix has been implicated in PD. The appendix is a tissue rich in immune cells, serving as part of the gut-associated lymphoid tissue and as a storehouse for the gut microbiome. The functions of the appendix converge with recent evidence demonstrating that gut inflammation and shifts in the microbiome are linked to PD. Some epidemiological studies have linked removal of the appendix to lowered PD risk, though there is controversy among these associations. What is apparent is that there is an abundance of aggregated forms of α-synuclein in the appendix relevant to PD pathology. α-Synuclein pathology is thought to propagate from gut to brain via the vagus nerve, which innervates GI tract locations, including the appendix. Remarkably, α-synuclein aggregates in the appendix occur not only in PD patients, but are also present in healthy individuals. This has led to the proposal that in the appendix α-synuclein aggregates are not unique to PD. Moreover, the molecular events leading to PD and the mechanisms by which α-synuclein aggregates transfers from gut to brain may be identifiable in the human appendix. The influence of the appendix on GI inflammation, autoimmunity, microbiome storage, and the lymphatic system may be yet unexplored mechanisms by which the appendix contributes to PD. Overall, the appendix represents a promising tissue site to advance our understanding of PD pathobiology.

18F-FDG uptake in the normal appendix in adults: PET/CT evaluation.

OBJECTIVE: This study aimed to determine the level of 18F fluorodeoxyglucose (18F-FDG) activity in the normal adult appendix using positron emission tomography/computed tomography (PET/CT). MATERIALS AND METHODS: We performed a retrospective review of PET/CT images using 18F-FDG in 563 consecutive asymptomatic adult patients without appendiceal pathology. We excluded 257 patients for an undetected or obscured appendix and three patients for appendicitis found on CT imaging. FDG uptake in the appendix was qualitatively and quantitatively assessed. The maximum standardized uptake value (SUVmax) was calculated for quantitative analysis with SUVmax of the normal liver for comparison. A total of 303 patients (200 males, 103 females, mean age of 66 years) were included in this study. Medical charts and histories were evaluated for patients who showed positive FDG accumulation. Pearson's correlations between appendiceal SUVmax and age, body mass index, and blood glucose levels were analyzed. RESULTS: The mean appendiceal SUVmax was 1.14 (range 0.52-5.12) with an appendix-to-liver SUVmax ratio of 0.34 (range 0.06-1.28). Three patients qualitatively showed a positive FDG accumulation with appendiceal SUVmax greater than 3.00. There were no correlations between appendiceal SUVmax and age, body mass index, or blood glucose levels. CONCLUSIONS: FDG in the normal adult appendix shows a low activity level and is lower compared with normal liver. However, the normal appendix can rarely show high FDG accumulation. In such cases, differentiation from appendiceal pathology solely by PET/CT images would be difficult.

[Influence of bone marrow autotransplantation on neurotransmitter structures of appendix vermiformis in old rats.]

The influence of bone marrow autotransplantation on neurotransmitter structures of appendix vermiformis was studied. The study revealed that an increase in the number of neurotransmitter structures (mast and granular luminescent cells), having a high content of catecholamines and serotonin was noted in old rats in 40 min after bone marrow autotransplantation. In the center of vermiformis appendix lymphoid nodes reproduction cellular programmed differentiations having a high content of neuroamines are determined. In 2 hours after bone marrow autotransplantation their number decreases, the content of catecholamines and serotonin reduces as well. In the immunohistochemical reaction increase in proliferative activity of cells both in proper t. mucosa plate crypts and appendix vermiformis lymphoid nodules is observed up to 40 min of experiment. In 2 hours Ki-67 positive cells reduce both in t. mucosa and t. s/mucosa of appendix vermiformis.

Digestive and appendicular soft-parts, with behavioural implications, in a large Ordovician trilobite from the Fezouata Lagerstätte, Morocco.

Trilobites were one of the most successful groups of marine arthropods during the Palaeozoic era, yet their soft-part anatomy is only known from a few exceptionally-preserved specimens found in a handful of localities from the Cambrian to the Devonian. This is because, even if the sclerotized appendages were not destroyed during early taphonomic stages, they are often overprinted by the three-dimensional, mineralised exoskeleton. Inferences about the ventral anatomy and behavioural activities of trilobites can also be derived from the ichnological record, which suggests that most Cruziana and Rusophycus trace fossils were possibly produced by the actions of trilobites. Three specimens of the asaphid trilobite Megistaspis (Ekeraspis) hammondi, have been discovered in the Lower Ordovician Fezouata Konservat-Lagerstätte of southern Morocco, preserving appendages and digestive tract. The digestive structures include a crop with digestive caeca, while the appendages display exopodal setae and slight heteropody (cephalic endopods larger and more spinose than thoracic and pygidial ones). The combination of these digestive structures and the heteropody has never been described together among trilobites, and the latter could assist in the understanding of the production of certain comb-like traces of the Cruziana rugosa group, which are extraordinarily abundant on the shallow marine shelves around Gondwana.

The immunology of the vermiform appendix: a review of the literature.

This literature review assesses the current knowledge about the immunological aspects of the vermiform appendix in health and disease. An essential part of its immunological function is the interaction with the intestinal bacteria, a trait shown to be preserved during its evolution. The existence of the appendiceal biofilm in particular has proved to have a beneficial effect for the entire gut. In assessing the influence of acute appendicitis and the importance of a normally functioning gut flora, however, multiple immunological aspects point towards the appendix as a priming site for ulcerative colitis. Describing the immunological and microbiotical changes in the appendix during acute and chronic inflammation of the appendix, this review suggests that this association becomes increasingly plausible. Sustained by the distinct composition of cells, molecules and microbiota, as well as by the ever more likely negative correlation between the appendix and ulcerative colitis, the idea of the appendix being a vestigial organ should therefore be discarded.

Calprotectin could be a potential biomarker for acute appendicitis.

BACKGROUND: Acute appendicitis is a common cause for a visit to the emergency department and appendectomy represents the most common emergency procedure in surgery. The rate of negative appendectomy however has remained high despite modern diagnostic apparatus. Therefore, there is need for a better preoperative screening of patients with suspected appendicitis. Calprotectin represents a predominant protein in the cytosol of neutrophil granulocytes and has been extensively investigated with regard to bowel pathologies. This study investigates the expression of calprotectin in the lumen of the vermiform appendix of patients undergoing appendectomy for suspected appendicitis. METHODS: Appendix specimens from patients undergoing emergency appendectomy for suspected acute appendicitis were examined. Acute appendicitis was confirmed on histopathology. The qualitative expression of calprotectin in the vermiform appendix specimens was analyzed using specific calprotectin antibodies. RESULTS: Vermiform appendix specimens from 52 patients (22 female and 30 male) including 11 with uncomplicated and 41 with complicated appendicitis were analyzed. Strong immunostainings were achieved with calprotectin antibody in the lumen of all specimens irrespective of the extent of appendicitis. Immunostaining was negative in the uninflamed appendix. CONCLUSIONS: High calprotectin activity could be demonstrated within the lumen of vermiform appendix specimens following appendectomy for acute appendicitis. The high luminal accumulation of calprotectin-carrying cells could be interpreted as an invitation to study the expression of calprotectin in stool as a new diagnostic aid in patients with suspected appendicitis.

Hyperbilirubinemia as a predictive factor in acute appendicitis.

PURPOSE: Our aim was to establish the role of hyperbilirubinemia as a predictive parameter for the prediction of either acute, or gangrenous/perforated appendicitis as well as to compare other parameters in a similar role. METHODS: Medical files of the patients who underwent appendectomies between September 2013 and September 2014 were evaluated. Age, gender, preoperative white blood cell count (WBC), neutrophil count (NEU), neutrophil percentage (NEU%), C-reactive protein (CRP), total/direct/indirect bilirubin levels, and the postoperative histopathological findings were recorded. The Fisher's exact, Pearson's χ (2), ANOVA, and Kruskal-Wallis tests while logistic regression for multivariate analysis was performed. p < 0.05 was accepted as statistically significant. RESULTS: The study group of 162 patients consisted of 97 (60 %) men and 65 (40 %) women with a median age of 36 (18-90). Histopathological examinations revealed normal appendix in 21 (13 %) patients, non-complicated acute appendicitis in 100 (62 %), and appendiceal gangrene/perforation in 41 (25 %) patients. WBC, NEU, NEU%, and CRP levels were significantly higher in cases of acute and gangrenous/perforated appendicitis (p < 0.01). Total and direct bilirubin levels were also significantly elevated in patients with acute and gangrenous/perforated appendicitis (p < 0.01). According to multivariate analysis, elevated CRP levels were associated with 14 times, elevated total bilirubin levels were associated with five times, and elevated direct bilirubin levels were associated with 36 times greater risk for appendiceal gangrene/perforation (p < 0.01, p < 0.05, p < 0.01, respectively). CONCLUSIONS: Hyperbilirubinemia, especially with elevated direct bilirubin levels, may be considered as an important marker for the prediction of appendiceal gangrene/perforation.

Appendectomy may delay Parkinson's disease Onset.

BACKGROUND: Alpha-synuclein (α-Syn) is particularly abundant in the vermiform appendix, which makes this structure an anatomical candidate for the initiation of Parkinson's disease (PD) pathology. We hypothesized that history of appendectomy might affect PD clinical onset. METHODS: A total of 295 PD patients enrolled in a comprehensive observational study were asked about past history of appendectomy. Cox's regression, with a time-dependent covariate, explored the effects of appendectomy on age at PD onset. RESULTS: Thirty-four patients (11.5%) had appendectomy before PD onset. There was no significant effect of appendectomy on age at PD onset for the entire cohort (P = 0.153). However, among patients with late onset (≥55 years), we found evidence that those with past appendectomy had more years of life without PD symptoms than patients without appendectomy (P = 0.040). No association was found for the young-onset group (P = 0.663). CONCLUSIONS: An apparent relationship was observed between appendectomy and PD onset in the late PD cohort.

MeCP2 in the enteric nervous system.

BACKGROUND: Rett syndrome (RTT) is an intellectual deficit and movement disorder that develops during early childhood in girls. Affected children are normal until 6-18 months of age, after which symptoms begin to appear. Most cases of RTT are due to mutations in the MeCP2 gene leading to disruption of neuronal communication in the central nervous system. In addition, RTT patients show peripheral ailments such as gastrointestinal (GI), respiratory, and cardiac dysfunction. The etiology of intestinal dysfunction in RTT is not well-understood. Reports on the presence of MeCP2 in the peripheral nervous system are scant. As such we examined the levels of MeCP2 in human and murine GI tissue and assessed MeCP2 expression at various developmental stages. METHODS: Immunohistochemistry for MeCP2, HuC/D, juvenile beta tubulin, and GFAP was performed on human and murine intestine. Western blots of these same tissues were probed with MeCP2, vAChT, nNOS, and beta-actin antibodies. KEY RESULTS: MeCP2 is expressed throughout the GI tract. MeCP2 is expressed specifically in the enteric nervous system of the GI tract. MeCP2 is expressed in the GI tract throughout development with appearance beginning at or before E11.5 in the murine intestine. CONCLUSIONS & INFERENCES: The proof of MeCP2 expression in enteric neurons suggests that the GI dysmotility in Rett may arise from enteric network dysfunction secondary to MeCP2 mutation.

The appendix as a viable source of neural progenitor cells to functionally innervate bioengineered gastrointestinal smooth muscle tissues.

UNLABELLED: Appendix-derived neural progenitor cells (NPCs) have both neurogenic and gliogenic potential, but use of these cells for enteric neural cell therapy has not been addressed. The objective of this study was to determine whether NPCs obtained from the appendix would differentiate into enteric neural subsets capable of inducing neurotransmitter-mediated smooth muscle cell (SMC) contraction and relaxation. NPCs were isolated from the appendix and small intestine (SI) of rabbits. Bioengineered internal anal sphincter constructs were developed using the same source of smooth muscle and innervated with NPCs derived from either the appendix or SI. Innervated constructs were assessed for neuronal differentiation markers through Western blots and immunohistochemistry, and functionality was assessed through force-generation studies. Expression of neural and glial differentiation markers was observed in constructs containing appendix- and SI-derived NPCs. The addition of acetylcholine to both appendix and SI constructs caused a robust contraction that was decreased by pretreatment with the neural inhibitor tetrodotoxin (TTX). Electrical field stimulation caused relaxation of constructs that was completely abolished in the presence of TTX and significantly reduced on pretreatment with nitric oxide synthase inhibitor (Nω-nitro-l-arginine methyl ester hydrochloride [l-NAME]). These data indicate that in the presence of identical soluble factors arising from intestinal SMCs, enteric NPCs derived from the appendix and SI differentiate in a similar manner and are capable of responding to physiological stimuli. This coculture paradigm could be used to explore the nature of the soluble factors derived from SMCs and NPCs in generating specific functional innervations. SIGNIFICANCE: This study demonstrates the ability of neural stem cells isolated from the appendix to differentiate into mature functional enteric neurons. The differentiation of neural stem cells from the appendix is similar to differentiation of neural stem cells derived from the gastrointestinal tract. The appendix is a vestigial organ that can be removed with minimal clinical consequence through laparoscopy. Results presented in this paper indicate that the appendix is a potential source of autologous neural stem cells required for cell therapy for the gastrointestinal tract.