Metrics details Needle knives are the most commonly used instrument during endoscopic treatment for gastric submucosal tumors (SMTs) The conventional resection method involves fully extending the needle-shaped knife head which allows it to more easily penetrate the muscularis propria while stripping the muscle layer of the tumor We propose a semi-blunt dissection method that can effectively reduce damage to the muscularis propria A total of 113 patients who underwent endoscopic resection of gastric SMTs originating from the muscularis propria were retrospectively analyzed The conventional method consisted of 73 patients; The other group consisted of 40 patients underwent the semi-blunt dissection method There was no significant difference between the two groups in age the maximum diameter of gastric muscularis propria damage was significantly greater in conventional method group than the other group (1.06 ± 0.48 cm vs There was also no significant difference between the two groups in terms of histological diagnosis postoperative complications and the percentage of histologically positive resection margins The semi-blunt dissection method has certain advantages in the endoscopic resection of gastric tumors originating from the muscularis propria including a small extent of gastric muscularis propria damage and a shorter postoperative hospital stay Needle knives are the most commonly used instrument during endoscopic treatment Based on the growth characteristics of the tumors originating from gastric muscularis propria the conventional resection method involves fully extending the needle-shaped knife head the metal surface of the knife tip can be placed against the loose tissue for high-frequency electric dissection while the endoscope can be used to carry the head end of the plastic knife handle along the fissure created by the high-frequency electric incision for blunt push dissection This method is named the semi-blunt dissection method No studies have compared the treatment efficacy and safety of conventional methods and semi-blunt dissection This study compared the treatment efficacy and safety of the two methods for treating gastric tumors originating from the muscularis propria A total of 113 patients who underwent endoscopic resection of gastric SMTs originating from the muscularis propria between 2017 and 2022 were retrospectively analyzed This study was approved by the Ethics Committee of Peking University People’s Hospital The inclusion criteria for the study were as follows: (1) age ≥ 18 years; (2) SMT evaluated by endoscopy EUS or CT assessment revealing that the tumor originated from the muscularis propria that at least half of the tumor was protruding into the gastric cavity and (3) the tumor had a diameter ≥ 10 mm or ≤ 40 mm The exclusion criteria for the study were as follows: (1) Upper gastrointestinal lesions measured by EUS < 10 mm or > 40 mm; (2) ≥ 1/2 of the tumor protruded out of the gastric cavity; (3) Portal hypertension; (4)A history of upper gastrointestinal surgery (5) Patients that took c-kit inhibitor (for GISTs) were excluded The knife head was fully extended during the cutting and peeling process; B The contact surface of the metal tip of the knife head was pressed to the cutting surface with slight pressure and a high-frequency current was applied to create a blunt separation fissure on the cutting surface while the plastic knife handle was used for blunt pushing and dissection it was divided into 3 mm sections to determine the maximum tumor diameter and resection margin Histopathological results were confirmed by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining the tumor risk was categorized according to the modified Fletcher classification The histological examinations were performed by a pathologist with more than 8 years of experience (1) Histological resection: R0 resection was defined as a resection with a clear edge under the microscope; R1 resection was defined as a gross tumor resection with a positive tumor edge under the microscope; R2 resection was defined as residual tumor visible to the naked eye (2) Complete endoscopic resection was defined as resection of the entire tumor without residual tumor; this included endoscopic R0 and R1 resection (3) Delayed bleeding was defined as postoperative bleeding (4) Recurrence: a submucosal tumor-like bulge found under endoscopy; a clearly visible tumor on CT scan; and biopsy results at the resection site suggestive of recurrent tumor cells Patients with a pathological diagnosis of GIST underwent endoscopy at 4 and 12 weeks after surgery endoscopy and CT examination at 24 weeks after surgery and endoscopy and CT examination every year thereafter Patients with pathologically diagnosed leiomyoma underwent endoscopy at 4 and 12 weeks and then endoscopy and CT examination every year thereafter SPSS 22.0 software was used for the statistical analysis Categorical variables were expressed as counts and composition ratio and were compared using the Chi-square test or Fisher exact test as appropriate and the t test was used to compare variables depicted as mean values P < 0.05 was considered to indicate statistical significance Perioperative patient characteristics and histology of gastric submucosal tumors (Table 1) The conventional method was used for Group A underwent the semi-blunt dissection method was significantly greater in Group A than in Group B (1.06 ± 0.48 cm vs p < 0.001); there was no statistically significant difference between the two groups in terms of endoscopic or laparoscopic suturing methods the average length of hospitalization in Group A was longer than that in Group B (7.66 ± 2.90 days vs p < 0.001); there was no significant difference between the two groups in terms of postoperative fever duration; there was no incidence of delayed bleeding or perforation in either group No recurrence was observed during the follow-up period the maximum pathological size of the resected lesions in Group B was significantly greater than that in Group A (1.95 ± 1.43 cm vs p = 0.006); there was no significant difference between the two groups in terms of histological diagnosis and the percentage of histologically positive resection margins Perioperative patient characteristics and histology of GISTs (Table 2) which consisted of 37 patients; Group B underwent the semi-blunt dissection method consisted of 16 patients there was no significant difference in age The maximum diameter of gastric muscularis propria damage in Group A was significantly greater than that in Group B (1.20 ± 0.49 cm vs There was no significant difference between the two groups in terms of the use of endoscopic or laparoscopic suture methods The average duration of hospitalization in Group A was significantly longer than that in Group B (8.43 ± 3.55 days vs There was no significant difference in terms of postoperative fever duration between the two groups There was no occurrence of delayed bleeding or perforation and no recurrence during the follow-up period there was no significant difference between the two groups in terms of histological diagnosis maximum pathological diameter of the resected lesion or the percentage of histologically positive resection margins the semi-blunt dissection group has smaller gastric muscularis propria damage few reports have compared different endoscopic resection methods the semi-blunt dissection method used in this study involves high-frequency shallow electrocautery to create a gap for blunt dissection with the plastic knife handle facilitating rapid blunt dissection; even at the edge of the tumor slightly compact connective tissue can also be isolated using this method appropriate selection of parameters enables coagulation of the small blood vessels at the cutting surface which reduces the risk of bleeding relative to blunt dissection alone and reduces the difficulty of subsequent dissection and the risk of perforation the maximum diameter of the resected lesions in Group A was smaller than that in Group B (1.26 ± 0.70 cm vs but the maximum diameter of gastric muscularis propria damage in Group A was greater than that in Group B (1.06 ± 0.48 cm) there was less gastric muscularis propria damage in Group B than in Group A and the difference was statistically significant The procedure used for Group A patients involved the creation of a sharp incision Because the tumor was embedded in the muscularis propria of the stomach with little tissue between the tumor margin and the healthy tissue as the needle-like knife was kept protruded to make the sharp incision use of an insulated-tip (IT) knife with a magnetic tip could reduce the risk of perforation of the muscularis propria but it will increase the cost for the patient sharp incisions were performed when the tumor boundary was not clear When the tumor boundary was clear and the tissues beneath the tumor were loose which not only reduced the risk of the knife tip piercing the muscularis propria but also allowed the wound to close easily; furthermore there was no need to substitute for an IT knife keeping the costs to the patient relatively low The average postoperative hospitalization time for Group B was shorter than that for Group A (5.80 ± 1.96 days vs Because of the small wound area in group B the patient did not need to be on the postoperative diet as long Group B patients also experienced less damage to the gastric wall (1.20 ± 0.49 cm vs p < 0.001) and a shorter mean duration of hospitalization (8.43 ± 3.55 days vs suggesting that the semi-blunt dissection method for GISTs can also reduce the wound surface area the risk of perforating the muscularis propria during surgery is low the generation of a large amount of pneumoperitoneum is avoided and the length of hospital stay is reduced none of the patients who underwent R1 resection experienced recurrence or metastasis during follow-up which is consistent with the findings of previous studies all the patients with GIST pathologies in this study had a very low risk of recurrence the results of this study suggest only that the effects of the two endoscopic treatment methods on the resection margin in patients with very low recurrence risk including those with low recurrence-risk GISTs Higher recurrence-risk GIST undergone R1- endoscopic resection were needed to be included in further research There are still some limitations in this study This was a single-center retrospective study; future prospective studies are needed to further evaluate the efficacy and safety of semi-blunt dissection GISTs are more likely to be malignant than leiomyomas and therefore should be the greater focus of studies on the effect of endoscopic treatment The number of GIST patients enrolled in this study was small and should be increased in future studies the GIST patients enrolled in this study all had a low or very low risk of recurrence so it is difficult to fully assess the true treatment efficacy and recurrence risk of the two treatment methods for resecting intermediate-risk GISTs Follow-up studies are needed to further enroll patients who have undergone endoscopic resection for intermediate-risk GISTs and compare the two endoscopic treatment methods the semi-blunt dissection method has certain advantages in the endoscopic resection of gastric tumors originating from the muscularis propria The dataset data used to support the findings of this study are available from the corresponding author at email address upon request Casali PG, Blay JY, Abecassis N, Bajpai J, Bauer S, Biagini R, et al. 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JGH Open. 2024;8(7):e70002. https://doi.org/10.1002/jgh3.70002 Meng Y, Li W, Han L, Zhang Q, Gong W, Cai J, et al. Long-term outcomes of endoscopic submucosal dissection versus laparoscopic resection for gastric stromal tumors less than 2 cm. J Gastroenterol Hepatol. 2017;32(10):1693–7. https://doi.org/10.1111/jgh.13768 He Z, Sun C, Zheng Z, Yu Q, Wang T, Chen X, et al. Endoscopic submucosal dissection of large gastrointestinal stromal tumors in the esophagus and stomach. J Gastroenterol Hepatol. 2013;28(2):262–7. https://doi.org/10.1111/jgh.12056 Andalib I, Yeoun D, Reddy R, Xie S, Iqbal S. Endoscopic resection of gastric gastrointestinal stromal tumors originating from the muscularis propria layer in North America: methods and feasibility data. Surg Endosc. 2018;32(4):1787–92. https://doi.org/10.1007/s00464-017-5862-9 Huang LY, Cui J, Liu YX, Wu CR, Yi DL. Endoscopic therapy for gastric stromal tumors originating from the muscularis propria. 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Ann Surg. 2000;231(1):51–8. https://doi.org/10.1097/00000658-200001000-00008 Download references This study was supported by Peking University People’s Hospital Scientific Research Development Fund (RDL2023-13) (RDL2024-04) Liming Zhang and Rui Zhao co-first authors LZ was involved in the concept and design of study; LZ and RZ were involved in the data curation; LZ RZ and JZ were involved in statistical analysis; LZ and RZ were involved in drafting the manuscript; all authors were involved in writing–review and editing; LZ was involved in the supervision of study All authors have made an intellectual contribution to the manuscript and approved the submission All authors read and approved the final manuscript The study protocol has been approved by the Ethical Committee of the Peking University People’s Hospital with an approval number (2024PHB480) and performed according to the Declaration of Helsinki Ethical Committee of the Peking University People’s Hospital waived need for written informed consents due to the retrospective nature of this study All authors have no have no conflicts of interest to disclose The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1186/s12876-025-03669-6 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Metrics details no prospective study has been conducted to compare the safety and effectiveness of endoscopic snare resection with an elastic band (ESR-EB) and endoscopic snare resection with a transparent cap (ESR-C) for treating gastric muscularis propria lesions We aimed to compare the safety and effectiveness of ESR-EB with those of ESR-C for gastric muscularis propria lesions less than 10 mm in diameter A total of 64 patients were enrolled prospectively from May 2023 to November 2023 at Shenzhen Hospital of Southern Medical University the First Affiliated Hospital of Shantou University and the People’s Hospital of Zhongshan City The study compared clinical characteristics and surgical outcomes between the two groups Complete resection was achieved in all the patients The operation time was significantly greater in the ESR-C group than in the ESR-EB group (41.31 ± 9.87 min vs The complication rate varied significantly between the two groups (P = 0.000) No recurrence or metastasis was observed in either group during the follow-up period Both ESR-C and ESR-EB achieved a 100% complete resection rate for gastric muscularis propria lesions less than 10 mm in diameter.ESR-EB had the potential to reduce the operation time and lower the occurrence of complications Chinese Clinical Trial Registry Identifier ChiCTR2300072856 This was a multicentre prospective cohort study performed in three cities: Southern Medical University Shenzhen Hospital (Shenzhen) the First Affiliated Hospital of Shantou University (Shantou) and Zhongshan People’s Hospital (Zhongshan) The study was conducted in accordance with the Declaration of Helsinki (revised in 2008) and approved by the Institutional Review Board and Ethics Committee of Shenzhen Second People’s Hospital (2023-091-01PJ) The three centres enrolled patients beginning in May 2023 and ending in November 2023 Informed consent for endoscopic resection was obtained from each patient The endoscopists decided on the procedure to be performed on each patient according to their own experience Patient data were calculated based on the complication rate =\(\:\left({p}_{A}\left(1-{p}_{A}\right)+{p}_{B}\left(1-{p}_{B}\right)\right){\left(\frac{{{z}}_{1-\alpha/2}+{{z}}_{1-\beta\:}}{{p}_{A}-{p}_{B}}\right)}^{2}\)= \(\left(0.15\times\:\left(1-0.15\right)+0.55\times\:\left(1-0.55\right)\right)\times\:{\left(\frac{1.96+1.28}{0.15-0.55}\right)}^{2} = 25\: (\text {patients}).\) more than 25 patients were included in each of the ESR-C and ESR-EB groups The following instruments were used in this study: an endoscopic image processor (Olympus CLV-290SL); an operating gastroscope (Olympus HQ260J); an endoscopic ligation device (Boston Scientific Corporation M00542251); a high-frequency electric generator (ERBE VIO300D); a snare (Boston Scientific Corporation 20 mm); a rotatable and repeated opening and closing of a soft tissue clip (Nanwei Medical Technology Co. POCC-D-26–195); and an endoscopic transparent cap (Olympus Procedure of endoscopic snare resection with a transparent cap (ESR-C) A: The lesion was found by white-light endoscopy; B: The snare was opened at the transparent cap slot C: The lesion is attached to the transparent cap The procedure of endoscopic snare resection with an elastic band (ESR-EB) A:The lesion is found by white-light endoscopy; B: Ligation of the lesion by using an elastic band; C: The snare is placed under the elastic band to resect the lesion; D: The wound surface is observed; E: The wound is sutured using clips; F: The specimen is recovered We analysed the endoscopic complete resection rate and complication rate (including intraoperative bleeding and intraoperative perforation) The patients were followed to observe whether recurrence and/or metastasis occurred Complete resection was defined as one-time resection of the lesion and the histological diagnosis was margin negative The operation time was defined as the time from endoscopic entry into the oesophagus to endoscopic exit from the oesophagus The resection time was defined as the time from the start of contact between the transparent cap and the lesion to the completion of the wound being sutured by clips Intraoperative bleeding was defined as active bleeding that required endoscopic haemostasis Intraoperative perforation was defined as the visualisation of intraabdominal organ tissue or the omentum Recurrence was defined as the presence of new bulging masses at the original location during follow-up Metastasis was defined as lymphatic metastasis or organ metastasis detected during follow-up imaging Each patient was instructed to undergo an endoscopy or CT scan 1 year after surgery All the data were statistically analyzed using SPSS28 statistical software and the sample size was calculated based on the data from published articles The number of patients in both groups was greater than or equal to the calculated sample size the data were fitted to a normal distribution using the t-test for two independent samples The Mann‒Whitney U test was used for nonnormally distributed data A total of 64 patients were enrolled; 29 patients underwent ESR-C, and 35 patients underwent ESR-EB. There were no differences in age, gender, location, tumour size, or growth pattern (P > 0.05); therefore, it was not necessary to match the two groups (Table 1) To further confirm the safety of these two endoscopic resection methods a multicentre prospective cohort study was conducted in three cities in southern China This is the first study to compare the safety and effectiveness of these two endoscopic methods for the resection of gastric muscularis propria lesions The results showed that the adverse event rate of the ESR-C group was much greater than that of the ESR-EB group The reason for this result is still unclear and we speculate that the cause may be that ESR-EB uses ligation with an elastic band Due to the flexibility of the elastic band the lamina propria will slowly break away from the elastic band during peristalsis or expansion thus reducing full-thickness resection of the gastric wall ESR-C tightens the root of the lesion with a snare Because the snare is inelastic and the time from tightening to resection is short the muscularis propria is not able to easily break free of the snare resulting in full-thickness resection of the gastric wall Although the incidence of perforation in ESR-C patients is high and all the perforations can be stitched by clips None of the patients developed peritonitis which may be related to the short perforation exposure time The difference in resection time between ESR-C and ESR-EB has not been reported in the literature the operation time in the ESR-C group was significantly greater than that in the ESR-EB group This is because the ESR-C requires opening of the snare at the front of the transparent cap Because this procedure is performed inside the gastric cavity it is difficult to access the front end of the snare when the snare is stuck into the transparent cap slot which requires repeated attempts and sometimes needs to be positioned against the gastric wall This results in a significant amount of time being spent in the preresection preparatory phase in ESR-C the ligation device is installed in vitro and has a simple installation operation that saves overall operation time Although ESR-C resection time was shorter than ESR-EB resection time due to the limitations of the transparent cap and ligation device size these two endoscopic procedures are suitable only for resection of gastric muscularis propria lesions less than 10 mm in diameter the mean follow-up time of this study was short which prevents us from evaluating postoperative recurrence and metastasis It is necessary to extend the follow-up time in future studies Three endoscopists performed endoscopic resection in this study We cannot avoid the difference in the ability of our staff to perform endoscopic evaluation and treatment; however each of the endoscopists had more than 5 years of working experience and all the staff members had completed more than 100 cases of ESR-EB and more than 100 cases of ESR-C this study compared only ESR-C and ESR-EB and not common methods such as ESE or EFTR Large-scale randomised controlled studies are needed to further explore the advantages and disadvantages of these endoscopic procedures and our team is currently working on these studies both ESR-C and ESR-EB are effective and safe for the resection of gastric muscularis propria lesions which is a safer and time-saving endoscopic operation technique can significantly reduce the incidence of adverse events and shorten the operation time All data generated or analysed during this study are included in this published article Mehren MV,Antonescu CR (2012) Update on the management of patients with gastrointestinal stromal tumors & Moreels Els Nieveen van Dijkum,Jean-Yves Blay van Hooft (2022) Endoscopic management of subepithelial lesions including neuroendocrine neoplasms European Society of Gastrointestinal Endoscopy (ESGE) Guideline.Endoscopy 54: 412–429 Xin-Li Mao (2015) Endoscopic treatments for small gastric subepithelial tumors originating from muscularis propria layer Rui Li (2023) Comparison analysis of two different types of endoscopic resection procedures in small gastric subepithelial tumours originating frommuscularis propria Haoyue Ouyang (2016) Discussion of the clinical efficacy of endoscopic mucosal resection for gastric stromal tumor Xianghong Yang (2013) Ligation-assisted endoscopic enucleation for the diagnosis and resection of small gastrointestinal tumors originating from the muscularis propria: a preliminary study Chunli Cao,Shujie Song,Yue Li,Side Liu(2016)Endoscopic band ligation versus endoscopic submucosal dissection and laparoscopic resection for small gastric stromal tumors Weijin Pan, Ding Shi. Band-assisted endoscopic mucosal resection for small (≤ 1.5 cm) submucosal tumors originating from the muscularis propria in the gastric fundus: a prospective study.Surgical Endoscopy. https://doi.org/10.1007/s00464-022-09688-8 (2022) Endoscopic Enucleation Is Effective and Relatively Safe in Small Gastric Subepithelial Tumors Originating from Muscularis Propria Byoung Wook Bang,Kye Sook Kwon,Yong Woon Shin,Hyung KilKim Side Liu (2021) Safety analysis of transparent cap-assisted endoscopic resection for ≤ 10 mm gastric submucosal tumors[J] Fachao Zhi (2015) Cap-aspiration lumpectomy for small submucosal tumors originating from the muscularis propria of the gastric fundus: a preliminary study (with videos) Xu(2022)Comparison of endoscopic full-thickness resection and cap-assisted endoscopic full-thickness resection in the treatment of small (≤ 1.5 cm) gastric GI stromal tumors ScienceDirect Gastrointest & Buscaglia JM (2014) Endoscopic approach to subepithelial lesions[J] and clinical impact of endoscopic ultrasound (EUS)-guided sampling in gastroenterology: European Society of Gastrointestinal Endoscopy (ESGE) Clinical Guideline ESMO/European Sarcoma Network Working Group Gastrointestinal stromal tumours: ESMO clinical practice guidelines for diagnosis Expert Committee of gastrointestinal stromal tumor of the Chinese Clinical Oncology Society The Chinese consensus on the diagnosis and treatment of gastrointestinal GISTs Weiss (2002) Diagnosis of gastrointestinal stromal tumors: a consensus approach.Hum Pathol 33:459–465 (2003) Endoscopic snare resection of benign ampullary tumor: can intraductal growth be treated endoscopically?Gastrointestinal Siyu Sun (2020) Direct endoscopic full-thickness resection for submucosal tumors with an intraluminal growth pattern originating from the muscularis propria layer in the gastric fundus C.–S.Wu (2006) Endoscopic submucosal dissection for the treatment of intraluminal gastric subepithelial tumors originating from the muscularis Efficacy of Endoscopic Submucosal Excavation for Gastrointestinal Stromal Tumors in the Cardia.Surg Laparosc Chinese Society of Digestive Endoscopology Download references This work was supported by the Shenzhen Second People’s Hospital Clinical Research Fund of Shenzhen High-level Hospital Construction Project (Nos.2023yjlcyj018 Medical Scientific Research Foundation of Guangdong Province of China (No.A2022329) Clinical Scientific Research Foundation of Guangdong Provincial Medical Association (No.A202302031) and Teaching Reform Research Project of Shenzhen University Medical Science Center (No.YXBJG202302) Xiangyu Wang and Genhua Yang contributed equally to this work The First Affiliated Hospital of Shenzhen University Marshall Laboratory of Biomedical Engineering Shenzhen Hospital of Southern Medical University Shenzhen Clinical Research Center for Digestive Disease The First Affiliated Hospital of Shantou University Zhaohui Liu and Xiangyu Wang analyzed the data and wrote the main manuscript text Jiefeng Li and Yongsheng Lu provided the original data Dayong Sun and Ruinuan Wu have no conflicts of interest or financial ties to disclose Download citation DOI: https://doi.org/10.1038/s41598-024-83203-y Sign up for the Nature Briefing newsletter — what matters in science The satirical student magazine Propria Cures - popularly known as PC - has been living in discord with the UvA for a year The university no longer wants the magazine on its campuses What happened and what does this mean for the oldest student magazine in the Netherlands “Wanted,” headlined the front page of student magazine Propria Cures (PC) in February 2024 Below it was a black-and-white photo of a librarian with her full name and the word “Newspaper thief” as the subheadline The librarian allegedly systematically removed PCs from the shelves The piece calls her a “walking pot of day cream” and “customer of Adolf Hitler’s barber” This is one of the “incidents” that have caused the oldest student magazine in the Netherlands, which presents itself as literary satirical, to be at odds with the UvA for a year now. The UvA has had enough of it, Het Parool reported last weekend The paper version of PC is no longer available at UvA locations,” says a UvA press officer but we wouldn’t be worth a damn as an employer if we didn’t stand up for our colleagues.” Propria Cures’ five-member editorial team gathered in a deserted building in Amsterdam-Noord PC has recently been holding offices and will be working nights on their fortnightly magazine for the foreseeable future they would like to talk to Folia about the struggles with the university “The UvA seems to have tolerated writing the most terrible things about minorities majorities and human tragedies for 135 years but now that we have called someone a newspaper thief we have suddenly gone too far,” says PC editor Maarten van Dorp Propria Cures has profiled itself as “literary satirical” since its inception in 1890, and once in a while it raises a stir. This happened, for instance, after an interview in 2008 with Albert Verlinde’s anus The Leiden introduction committee thought this inappropriate and therefore decided to remove the PC in question from the bags of first-year students The presenter himself could laugh about it Although many Dutch celebrities have been ridiculed in PC in the past (literary) writers were often the target of ridicule such as Leon de Winter and the late Harry Mulisch and Joost Zwagerman Infamous is the montage photo of Leon de Winter in a mass grave posted at the time (1992) because he was said to have “exploited his Jewish background too much” It landed the editors at the time with €10,000 in damages and a forced rectification The UvA also refers to another incident of threatening an employee A PC from 2023 contained the following sentence: “Nothing should you may die.” How do you view this comment isn’t this a death threat?“That sentence must be understood within context,” Van Dorp responded “The comment was posted in the regular correspondents column in which the editors respond to readers letters and only had the connotation of an utterance like ‘walk to the moon’ you can only interpret it that way too.” Jokingly “I can guarantee the college board that we don’t have an armed branch at PC or anything.Where do you draw the line for satire you implicitly approve of everything that is on the right side of the line where there is a joke in almost every sentence and so nothing carries any real weight he points to a 2023 article full of ‘satirical’ death threats to politician Pieter Omtzigt written by his colleague Alexandra Philippa It begins with: “I’m going to kill Pieter Omtzigt I’m going to run him over with a Peugeot 205 I’m going to stir novichok through his peasant boys and peasant boys through his novichok.” Article title: “This is not a joke” “Everyone understands it’s a joke precisely because it’s in Propria Cures,” Van Dorp continues the clearer it becomes,” Philippa adds.Omtzigt could not laugh about it at the time He responded on X (then Twitter) that he thought it was “not a successful joke” because of the climate in which politicians are much threatened What did you guys think of that?Philippa: “I maintain I thought it could be done.”Meeus: “The point is also that it became news because he shared the article himself in front of his tens hundreds of thousands of followers on Twitter it was only in a magazine for subscribers and UvA students.”Van Dorp: “What I do appreciate is that he simply said: not a successful joke He didn”t call for the magazine to be banned or anything like that.”How do you look back on your publication “Wanted Newspaper Thief” about the UvA librarian?Meeus: “In retrospect maybe we should have taken out either the name or the photo Especially because it is a librarian and not a writer politician or presenter of a television programme if you are going to throw away magazines of ours you can expect it back.”Aren’t riots exactly what you want?Philippa: “A riot is good But a riot where you are no longer allowed to lie with your magazine at the UvA is extremely annoying.”Does that affect your viability?“Not really because we find that many people still read it or send in something It is also still available in all kinds of other places it could be that in time it will decline a bit because of this the approachability for students is now gone that moment when you can just get a leaf from the canteen of the UB.” “The magazine bins in which PC was put by editors of the magazine itself were once intended for printed UvA media and Folia which is now fully digital until PC started insulting individual staff members several times PC was urged several times to stop doing that we have been discouraging the use of prints The magazine bins have therefore been removed.” Booij missed out on the 125 euros per hour (in a 20-hour working week) he was supposed to receive for this job.According to the UvA this did not play any role in the decision-making process “PC has been urged several times to stop naming/shaming individual employees,” the UvA responded when asked and later also in March and April of 2024.”“Propria Cures is a satirical magazine but insulting doxxing and slandering employees has nothing to do with satire,” continues a UvA press officer “That was cross-border as far as we are concerned It affects colleagues personally who did not ask for it and are not in a position that could justify it Metrics details Bacterial toxins are well-studied virulence factors; however recent studies have revealed their importance in bacterial niche adaptation Enterotoxigenic Bacteroides fragilis (ETBF) expresses B fragilis toxin (BFT) that we hypothesized may contribute to both colonic epithelial injury and niche acquisition We developed a vertical transmission model for ETBF in mice that showed that BFT enabled ETBF to access a lamina propria (LP) niche during colonic microbiome development that was inaccessible to non-toxigenic B LP entry by ETBF required BFT metalloprotease activity and showed temporal restriction to the pre-weaning period dependent on goblet-cell-associated passages In situ single-cell analysis showed bft expression at the apical epithelial surface and within the LP BFT expression increased goblet cell number and goblet-cell-associated passage formation These findings define a paradigm by which bacterial toxin expression specifies developmental niche acquisition suggesting that a selective advantage conferred by a toxin may impact long-term host health Prices may be subject to local taxes which are calculated during checkout No specific code was used in the analysis of data in this article Sensitive quantitative analysis of the meconium bacterial microbiota in healthy term infants born vaginally or by cesarean section The person-to-person transmission landscape of the gut and oral microbiomes Bacterial colonization factors control specificity and stability of the gut microbiota and IgA response to species of the order Bacteroidales in the human gut Adaptive evolution within gut microbiomes of healthy people The landscape of type VI secretion across human gut microbiomes reveals its role in community composition Bacteroides fragilis subverts mucosal biology: from symbiont to colon carcinogenesis Induction of persistent colitis by a human commensal Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria Non-toxigenic Bacteroides fragilis (NTBF) administration reduces bacteria-driven chronic colitis and tumor development independent of polysaccharide A Strain competition restricts colonization of an enteric pathogen and prevents colitis Human gut microbiome viewed across age and geography The long-term stability of the human gut microbiota Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features Spatially distinct physiology of Bacteroides fragilis within the proximal colon of gnotobiotic mice A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species Cholera toxin promotes pathogen acquisition of host-derived nutrients difficile exploits a host metabolite produced during toxin-mediated disease Clostridioides difficile exploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses Bacteroides fragilis toxin exhibits polar activity on monolayers of human intestinal epithelial cells (T84 cells) in vitro Antibiotics promote inflammation through the translocation of native commensal colonic bacteria Human symbionts inject and neutralize antibacterial toxins to persist in the gut Diversification of type VI secretion system toxins reveals ancient antagonism among bee gut microbes Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host Microbial antigen encounter during a preweaning interval is critical for tolerance to gut bacteria Live imaging reveals listeria hijacking of E-cadherin recycling as it crosses the intestinal barrier Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes to anaerobic sepsis in mice A two-component system Regulates Bacteroides fragilis toxin to maintain intestinal homeostasis and prevent lethal disease Salmonella finds a way: metabolic versatility of Salmonella enterica serovar Typhimurium in diverse host environments The Bacteroides fragilis pathogenicity island links virulence and strain competition function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog Tunable expression tools enable single-cell strain distinction in the gut microbiome DeconvolutionLab2: an open-source software for deconvolution microscopy Download references This work was supported by a Pilot and Feasibility Award from the Digestive Diseases Research Core Center at the University of Chicago (NIDDK P30DK42086) National Institutes of Health (NIH) Award DK085025 to J.L.S. and NIH Awards AI138565 and AI157196 to J.B.W was supported by the National Institute of Allergy and Infectious Diseases of the NIH (F30AI126791) were trainees of the NIH Medical Scientist Training Program at the University of Chicago (GM007281) Math1fl/fl Vil-Cre-ERT2 mice were a gift from R Present address: Department of Dermatology Present address: Division of Gastroenterology and Hepatology University of Pennsylvania School of Medicine These authors contributed equally: Craig A Ezequiel Valguarnera & Juliane Bubeck Wardenburg Interdisciplinary Scientist Training Program generated the fluorescent bacterial strains Sarkis Mazmanian and Benjamin Ross for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations (a) Timeline of vertical transmission experiments decolonized with clindamycin in drinking water beginning on E14 and assayed for faecal colonization weekly Offspring (closed circles) were weaned at age 3 weeks (P21) and feces were collected for CFU analysis at the indicated time points Average CFU ± standard deviation of n = 14 litters is shown (c) Weight of P21 mice colonized with NTBF each data point represents a litter average Source data and distal colons were extracted at eight weeks from adult mice that were gavaged at six weeks with (a) ETBF or (d) ETBF in the absence of antibiotic treatment (e) Quantification of LP burden of each strain as in a-d stained with phalloidin (grey) and DAPI (blue) Source data tissues were harvested from pups born to dams colonized with (a) ETBF or ETBF Δccf Sections were stained with DAPI (blue) and phalloidin (gray) and visualized by confocal microscopy f) To normalize crypt infiltration across strains an infiltration ratio was calculated per strain by dividing the distance bacteria invaded by the total length of the crypt (g) To illustrate total abundance in tissue versus crypt the total number of bacteria present in crypts was divided by the total number of bacteria in adjacent lamina propria (h) Analysis of the ratio of LP burden to total tissue burden (TTB = crypt + LP burden) of the noted strains Data are representative of 4 biological replicates Displayed images are representative of a minimum of 6 independent images Source data Pups born to ETBF-colonized dams were inoculated with NTBF WT or ΔtssC on (a f) P20 and tissues were harvested from P23 mice to visualize ETBF (green) and NTBF or its ΔtssC variant (red) in the proximal colon (left and center panel) and enumerate CFU (right panel) and **** p < 0.0001 in pairwise comparisons (two-tailed t-test) Data represents n = 11-14 mice in total per group (b) Source data f) P20 and tissues were harvested P23 mice to visualize ETBF (green) and NTBF or its ΔtssC variant (red) in the distal colon (left and center panel) and enumerate CFU (right panel) Source data Pups born to (a) ETBF (ATCC 43859) or (b) ETBF Δbft-colonized dams were inoculated with NTBF (TM4000/638 R) on P16 and tissues were harvested from P23 mice to enumerate CFU (right panel) No statistically significant differences were found in pairwise comparisons (two-tailed t-test) Data represents n = 7–10 mice in total per group Source data Five-week-old mice born to dams colonized with ETBF Δbft were subjected to either apical (a d) exposure of active BFT (BFTWT) or inactive toxin (BFTE349A) before tissue harvest 24-48 hours later Sections were stained with AB-PAS and visualized with brightfield microscopy for enumeration of GCs and measurement of crypt length Data represents 2-4 biological replicates with n = 5–7 mice in total per group Source data (a) Survival data for Math1fl/flVil-Cre-ERT2 mice either homozygous null (−/−) or heterozygous (+/−) for Vil-Cre-ERT2 Math1 deletion was induced by daily tamoxifen injections starting at P5 and ending at P10 Ear tissue was collected from dead neonates for genotyping mice were sacrificed at P23 for genotyping and to assess faecal ETBF CFUs (b) Math1fl/fl Vil-Cre-ERT2+/− = 14 mice; Math1fl/fl Vil-Cre-ERT2−/− = 6 mice Gehan-Breslow-Wilcoxon test showed statistically significant differences between survival curves Data representative of two independent experiments Source data RNA in situ hybridization analysis with probes for gfp (green) and bft (red) was performed on 14 µm fixed-frozen sections of ceca that were extracted at eight weeks from adults gavaged at six weeks with (a) ETBF (b) ETBF in the absence of antibiotic treatment Sections were stained with phalloidin (gray) and DAPI (blue) and visualized with Airyscan images are representative of six adults over two independent replicates (24 mice total) Five-week-old mice born to dams colonized with ETBF Δbft were subjected to either apical (a–c) or basolateral (d–f) exposure of active (BFTWT) or inactive toxin (BFTE349A) before tissue harvest 24-48 hours later e) Tissue infiltration was assessed as bacilli per cubic micron of lamina propria (L.P.) f) crypt lengths were measured across all four groups n = 8 mice per strain.** p < 0.01 and **** p < 0.0001 in pairwise comparisons (two-tailed t-test) Source data ETBF can enter the LP in antibiotic-treated adult mice ETBF Δbft is restricted from the LP in antibiotic-treated adult mice ETBF and the bft transcript are visualized in the LP a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law Download citation DOI: https://doi.org/10.1038/s41564-023-01559-9 Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research Metrics details Multiple mechanisms exist in regulation of host responses to massive challenges from microbiota to maintain immune homeostasis in the intestines Among these is the enriched Th17 cells in the intestines which regulates intestinal homeostasis through induction of antimicrobial peptides and secretory IgA among others the means by which Th17 cells develop in response to microbiota is still not completely understood Although both TLR5 and CD172α+ lamina propria dendritic cells (LPDC) have been shown to promote Th17 cell development it is still unclear whether TLR5 mediates the CD172α+LPDC induction of Th17 cells By using a microbiota antigen-specific T cell reporter mouse system we demonstrated that microbiota antigen-specific T cells developed into Th17 cells in the intestinal LP but not in the spleen when transferred into TCRβxδ−/− mice LPDCs expressed high levels of TLR5 and most CD172α+LPDCs also co-expressed TLR5 IL-6 and TGFβ when stimulated with commensal flagellin and promoted Th17 cell development when cultured with full-length CBir1 flagellin but not CBir1 peptide whereas TLR5-deficient LPDC did not induce Th17 cells Our data thereby demonstrated that TLR5 mediates CD172α+LPDC induction of Th17 cells in the intestines both of which could contribute to the maintenance of intestinal homeostasis the cells and environmental factors which promote Th17 cell development in intestines are still not completely understood it is still unclear whether TLR5 mediates CD172α+ LPDC induction of Th17 cells in the intestine in response to commensal bacteria and thus controls immune homeostasis and intestinal inflammation We report here by using a microbiota antigen-specific T cell reporter system that LPDCs express high levels of TLR5 CD172α+ but not CD172α− LPDCs induce microbiota antigen-specific Th17 cells Intestinal LPDCs induce Th17 cell development (A) CBir1 Tg CD4 T cells were labeled with CFSE and transferred into TCRβ×δ−/− mice The mice were gavaged with CBir1 flagellin the same day Cytokine production of CBir1 Tg CD4 T cells in spleen and intestinal LP was determined by intracellular staining 3 days later FACS plots are representative of 3 independent experiments Bar charts of cytokine expression from pool of 3 experiments (B) CBir1 Tg CD4 T cells were cultured with splenic DC or LPDC isolated from C57BL/6 mice in the presence of full-length CBir1 flagellin or CBir1 peptide for 5 days Cytokine production of CBir1 Tg CD4 T cells was determined by intracellular staining indicating that spleen DC and LPDC differentially induce Th1 and Th17 cell development in response to stimulation of full-length CBir1 flagellin MLN DC and LPDC take up and present CBir1 flagellin to T cells after mucosal challenge in vivo Wild-type and TCRβ×δ−/− mice were gavaged with Alexa 647-labeled CBir1 flagellin Uptake of CBir1 flagellin by MLN DC and LPDC was determined 2 hrs later by flow cytometry (A) When cultured with CD4 T cells of CBir1 Tg mice Data are reflective of 3 independent experiments *p<0.05 and **p<0.01 compared to controls More LPDCs express TLR5 than spleen DCs and most CD172α+ LPDCs are also TLR5+ Spleen cells and LP cells were isolated from C57BL/6 mice CD103 and TLR5 and analyzed by flow cytometry (A) CD11c+ cell expression of TLR5 was shown (B) TLR5 expression by CD11b+ and CD11b− DCs by gating on CD11c+ population (C) CD11c+ DC expression of CD103 and CD11b by gating on CD11c+ population (D) Expression of TLR5 and CD172α by CD103+ DC and CD103− DCs in LP of wild-type C57BL/6 mice by gating on CD11c+CD11b+ population (E,F) CD172α expression by CD103+ DC and CD103− DCs in LP of wild-type (E) and TLR5 KO C57BL/6 mice (F) by gating on CD11c+ population FACS plots are reflective of 2–4 independent experiments Different cytokine profiles and antigen-presenting ability of spleen DC and LPDC in response to CBir1 flagellin stimulation (A) Spleen DC and LPDC isolated from C57BL/6 mice were stimulated with full-length CBir1 flagellin IL-23 and IL-6 production in the supernatant was measured by an ELISA 24 hrs later (B) CBir1 Tg T cells were cultured with spleen DC or LPDC with or without anti-TGFβ Cytokine production in the supernatant was determined by an ELISA 3 days later Data are reflective of 4 independent experiments CD172α+ LPDCs induce Th17 cell development (A) CD172α+ and CD172α− LPDCs were isolated from C57BL/6 mice and cultured with CBir1 Tg T cells in the presence of full-length CBir1 flagellin or CBir1peptide Cytokine production of CBir1 T cells was determined by intracellular staining 5 days later FACS plots are reflective of 2 independent experiments (B) LPDCs were isolated from wild type or TLR5 KO mice and cultured with CBir1 Tg T cells in the presence of full-length CBir1 flagellin or CBir1peptide These data further demonstrated that TLR5 mediates CD172α+ LPDCs promotion of Th17 cell development in response to microbiota antigen stimulation the means by which a T cell response to microbiota is regulated is still not completely clear We demonstrated by using a micobiota antigen-specific TCR Tg reporter mice that DCs instructed T cells to develop into Th17 cells in response to microbiota at the mucosal surface which was mediated by LPDC production of IL-6 Among multiple subsets of lamina propria DCs CD172α+ DCs were able to induce Th17 cell development flagellin can function as an antigen to induce adaptive T cell response and as a TLR5 ligand to induce innate response which are specific for an immunodominant commensal antigen CBir1 flagellin developed into Th17 cells in intestines but not in spleens when transferred into TCRβ×δ−/− mice expressed high levels of TLR5 and produced IL-23 IL-6 and TGFβ in response to stimulation of CBir1 flagellin but not LPS and thus could provide a cytokine milieu favoring Th17 cell development which is consistent with a previous report when cultured with microbiota antigen CBir1-specific Tg T cells whereas splenic DC induced a Th1-dominant response indicating that LPDC were able to induce Th17 cells in vitro LPDC induced Th17 cell development only in the presence of full-length CBir1 flaggelin indicating that LPDC TLR5 signaling is crucial for Th17 cell development in response to microbiota antigen stimulation These findings may mean that CD172α+ populations could be the LPDC subset inducing Th17 cell responses among the CD103+CD11b+ subset of LPDCs LPDC induced Th17 cell differentiation in response to microbiota antigen stimulation further confirming CD172α+ LPDC induction of microbiota antigen-specific Th17 cells most CD172α+ LPDCs also co-expressed TLR5 and TLR5-deficient LPDC did not induce Th17 cell development TLR5 deficiency did not affect development of CD172α+ LPDC our data further argue that TLR5 signaling mediates induction of Th17 cells by various LPDC subsets TCRβ×δ−/− mice and TLR5−/− mice were purchased from Jackson Laboratory CBir1 Tg mice were housed and maintained in the animal facilities of the University of Texas Medical Branch All experiments were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Texas Medical Branch All the methods were carried out in accordance with the approved guidelines Flow cytometry antibodies anti-mouse CD4-magnet beads were purchased from BD Bioscience anti-CD172α were purchased from Biolegends Anti-TLR5 antibody was purchased from NOVUS Biologicals single cells isolated from spleen were incubated with anti-CD4 beads for 30 min at 4 °C The cells were then placed on a magnetic column to isolate CD4+ T cells Small and large intestinal segments were prepared from the mice and treated with PBS containing 10% FCS 1 mM sodium pyruvate and 10 mM EDTA for 30 min at 37 °C to remove epithelial cells and were washed extensively with PBS Intestinal segments were digested with 400 Mandl units/ml collagenase D (Roche) and 10 μg/ml DNase I (Roche) in RPMI 1640/10% FCS with continuous stirring at 37 °C for 45–90 min Cells were spun through a 40%/70% Percoll gradient to enrich for DCs The obtained cells were incubated with PE-conjugated anti-CD11c after FcR blocking and isolated by MACS Purified DCs were then stained with different antibodies and subsets were sorted by FACSVantage SE (BD Biosciences) The purity of the sorted DCs was routinely >95% The FACS was performed as previously described52 after washing with PBS having 0.1% sodium azide plus 2% heat-inactivated newborn calf serum the cells were incubated with various conjugated mAbs washed and fixed in 1% buffered paraformaldehyde and quantitated by using an LSRII/Fortessa and FACSDiva software (Becton Dickinson Further analysis was carried out by using FlowJo A mAb of the same isotype but irrelevant specificity was used as a negative control As described previously52 5 × 105 cells were stimulated for 6 hrs with PMA and ionomycin and monensin were added for the last 2 hrs of culture the cells were fixed and permeablized by using Cytofix/cytoperm solution (BD Pharmingen) IL-17 and IFNγ were stained by using conjugated antibodies (Biolegend) CBir1 CD4 T cells were cultured with LPDC or spleen DC in the presence of various antigens the cells were collected for intracellular staining CBir1 CD4 T cells were cultured with APC in the presence of various antigens and TGFβ (5 ng/ml) The data from all experiments were analyzed by using Prism (GraphPad) Statistical significance was determined by using Student’s t test with paired tests The results were considered significant at a P value of less than 0.05 TLR5 mediates CD172α+ intestinal lamina propria dendritic cell induction of Th17 cells Host-microbiota interactions in inflammatory bowel disease Genetics and environmental interactions shape the intestinal microbiome to promote inflammatory bowel disease versus mucosal homeostasis The orphan nuclear receptor ROR gammat directs the differentiation program of proinflammatory IL-17+ T helper cells Induction of intestinal Th17 cells by segmented filamentous bacteria Involvement of IL-17A in the pathogenesis of DSS-induced colitis in mice A protective function for interleukin 17A in T cell-mediated intestinal inflammation Neutralization of interleukin-17 aggravates dextran sulfate sodium-induced colitis in mice Critical role of IL-17 receptor signaling in acute TNBS-induced colitis Cytokine-mediated regulation of antimicrobial proteins Interleukin-17 and its target genes: mechanisms of interleukin-17 function in disease Th17 cells upregulate polymeric Ig receptor and intestinal IgA and contribute to intestinal homeostasis Interleukin (IL)-21 promotes intestinal IgA response to microbiota Cutting edge: lung mucosal Th17-mediated responses induce polymeric Ig receptor expression by the airway epithelium and elevate secretory IgA levels Immunomodulatory dendritic cells in intestinal lamina propria Selective generation of gut tropic T cells in gut-associated lymphoid tissue (GALT): requirement for GALT dendritic cells and adjuvant Involvement of intestinal dendritic cells in oral tolerance immunity to pathogens and inflammatory bowel disease Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells Regional specialization within the intestinal immune system Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance Tissue-tropic effector T cells: generation and targeting opportunities Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing Differential antigen processing by dendritic cell subsets in vivo Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens Differential production of inflammatory chemokines by murine dendritic cell subsets The CD8+ dendritic cell subset selectively endocytoses dying cells in culture and in vivo Matrix protein mediated shutdown of host cell metabolism limits vesicular stomatitis virus-induced interferon-alpha responses to plasmacytoid dendritic cells Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation Focused specificity of intestinal TH17 cells towards commensal bacterial antigens A new subset of CD103+CD8alpha+ dendritic cells in the small intestine expresses TLR3 TLR7 and TLR9 and induces Th1 response and CTL activity Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5 Signal regulatory protein alpha (SIRPalpha) regulates the homeostasis of CD103(+) CD11b(+) DCs in the intestinal lamina propria Intestinal lamina propria dendritic cells maintain T cell homeostasis but do not affect commensalism Signal regulatory protein alpha (SIRPalpha)/CD47 interaction and function coordinated T regulatory cell-IgA response to the intestinal microbiota CD4+ T-cells in the regulation of inflammatory responses in the intestine Bacterial flagellin is a dominant antigen in Crohn disease Functional specializations of intestinal dendritic cell and macrophage subsets that control Th17 and regulatory T cell responses are dependent on the T cell/APC ratio source of mouse strain and regional localization Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens IRF4 promotes cutaneous dendritic cell migration to lymph nodes during homeostasis and inflammation Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages IRF4 transcription-factor-dependent CD103(+)CD11b(+) dendritic cells drive mucosal T helper 17 cell differentiation IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses CCR2(+)CD103(−) intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells ATP drives lamina propria T(H)17 cell differentiation Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17-producing T cell responses Steady-state migrating intestinal dendritic cells induce potent inflammatory responses in naive CD4+ T cells Comparative transcriptional and functional profiling defines conserved programs of intestinal DC differentiation in humans and mice Microbiota innate stimulation is a prerequisite for T cell spontaneous proliferation and induction of experimental colitis Download references This work was supported by NIH grants DK098370 and DK105585 and John Sealy Memorial Endowment Fund HL is a recipient of China Scholarship Council (CSC) fellowship designed the project and wrote the manuscript The authors declare no competing financial interests Download citation Metrics details The prognosis difference based on the depth of tumor muscularis propria invasion in gastric cancer (GC) was still debated and therapy strategy for stage IB GC patient required further investigation A total of 380 patients with pT2 GC after radical surgery were retrospectively analyzed including 185 in superficial muscularis propria (sMP) group and 195 in deep muscularis propria (dMP) group The overall survival (OS) was significantly better for patients in sMP group than for patients in dMP group (P = 0.007) elevated initial CA19-9 and AFP level were independent prognostic factors for OS The sMP group had a significantly better OS than dMP group (P = 0.014) in pN0 stage the survival outcomes were not significantly different between deep muscularis propria tumor invasion without lymph node metastasis (dMPN0) group (stage IB) and superficial muscularis propria tumor invasion with stage 1–2 lymph node metastasis (sMPN1–2) group (stage II) (P = 0.100) Patients with adjuvant chemotherapy had a statistically better survival than those without in dMPN0 group (P = 0.045) and dMPN0 patients with adjuvant chemotherapy had better OS than sMPN1–2 patients (P = 0.015) greater postoperative survival could be observed in sMPN0 patients than dMPN0 patients in p53-positive group (P = 0.002) and similar OS could be seen between dMPN0 patients with p53-positive and T2N1–2 patients (P = 0.872) As a unique subclassification of stage IB GC appropriate adjuvant chemotherapy should be considered for patients with dMPN0 stage elevated LDH could be potential factors in identifying the different prognoses for stage IB GC patients Accurate staging of gastric cancer is the basis for guiding treatment strategy and judging the prognosis of patients TNM staging system introduced by the Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) is adopted internationally in recent years which could not be simply explained by the TNM staging and postoperative therapy regimens the muscularis propria of the stomach is histologically subdivided further into two layers: the inner circular layers the newest edition of TNM stage system does not specify details for definition of subclassification of pT2 stage (sMP vs we believe that it is reasonable to consider that the subclassification of the pT2 stage can be used as a new standard for prognostic prediction and clinical decision-making we conduct present study to identify the relations between subclassification of pT2 gastric cancer and survival outcomes and clinicopathological features according to the depth of tumor involvement and further analyze potential markers to reinforce the prognostic and therapy-guided ability of the TNM staging system The study was approved by the clinical research ethics committee of the Jiangsu Cancer Hospital and was conducted in accordance with the Declaration of Helsinki Flow diagram of the patient selection process According to the treatment guidelines and preoperative examination (radiological imaging tests and pathological tests) patients who matched surgical indications in our hospital underwent gastrectomy with standard lymph node dissection The decision as to whether patients received postoperative adjuvant chemotherapy depended on the integrated evaluation of pathological examination Adjuvant chemotherapy regimens were single-agent fluoropyrimidine or platinum combined with fluoropyrimidine Clinicopathologic features include gender (male Initial tumor markers were detected within seven days before surgery and maximal LDH was defined as the maximum of LDH during follow-up Pathological tumor staging was based on the TNM staging system of UICC/AJCC (eighth edition) Continuous and categorical variables were assessed through the t-test Kaplan–Meier method and log-rank test were performed to distinguish univariate survival outcomes Variables with P value < 0.1 in the univariate survival analysis were included in multivariate Cox’s proportional hazard model to identify independent prognostic factors Statistical differences were considered as significant at two-sided P values < 0.05 All statistical analyses were estimated through SPSS software (version 22 By December 31, 2020, 10 of the 390 cases were lost to follow up during the follow-up. The follow-up period ranged from 2 to 166 months with a median follow-up duration of 99 months. For survival outcomes, patients in sMP group had a statistically better 5-year OS rate than those in dMP group (91% vs. 84%, P = 0.007) (Fig. 2). Comparison of the survival outcomes for patients with sMP and dMP gastric cancers Univariate and multivariate survival analyses were used to find prognostic factors (Table 2) CEA and AFP level were statistical prognostic factors in the univariate analysis Multivariate Cox regression analysis confirmed that depth of tumor invasion elevated initial CA19-9 and AFP level were independent prognostic factors Survival curves of pT2 cancers according to the pN0 staging and pN+ staging, respectively. A For patients with pN0 stage tumor, prognosis of the sMP group were significantly different with that of the dMP group (P = 0.014). B For patients with pN+ stage tumor, prognosis of the sMP group were not significantly different with that of the dMP group (P = 0.384) Survival curves of gastric cancer patients in dMP,N0 group and sMP,N1-2 group according to the adjuvant chemotherapy status A The survival outcome of patients were not significantly different between dMP,N0 group and sMP,N1-2 group (P = 0.100) B The survival outcome of patients with and without adjuvant chemotherapy were significant difference in dMP,N0 group (P = 0.045) In comparison to patients in sMP,N1–2 group patients in dMP,N0 group who accepted adjuvant chemotherapy had better postoperative survival (P = 0.015) (C) but not significance in that without adjuvant chemotherapy (P = 0.599) (D) Survival curves of gastric cancer patients in dMP,N0 group and T2,N1–2 group according to other potential prognosis factors A The survival outcome of sMP,N0 patients were better than dMP,N0 patients in p53-positive group (P = 0.002) B The survival outcome of patients in p53+,dMP,N0 group were not significantly different to patients in T2,N1–2 group (P = 0.872) C The survival outcome of sMP,N0 patients were greater than dMP,N0 patients in elevated maximal LDH level group (P = 0.029) D The survival outcome of patients in elevated maximal LDH,dMP,N0 group were not significantly different to patients in T2,N1–2 group (P = 0.514) E There were statistically better overall survival of sMP,N0 patients than dMP,N0 patients in elevated initial CEA level group (P = 0.011) F The overall survival of patients in elevated initial CEA,dMP,N0 group were similar to patients in T2,N1–2 group (P = 0.935) and the latest 8th TNM staging system did not define the detailed subclassification of the pT2 stage it was necessary to investigate prognostic differences based on the depth tumor muscularis propria infiltration this was the first study to evaluate differences in the clinicopathologic features and the prognoses of pT2 subclassification based on the depth of tumor muscularis propria infiltration in a large cohort of Chinese gastric cancer patients Molecular markers and serum tumor markers also were firstly investigated in the pT2 subclassification We also found that neural invasion and elevated initial AFP levels were more likely to appear in the dMP group than the sMP group when patients were grouped depending on the depth of tumor invasion and pN stage we further found that the survival outcomes were not significantly different between patients with the dMPN0 stage and with the sMPN1–2 stage After further comparison according to postoperative adjuvant chemotherapy status we observed that significantly improved survival outcomes for patients who had received the adjuvant chemotherapy in the dMPN0 staging group We also observed that the dMPN0 patients with the adjuvant chemotherapy had a better postoperative survival compared to sMPN1–2 patients significantly different survival could be seen between sMPN0 group (stage IB) and dMPN0 group (stage IB) but not dMPN0 group (stage IB) and sMPN1–2 group (stage II) and dMPN0 patients who received the adjuvant chemotherapy had obviously improved postoperative survival compared to sMPN1–2 patients These results showed that the dMPN0 stage should be divided from stage IB as a special subclassification and the dMPN0 patients should receive appropriate adjuvant chemotherapy and follow-up strategy we demonstrated that positive expression of p53 protein was a negative prognostic factor for the OS in the early stages of GC the sMPN0 group had better survival outcomes than dMPN0 group similar survival outcomes could be seen between the dMPN0 patients (stage IB) with p53-positive and T2N1–2 patients (stage II) Although we not found significant survival difference between dMPN0 patients with p53+ receiving and those not receiving adjuvant chemotherapy but due to the small sample size (55 patients) of the subgroup analysis the result remained open to question and needed to be validated by a larger sample size research the above results might unveil that dMPN0 patients (stage IB) with positive expression of p53 might potentially benefit from adjuvant chemotherapy like patients in stage II overexpression of HER-2/neu protein was not an adverse prognostic predictor we noticed that patients with the sMPN0 had a higher overall survival than those with the dMPN0 in the elevated maximal LDH level group and there was similar OS between the dMPN0 patients with the elevated maximal LDH level and T2N1–2 patients Similar results also were shown in the dMPN0 patients with elevated maximal CEA levels we confirmed that serologic tumor markers could be used to identify individual heterogeneity and improve the survival prediction ability of the TNM staging our present study still has several limitations although the sample size of this study is the largest amongst other studies focusing on the T2 subclassification to date the results are susceptible to selection bias prospective studies are therefore required to verify the findings of this study IHC has been applied to detect molecular markers of cancer in our present study concentration and evaluation standard of positivity used in IHC might produce potential bias All data generated or analyzed during this study are included in this published article The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request Cancer survival in Europe 1999–2007 by country and age: results of EUROCARE-5—a population-based study The clinical and prognostic significance of paraoxonase-2 in gastric cancer patients: immunohistochemical analysis Clinical significance of serum tumor markers for gastric cancer: a systematic review of literature by the Task Force of the Japanese Gastric Cancer Association A novel subclassification of pT2 gastric cancers according to the depth of muscularis propria invasion: superficial muscularis propria versus deep muscularis propria/subserosa New molecular staging with G-factor supplements TNM classification in gastric cancer: a multicenter collaborative research by the Japan Society for Gastroenterological Carcinogenesis G-Project committee EGFR gene amplification is relatively common and associates with outcome in intestinal adenocarcinoma of the stomach gastro-oesophageal junction and distal oesophagus Assessment of a HER2 scoring system for gastric cancer: results from a validation study Outcomes of surgical treatment for gastric cancer patients: 11-year experience of a Chinese high-volume hospital Validation of the new AJCC TNM staging system for gastric cancer in a large cohort of patients (n = 2,155): focus on the T category Prognostic value of subclassification of T2 tumours in patients with gastric cancer Comparison between superficial muscularis propria and deep muscularis propria infiltration in gastric cancer patients: a retrospective cohort study Phase III trial comparing capecitabine plus cisplatin versus capecitabine plus cisplatin with concurrent capecitabine radiotherapy in completely resected gastric cancer with D2 lymph node dissection: the ARTIST trial Adjuvant capecitabine and oxaliplatin for gastric cancer after D2 gastrectomy (CLASSIC): a phase 3 open-label The regulation of p53 function: Steiner Award Lecture The significance of p53 in clinical practice An evaluation of six antibodies for immunohistochemistry of mutant p53 gene product in archival colorectal neoplasms The prognostic significance of p53 expression in gastric cancer: a meta-analysis The prognostic significance of the accumulation of p53 tumour-suppressor gene protein in gastric adenocarcinoma Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3 Evaluation of HER2 protein expression in gastric carcinomas: comparative analysis of 1,414 cases of whole-tissue sections and 595 cases of tissue microarrays ADAM 10 is associated with gastric cancer progression and prognosis of patients Overexpression of Grb2/HER2 signaling in Chinese gastric cancer: their relationship with clinicopathological parameters and prognostic significance Prognostic significance of immunohistochemical expression of EGFR and C-erbB-2 oncoprotein in curatively resected gastric cancer The correlation between pre-operative serum tumor markers and lymph node metastasis in gastric cancer patients undergoing curative treatment Prognostic role of lactate dehydrogenase in solid tumors: a systematic review and meta-analysis of 76 studies Prognostic factors in 868 advanced gastric cancer patients treated with second-line chemotherapy in the real world Download references This study was supported by the program of Jiangsu Cancer Research (ZN201605) and the general program of Jiangsu Commission of Health (H2017034) Kang He and Cheng Chen have contributed equally to this work The Affiliated Cancer Hospital of Nanjing Medical University Jiangsu Cancer Hospital and Jiangsu Institute of Cancer Research SYW and JH analyzed the study data through statistics software informed consent from all patients was obtained verbally and confirmed by the clinical research ethics committee of the Jiangsu Cancer Hospital The authors declare that they have no competing interests : Distinction of sMP (inner circular muscle bandsand) and dMP (outer longitudinal muscle bands) invasion according to the maximal invasive depth of tumor : Comparison of the prognoses for sMPN1-2 patients according to the status of adjuvant chemotherapy : Comparison of the prognoses for dMPN0 patients with positive expression of p53 according to the status of adjuvant chemotherapy unless otherwise stated in a credit line to the data Download citation DOI: https://doi.org/10.1186/s12876-021-02090-z Metrics details Crohn’s disease causes chronic inflammation in the gastrointestinal tract and its pathogenesis remains unclear In the intestine of Crohn’s disease patients CD14+CD11+CD163low macrophages contribute to inflammation through the induction of Th17 cells and production of inflammatory cytokines; the CD14+CD11c+163high fraction is anti-inflammatory through the production of IL-10 in normal cases the 16S rRNA gene amplicon sequencing method was used to identify bacteria that are specifically present in intestinal CD14+CD11c+ macrophages of Crohn’s disease patients Bacteria present in intestinal CD14+CD11c+ macrophages and mucus of Crohn’s disease patients were separated into different clusters in principal coordinates analysis There was a statistically significant increase in the relative composition of CD14+CD11c+ macrophages from mucus in two phyla (Proteobacteria [p = 0.01] and Actinobacteria [p = 0.02]) and two families (Moraxellaceae [p < 0.001] and Pseudomonadaceae [p = 0.01]) OTU-1: Acinetobacter and OTU-8: Pseudomonadaceae tended to concentrate in the CD14+CD11c+CD163low subset OTU-15: Collinsella tended to concentrate more in the CD14+CD11c+CD163high subset than the other subset and mucus These reports suggest that some bacteria from Crohn’s disease patients may survive in the host intestinal macrophages and activate an inflammatory response there is limited knowledge about the bacterial flora present in macrophages and in particular regarding the microbiota in macrophages from the intestinal lamina propria of Crohn’s disease patients we illuminated the intracellular microbiota in CD14+CD11c+ macrophages from the intestinal lamina propria using the 16S rRNA gene amplicon sequencing method to identify bacteria that are specifically present in intestinal CD14+CD11c+ macrophages of Crohn’s disease patients and compared bacterial flora between CD14+CD11c+CD163low and CD14+CD11c+CD163high macrophages to investigate whether there is a difference in bacterial flora Intestinal samples were obtained through ileum resection at Hyogo College of Medicine Hospital from patients with a confirmed diagnosis of Crohn’s disease endoscopically Cases that were difficult to distinguish from other disease such as ulcerative colitis or Behçet’s disease were excluded Parts of the samples with macroscopically and histologically inflammatory finding unique to Crohn’s disease were used This was a clinical study in which Osaka University received samples from Hyogo College of Medicine Hospital with All samples were obtained with written informed consent according to the principles of the declaration of Helsinki after approval by the ethics committee of the Osaka University Medical Hospital (Approval number: 10261) resected intestinal samples were washed in PBS to remove feces The mucus surface was wiped with a scalpel blade and the exposed lower mucus was again wiped with a new scalpel blade to obtain mucus samples The intestinal samples after wiping off all the remaining mucus were put into HBSS containing 5 mM EDTA and incubated for 6 min under shaking conditions After removing muscle layer with Cooper scissors the mucosal layer was cut into small pieces and incubated in RPMI 1640 containing 4% fetal bovine serum (FBS) and 80 U/ml DNase I (Roche) for 60 min in a 37 °C shaking water bath The digested tissues were resuspended with HBSS containing 5 mM EDTA and passed through a 40 μm cell strainer The isolated cells were resuspended in 7 ml of 20% Percoll and 2 ml of 40% Percoll in a 15 ml tube Percoll gradient separation was performed at 500 × g for 30 min at 4 °C The LPCs were collected at the interface of the Percoll gradient and washed with PBS containing 4% FBS The cell suspension was stained with Fixable Viability Stain 450 (BD Horizon) these cells were stained with the lineage markers (FITC-conjugated anti-human CD3 (HIT3a and PerCP/Cy5.5-conjugated anti-human CD163 (GHI/61 BioLegend) Flow cytometry was performed using a FACS Aria II system (BD Biosciences) to collect CD14+CD11c+CD163low subset and CD14+CD11c+CD163high subset The sorted cell suspension and mucus samples collected in advance were lysed in BufferT1 (NucleoSpin Tissue XS MACHEREY-NAGEL) and incubated 10 min at 95 °C DNA was extracted using the NucleoSpin Tissue XS (MACHERRY-NAGEL) kit according to the kit protocol with the modification of adding 90 mg lysozyme (L6876 Sigma-Aldrich) and 12000 U achromopeptidase (TBL-1 Wako Pure Chemical) and incubating 90 min at 37 °C prior to the kit-specified proteinase K treatment of 30 min at 56 °C Only the samples that passed a minimum read depth of 3,000 were included in downstream statistical analyses The univariate differential abundance of OTUs was tested using a negative binomial noise model for the overdispersion and Poisson process intrinsic to this data 16S rRNA gene sequencing and microbiome data analysis were provided by Second Genome Twelve patients were included in this study: 9 males and 3 females Median age at the operation were 45.5 (range 20–67) years old Twelve macrophage samples from the CD14+CD11c+CD163low fraction 11 macrophage samples from the CD14+CD11c+CD163high fraction and 12 mucus samples met the read depth criteria mentioned above the mean sequencing depth of the CD14+CD11c+CD163low subset was 24,674 reads (range that of CD14+CD11c+CD163high subset was 23,721 reads (range Diversity and similarity of bacteria present in CD14+CD11c+ macrophage of lamina propria and mucus from Crohn’s disease patients (a) The OTU richness and Shannon Diversity Index of bacteria present in CD14+CD11c+ macrophage and mucus OTU richness represents the number of OTUs in each sample The Shannon Diversity Index takes into account the richness and evenness of OTUs within samples (b) Weighted principal coordinates analysis of microbiome samples of CD14+CD11c+ macrophage and mucus This analysis uses the sample-to-sample dissimilarity values to position the points relative to each other by maximizing the linear correlation between the dissimilarity values and the plot distances Next, weighted principal coordinates analysis was performed to evaluate the homology of each sample (Fig. 1b) Samples were separated into different clusters in general by CD14+CD11c+ macrophages and mucus by Axis 2 indicating that the similarity of the bacterial flora differed between mucus and CD14+CD11c+ macrophages Composition of the microbiome present in CD14+CD11c+ macrophage of lamina propria and mucus from Crohn’s disease patients The relative abundance ratios of each microorganism group are shown in different colors Diversity and similarity of bacteria present in CD14+CD11c+CD163low and CD14+CD11c+CD163high macrophage of lamina propria from Crohn’s disease patients (a) The OTU richness and Shannon Diversity Index of bacteria present in CD14+CD11c+CD163low and CD14+CD11c+CD163high macrophage (b) Weighted principal coordinates analysis of microbiome samples of CD14+CD11c+CD163low and CD14+CD11c+CD163high macrophage We performed weighted principal coordinates analysis and examined their homology, but no significant cluster formation was observed in either the CD14+CD11c+CD163low fraction or the CD14+CD11c+CD163high fraction (Fig. 3b) This study is the first report to comprehensively analyze the bacterial flora in macrophages in the human intestinal lamina propria of Crohn’s disease patients by NGS we showed that the bacterial flora of CD14+CD11c+ macrophages in intestinal lamina propria differed in alpha diversity and homology from that of mucus when OTUs with higher average read number between CD163low and CD163high subsets of CD14+CD11c+ macrophages were compared there were OTUs showing different bacterial concentrations or dilutions versus mucus which suggests bacteria may have different distributions depending on the subset of macrophages and there are reports that in fact macrophages derived from Crohn’s disease patients have reduced ability to treat intracellular bacteria further detailed analysis of the bacteria present inside the macrophage may be the key to clarifying the pathology of Crohn’s disease bacteria abundantly found in the macrophage samples from this study include those that may have resistance to digestion by macrophages and those that may have immunological responses in patients with Crohn’s disease It is also interesting that bacteria about which pathogenicity has not been reported are abundantly found inside macrophages We hope that more detailed phylogenetic and pathogenicity analysis will be verified for these bacteria the expression of genes that negatively regulate the MyD88-independent TLR signaling pathway and genes that positively regulate prostaglandin E synthesis were enhanced in CD14+CD11c+CD163low cells compared to CD14−CD11c+ cells the expression of genes related to the type 1 interferon signaling pathway was enhanced in Crohn’s disease CD14+CD11c+CD163low cells compared with normal CD14+CD11c+CD163low cells there is a possibility that CD14+CD11c+CD163low macrophages from Crohn’s disease patients may have enhanced activation by LPS stimulation in endosomes and bacteria involved in this activation process may be obtained this time there was no significant difference between CD163low and CD163high fractions of CD14+CD11c+ macrophages regarding the diversity and homology of intracellular bacterial flora some OTUs showed contradictory changes in concentration or dilution versus mucus between the CD163low and CD163high subsets so there may be differences in the ability of the macrophage subset to handle certain intracellular bacteria It remains to be verified whether the bacteria found in the Crohn’s disease intestinal CD14+CD11c+ macrophage in this time are actually different in terms of persistence in macrophages and inflammation induction to macrophages The present study did not consider comparison between macrophages in the normal intestinal tract and macrophages in non-inflamed part of intestine from Crohn’s disease patients By combining these analyses with this study’s results it may be possible to identify the bacterial flora that contribute to the pathology of Crohn’ s disease Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-gamma axis Human CD14+ macrophages in intestinal lamina propria exhibit potent antigen-presenting ability Increased Th17-inducing activity of CD14+ CD163 low myeloid cells in intestinal lamina propria of patients with Crohn’s disease Identification of a human intestinal myeloid cell subset that regulates gut homeostasis Adherent invasive Escherichia coli strains from patients with Crohn’s disease survive and replicate within macrophages without inducing host cell death Monocyte-derived macrophages from Crohn’s disease patients are impaired in the ability to control intracellular adherent-invasive Escherichia coli and exhibit disordered cytokine secretion profile Crohn’s disease-associated Escherichia coli survive in macrophages by suppressing NFκB signaling Microbial Population Differentials between Mucosal and Submucosal Intestinal Tissues in Advanced Crohn’s Disease of the Ileum Reduced Abundance of Butyrate-Producing Bacteria Species in the Fecal Microbial Community in Crohn’s Disease Interplay of host genetics and gut microbiota underlying the onset and clinical presentation of inflammatory bowel disease Analysis of endoscopic brush samples identified mucosa-associated dysbiosis in inflammatory bowel disease Innate Myeloid Cell Subset-Specific Gene Expression Patterns in the Human Colon are Altered in Crohn’s Disease Patients Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample UPARSE: highly accurate OTU sequences from microbial amplicon reads An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea A new method for non‐parametric multivariate analysis of variance Gut microbiota and IBD: causation or correlation Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock A clinician’s guide to microbiome analysis New IBD genetics: common pathways with other diseases Microbiota profile in new-onset pediatric Crohn’s disease: data from a non-Western population Comparison of the virulence potential of Acinetobacter strains from clinical and environmental sources Concordance in Anti-OmpC and Anti-I2 Indicate the Influence of Genetic Predisposition: Results of a European Study of Twins with Crohn’s Disease Contextual control of skin immunity and inflammation by Corynebacterium The killing of macrophages by Corynebacterium ulcerans TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta CD14 controls the LPS-induced endocytosis of Toll-like receptor 4 Plasma membrane Toll-like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon-β Mechanisms of Toll-like Receptor 4 Endocytosis Reveal a Common Immune-Evasion Strategy Used by Pathogenic and Commensal Bacteria Autocrine-paracrine prostaglandin E2 signaling restricts TLR4 internalization and TRIF signaling Download references This work was supported by JSPS KAKENHI Grant Numbers JP17K16546 (Y.S.) JP17K10631 (T.M.) and Research Grant of the Princess Takamatsu Cancer Research Fund (T.M.) We thank San Francisco Edit for editing a draft of this manuscript Department of Therapeutics for Inflammatory Bowel Diseases conceived the idea and executed the research contributed to the creation and tested the method contributed substantially to the review of the manuscript The other authors declare no competing interests Download citation DOI: https://doi.org/10.1038/s41598-020-59937-w Metrics details This article has been updated Management of high-grade T1 (HGT1) bladder cancer represents a major challenge We studied a treatment strategy according to substaging by depth of lamina propria invasion In this prospective observational cohort study patients received initial transurethral resection (TUR) Subjects with shallower lamina propria invasion (HGT1a) were followed without further surgery whereas subjects with HGT1b received a second TUR Association of clinical and histological features with outcomes (primary: progression; secondary: recurrence and cancer-specific survival) was assessed using Cox regression with 89 (44.5%) cases T1a and 111 (55.5%) T1b disease progression was observed in 31 (15.5%) and in univariate analysis and tumour size remained significant for progression the strategy of performing a second TUR only in T1b cases results in a global low progression rate of 15.5% Tumours deeply invading the lamina propria (HGT1b) showed a three-fold increase in risk of progression with HGT1b cases being thoroughly evaluated for cystectomy Inclusion in the TNM system should also be carefully considered most are non-muscle invasive (NMIBC); that is either non-invasive and confined to the mucosa (Ta and carcinoma in situ (CIS)) or invading the lamina propria (LP) but not yet the detrusor muscle (T1) identifying those cases of HGT1 suitable for organ preservation and differentiating them from those that will most benefit from a timely cystectomy is a major challenge and key to improving outcomes we showed that tumour size >3 cm and associated CIS predict positive findings in the first evaluation at 3 months and speculated that HGT1a cases can safely forgo a second TUR we present the mature results of the first 200 cases of initial HGT1 treated with this optimised approach as well as evaluating clinical and histological features as prognostic factors All patients received a postoperative dose of intravesical mitomycin-C thick-walled blood vessels deep in LP served as a landmark or alternatively location of MM in tumour-free tissue of the same TUR was used for reference clearly identifiable and disease-free muscularis propria were included in this study Patients with LP invasion (T1) but without muscularis propria in the specimen represent ∼10% of all T1 in our centre did not fulfill the requirements to enter this protocol and For all patients with positive findings at 3 months was taken according to disease status and the patient’s preference Fourteen percent cases did not receive immediate MitC post-TUR BCG maintenance schedule consisted of three weekly instillations of Oncotice at 3 months and every 6 months thereafter (for 2–3 years depending on tolerance) *According to physician advice and patients choice Abbreviations: BRB=bladder random biopsies; TUR=trans urethral resection The primary objective was the assessment of progression Progression was defined as occurrence of an invasive tumour at post-BCG TUR the development of muscle invasive or more advanced stage carcinoma Time to progression was defined as time from diagnosis of BC to the date when disease progression was observed or censored on the last known date alive without progression Recurrence was defined as the first evidence of any NMIBC detected on follow-up Time to recurrence was defined as time from diagnosis of BC to the date when disease recurrence was observed or censored at the last known date alive without disease recurrence Cancer-specific survival was defined as death from the same cancer or related causes The secondary objective was the assessment of prognostic factors Eleven clinical and pathological variables were prospectively documented to evaluate their capacity to predict events Kaplan–Meier analysis was used to summarise median time to recurrence and time to progression and CSS with the variables of interest was assessed using Cox regression models in univariate and multivariable models Two hundred primary urothelial HGT1 cases have been identified at TUR and have entered the present protocol. Median age at diagnosis was 71 years; 89.5% were males, with 89 (44.5%) cases being T1a and 111 (55.5%) T1b. Patients and tumour characteristics are shown in Table 1 classified by substaging The median follow-up period was 71 months (range: 5–107 months) The second TUR (performed only in T1b) was negative in 67.5% of cases and positive in the remaining 32.4% (11 invasive and 25 non-invasive; primary histology being persistent HGT1) Tolerance and patient dropout of BCG was not documented specifically within the study group but for the broader population of high-risk BC at our hospital ∼30% do not complete the first year Recurrence was observed in 57 (28.5%) patients and disease progression in 31 (15.5%) and one of unknown cause) and 18 underwent cystectomy (2 pT0 and 3 pT4; one cystectomy at another centre; and 3 presented with positive lymph nodes) Ten potential candidates for cystectomy received palliative treatment because they were considered ineligible due to high surgical and/or anaesthetic risk (A ) Association of substaging and time to progression (B) Association of tumour pattern and time to progression (C) Association of tumour size and time to progression (D) Association of CIS and time to progression The low number of deaths due to BC precluded analysis of predictors of CSS Five-year CSS was 99% for T1a and 95% for T1b Of the 13 patients who died due to BC (3 of local progression (no cystectomy) and 10 from metastatic BC 6 of which had undergone cystectomy) all except one were originally T1b the association of TIS and tumour size in deeply invasive cases (T1b) gave an HR for progression of 4.81 with 95% CI (2.07 11.20) (P=0.0003) for patients with T1b+CIS+>3 cm compared with the rest the proposed strategy of deferring a second TUR until after induction of BCG and doing so only in T1b cases results in a global low progression rate of 15.5% Cases in which tumours invaded the MM (T1b) had a three-fold increased risk of progression compared with those without MM invasion (T1a) even though the latter group was treated more conservatively Our report goes on to confirm the role of CIS and tumour pattern as prognostic factors for worse outcomes in this group of high-risk non-muscle invasive BC Given the public nature of our health-care system we chose to differentially allocate patients to treatment using a more aggressive approach for T1b tumours A head-to-head comparison among these approaches in a large data set unfortunately is unavailable to date the fact that substaging remained a robust prognostic factor in our series (three-fold increase risk of progression in the adjusted multivariable model and five-fold increase in the univariate model) even though patients with HGT1a underwent a more conservative approach demonstrates the magnitude of negative impact associated with depth of invasion but the limitations mentioned previously should also be taken into account Mitomycin-C might have only played a minor role given that no impact on progression has been demonstrated with this agent in all randomised published trials Also the proposed approach in delaying the second TUR makes it impossible to accurately differentiate between true understaging and early invasive recurrence the findings of our study suggest that larger tumours and those with a solid pattern are associated with a worse outcome in HGT1 are consistent with the relevance of these parameters in the broader NMIBC population Combining these factors we observed that patients with T1b tumours that were larger than 3 cm and associated with CIS were 4.8 times more likely to progress than the rest our report proposes a novel approach for HGT1 BC based on the selection of patients who will most benefit from a second TUR specifically those with deep invasion of the MM (T1b) The main clinical implication of separately identifying almost half of HGT1 cases with T1a substaging as having a low (4%) progression rate is that they can be spared a second endoscopic procedure with no harm More accurate risk stratification incorporating depth of invasion and tumour size would enable improved resource allocation for reTUR It would also help identify optimal candidates for prompt cystectomy namely T1b tumours larger than 3 cm and with associated CIS If the one in five HGT1 patients who will eventually develop a muscle invasive BC could be identified after diagnostic TUR then cystectomy could be offered more selectively overtreatment and the morbidity and anxiety of undergoing additional procedures could be spared in patients with more indolent prognosis Our results further support the notion that the ‘rule of thirds for HGT1’ (i.e. one-third will require deferred cystectomy and one-third will die of disease) is currently outdated Based on the prognostic impact of T1 substaging in the present and previous studies we believe there is an argument for evolution of the current TNM classification Together with tumour size and the detection of CIS an updated classification would help to identify the patients most suitable for cystectomy Randomised control trials to evaluate the positive impact of this subclassification of tumours would also be beneficial With this contemporary approach for the management of HGT1 BC in which the strategy of complete initial TUR+MitC+BCG is only followed by a second TUR in T1b cases low recurrence and progression rates (28.5% and 15.5% tumour size and associated CIS are also prognostic factors for progression which should be contemplated in future strategies and aid in the selection of patients for cystectomy This paper was modified 12 months after initial publication to switch to Creative Commons licence terms American Urological Association (2007) Guidelines for the management of non-muscle invasive bladder cancer (stages Ta Roghmann F PROMETRICS 2011 research group (2013) Prediction of 90-day mortality after radical cystectomy for bladder cancer in a Prospective European Multicenter Cohort Rouprêt M (2013) EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2013 Hartmann A (2011) Substaging by estimating the size of invasive tumour can improve risk stratification in pT1 urothelial bladder cancer-evaluation of a large hospital-based single-centre series Wiegel T (2013) Phase 3 study of adjuvant radiotherapy versus wait and see in pT3 prostate cancer: impact of pathology review on analysis Pan CC (2012) Prognostic significance in substaging ofT1 urinary bladder urothelial carcinoma on transurethral resection Bostwick DG (1999) Predicting cancer progression in patients with stage T1 bladder carcinoma Hair M (2007) Consistency of microstaging pT1 bladder transitional cell carcinoma Burger M (2007) Bladder sparing approach for initial T1G3 bladder cancer: Do multifocality size of tumor or concomitant carcinoma in situ matter Ruffion A (2013) Microarray gene expression profiling and analysis of bladder cancer supports the sub classification of the T1 tumours in T1a and T1b stages Zorlu F (2010) Impact of routine second transurethral resection on the long-term outcome of patients with newly diagnosed pT1 urothelial carcinoma with respect to recurrence and disease-specific survival: a prospective randomised clinical trial Sesterhenn IA (2006) World Health Organization Classification of Tumors Tumors of the Urinary System and Genital Organs Bray F (2013) Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012 Palou J (2014) Prognostic factors and risk groups in T1G3 non-muscle-invasive bladder cancer patients initially treated with Bacillus Calmette-Guérin: results of a retrospective multicenter study of 2451 patients Dalbagni G (2007) Can restaging transurethral resection of T1 bladder cancer select patients for immediate cystectomy Oosterlinck W (2004) A second-look TUR in T1 transitional cell carcinoma: why Debruyne FM (1994) Should random urothelial biopsies be taken from patients with primary superficial bladder cancer Members of the Dutch South-East Co-Operative Urological Group Bellmunt J (2014) Improving selection criteria for early cystectomy in high-grade T1 bladder cancer: A meta-analysis of 15,215 patients Vicente-Rodriguez J (2000) Multivariate analysis of the prognostic factors of primary superficial bladder cancer Morote J (2010) Risk factors for positive findings in patients with high-grade T1 bladder cancer treated with transurethral resection of bladder tumour (TUR) and bacille Calmette-Guerin therapy and the decision for a repeat TUR Orsola I (2005) Initial high-grade T1 urothelial cell carcinoma: feasibility and prognostic significance of lamina propria invasion microstaging (T1a/b/c) in BCG-treated and BCG-non-treated patients Villavicencio H (2012) Female gender and carcinoma in situ in the prostatic urethra are prognostic factors for recurrence and disease-specific mortality in T1G3 bladder cancer patients treated with bacillus Calmette-Guérin Sternberg CN (2002) Long-term follow-up of G3T1 transitional cell carcinoma of the bladder treated with intravesical bacille Calmette-Guérin: 18-year experience Dalbagni G (2007) Treatment paradigm shift may improve survival of patients with high risk superficial bladder cancer Pfister C Comité de Cancérologie de l'Association Française d'Urologie (2013) Prognostic interest in discriminating muscularis mucosa invasion (T1a vs T1b) in nonmuscle invasive bladder carcinoma: French National Multicenter Study with central pathology review Herr HW (2014) The effect of restaging transurethral resection on recurrence and progression rates in patients with nonmuscle invasive bladder cancer treated with intravesical bacillus Calmette-Guérin Hollenbeck BK (2011) Use of restaging bladder tumor resection for bladder cancer among Medicare beneficiaries Kurth K (2006) Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: a combined analysis of 2596 patients from seven EORTC trials Alfred Witjes J (2011) Long-term cancer-specific survival in patients with high-risk non-muscle-invasive bladder cancer and tumour progression: a systematic review van der Kwast TH (2005) A new system for substaging pT1 papillary bladder cancer: a prognostic evaluation Zlotta AR (2012) Prognostic value of molecular markers sub-stage and European Organisation for the research and treatment of cancer risk scores in primary T1 bladder cancer Zlotta AR (2012) A new and highly prognostic system to discern T1 bladder cancer substage Zaak D (2014) Clinical and cost effectiveness of hexaminolevulinate-guided blue-light cystoscopy: evidence review and updated expert recommendations Eur Urol e-pub ahead of print 4 July 2014 doi:10.1016/j.eururo.2014.06.037 Shariat SF (2013) Accuracy of the EORTC risk tables and of the CUETO scoring model to predict outcomes in non-muscle-invasive urothelial carcinoma of the bladder Download references Dana-Farber/Brigham and Women’s Hospital Cancer Center Departments of Biostatistics and Computational Biology University of Massachusetts Medical School The authors declare no conflict of interest This work is published under the standard license to publish agreement After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License From twelve months after its original publication this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License visit http://creativecommons.org/licenses/by-nc-sa/3.0/ Download citation Metrics details Here we show that adenosine 5′-triphosphate (ATP) that can be derived from commensal bacteria activates a unique subset of lamina propria cells leading to the differentiation of TH17 cells Germ-free mice exhibit much lower concentrations of luminal ATP accompanied by fewer lamina propria TH17 cells Systemic or rectal administration of ATP into these germ-free mice results in a marked increase in the number of lamina propria TH17 cells A CD70highCD11clow subset of the lamina propria cells expresses TH17-prone molecules IL-23p19 and transforming-growth-factor-β-activating integrin-αV and -β8 and preferentially induces TH17 differentiation of co-cultured naive CD4+ T cells The critical role of ATP is further underscored by the observation that administration of ATP exacerbates a T-cell-mediated colitis model with enhanced TH17 differentiation These observations highlight the importance of commensal bacteria and ATP for TH17 differentiation in health and disease and offer an explanation of why TH17 cells specifically present in the intestinal lamina propria IL-17 family cytokines and the expanding diversity of effector T cell lineages TH-17 cells in the circle of immunity and autoimmunity The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides Essential autocrine regulation by IL-21 in the generation of inflammatory T cells IL-21 initiates an alternative pathway to induce proinflammatory TH17 cells IL-6 programs TH-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways The genetics of inflammatory bowel disease TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells Transforming growth factor-β induces development of the TH17 lineage Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells a ‘double agent’ in the immune pathology war Colonic patches direct the cross-talk between systemic compartments and large intestine independently of innate immunity Intestinal IgA synthesis: regulation of front-line body defences Extracellular nucleotide signaling by P2 receptors inhibits IL-12 and enhances IL-23 expression in human dendritic cells: a novel role for the cAMP pathway P2X receptors as cell-surface ATP sensors in health and disease Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells ATP level variations in heterotrophic bacteria during attachment on hydrophilic and hydrophobic surfaces Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-β and retinoic acid-dependent mechanism CD27 is required for generation and long-term maintenance of T cell immunity Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses Inflammatory bowel disease in C.B-17 scid mice reconstituted with the CD45RBhigh subset of CD4+ T cells A microbial symbiosis factor prevents intestinal inflammatory disease Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway Colonic epithelial functional phenotype varies with type and phase of experimental colitis Download references This work was supported by Grants-in-Aid from the Ministry of Education the Osaka Foundation for the Promotion of Clinical Immunology Senri Life Science Foundation and the Naito Foundation planned experiments and analyses and wrote the paper; K.A Koji Atarashi and Junichi Nishimura: These authors contributed equally to this work Yakult Central Institute for Microbiological Research Tohoku University Graduate School of Medicine Department of Cell Physiology and Pharmacology The file contains Supplementary Figures 1-12 with Legends Download citation a layer of cells forming part of the mucous membrane boasts a complicated mix of cell populations including the selective presence of TH17 or T helper 17 cells the subset of T helpers that produces interleukin 17 A study in mice now shows that commensal bacteria activate a unique subset of intestinal dendritic cells to induce interleukin-6 production and TGF-beta activation thereby promoting the local differentiation of TH17 cells It is the ATP that the bacteria produce that promotes this effect This finding highlights the importance of commensal bacteria and ATP in immuno-logical diseases and may help in determining the mechanisms by which aberrant TH17 cell responses result in immune disorders including inflammatory bowel diseases Metrics details The mechanisms underlying bladder contractile disorders such as overactive bladder are not fully understood and there is limited understanding of the receptor systems modulating spontaneous bladder contractions We investigated the potential for histamine to have a role in mediating contractility of the urothelium with lamina propria (U&LP) or detrusor via the H1-H4 histamine receptor subtypes Isolated strips of porcine U&LP or detrusor smooth muscle were mounted in gassed Krebs-bicarbonate solution and responses to histamine obtained in the absence and presence of selective receptor antagonists The presence of histamine increases the frequency of U&LP spontaneous phasic contractions and baseline tensions fexofenadine and cyproheptadine were effective at inhibiting contractile responses Cimetidine (H2-antagonist) enhanced increases in baseline tension in response histamine whereas amthamine (H2-agonist) induced relaxation Although thioperamide (H3/H4-antagonist) increased baseline tension responses to histamine selective H1/H2-receptor antagonism revealed no influence of these receptors fexofenadine and cyproheptadine were effective at inhibiting baseline tension increases in response to histamine Our findings provide evidence that histamine produces contractile responses both in the U&LP and detrusor via the H1-receptor and this response is significantly inhibited by activation of the H2-receptor in the U&LP but not the detrusor One alternative target involved in the pathogenesis of contractile dysfunctions such as OAB may be the inflammatory mediators released from mast cells at sites of inflammation the involvement of histamine is of great interest as it is the predominant mediator involved in the mechanisms of acute allergy and inflammation This study aims to investigate the effect of histamine on the contractility of U&LP and detrusor smooth muscle and identify the histamine receptor subtypes responsible for mediating contractile responses Adjacent pieces of U&LP and detrusor were suspended in pairs in 10 mL organ baths (Labglass Australia) containing Krebs-bicarbonate solution (NaCl 118.4 mM KH2PO4 1.18 mM and D-glucose 11.7 mM) at 37 °C and perfused with a gas mixture of 95% oxygen and 5% carbon dioxide Tissue strips were washed three times and tension readjusted to approximately 2 g where this was maintained as the baseline tension A single dose of agonist was added to the tissue both in the presence and absence of antagonists and concentrations selective for each receptor subtype determined using affinity values appearing in the literature Tissue strips that were exposed to antagonists and tissues treated with vehicle control were incubated for 30 minutes to allow full equilibration with the receptor Baseline tension and the frequency and amplitude of spontaneous phasic contractions were recorded simultaneously using isometric force transducers (MCT050/D Australia) on a Powerlab system using Labchart v7 software (ADInstruments) frequency was expressed as the number of spontaneous phasic contractions per minute (cycles/min) and amplitude was expressed in grams (lowest point of spontaneous phasic contraction to peak) Data was graphed and analysed using GraphPad Prism version 7.00 for Windows (GraphPad Software La Jolla California USA) and results shown as the mean change ± SEM Responses between control and experimental tissues were compared using Student’s t-test with p < 0.05 considered significant USA) was applied to the tissue after a 30-minute equilibration period amplitude and spontaneous phasic contractions of U&LP were measured before the application of the agonist and 2 minutes after The baseline tension for detrusor was measured at the same time-points as used for U&LP The H1 receptor antagonists pyrilamine (30 nM the H2 receptor antagonist cimetidine (1 µM USA) and the dual H3&H4 receptor antagonist thioperamide (1 µM After 30-minute incubation with antagonists a single dose of histamine (100 µM) was applied to the tissues Histamine responses to pyrilamine (30 nM) treated tissues were compared with a combination of pyrilamine (30 nM) and cimetidine (1 µM) treated tissues; cimetidine (1 µM) treated tissues were compared with a combination of pyrilamine (30 nM) and cimetidine (1 µM) treated tissues; combination of pyrilamine (30 nM) and cimetidine (1 µM) treated tissues were compared with a combination of pyrilamine (30 nM) cimetidine (1 µM) and thioperamide (1 µM) treated tissues In the absence of histamine or any antagonists strips of urothelium/lamina propria (U&LP) developed spontaneous phasic contractions at a frequency of 3.56 ± 0.13 cycles per min−1 (cpm) with an amplitude of 0.46 ± 0.03 g (n = 48) When histamine (100 µM) was added to the tissues U&LP baseline tension increased by 0.98 ± 0.13 g (p < 0.001 frequency of spontaneous phasic contractions increased by 1.32 ± 0.25 cpm (p < 0.001 n = 48) and amplitude decreased by 22.9 ± 5.8% (p < 0.001 n = 48) during the peak response to histamine (100 µM) frequency and amplitude of spontaneous phasic contractions in response to histamine was not affected by the presence of the muscarinic receptor antagonist atropine (1 µM When H2 agonist amthamine (1 μM) was added to strips of U&LP that were left to equilibrate at a tension of approximately 2 g in the absence of any agonists and antagonists resting baseline tension decreased by 0.13 ± 0.02 g (p < 0.01 n = 12) 5 minutes after the addition of agonist and by 0.22 ± 0.04 g after 20 minutes (p < 0.01 an increase in baseline tension of 0.99 ± 0.27 g (p < 0.001 n = 48) was observed in response to histamine (100 µM) The addition of H2 agonist amthamine (1 µM) had no influence on the baseline tension in detrusor preparations U&LP responses to histamine (100 µM) as control (left) and in the presence of histamine receptor antagonists pyrilamine None of the antagonists had any influence on the amplitude of phasic spontaneous contractions in response to histamine (100 µM) U&LP responses to histamine (100 µM) in the presence of more than one histamine receptor antagonist To isolate the potential influence of H3 and H4 receptors thioperamide (1 µM) was added to tissues pre-treated with pyrilamine (30 nM) and cimetidine (1 µM Responses to histamine (100 µM) in the presence of thioperamide (1 µM) exhibited a small but observable increase in baseline tension Detrusor responses to histamine (100 µM) as a control (left) and in the presence of pyrilamine (right is not involved in the contractile response to histamine 4) H2 receptor activation stimulates relaxation of U&LP in response to histamine our study is the first to identify and compare the responses to histamine in two distinct layers of the bladder that differ in structure and function: U&LP and detrusor U&LP is comprised of several layers of epithelial cells (urothelium) that line the lumen of the bladder and the underlying connective tissue layer (lamina propria) whereas detrusor is made of smooth muscle cells The role of the H2 receptor was established through agonist and antagonist studies which is a potent and highly selective H2 agonist a relaxation of baseline tension was observed in U&LP samples caused increases in baseline tension in U&LP however had no effect on the frequency of spontaneous phasic contractions The lack of responses to either the H2 antagonist cimetidine or agonist amthamine in detrusor preparation indicates that the H2 receptor subtype the lack of any inhibition from atropine to the histamine response or receptor antagonism in this study suggests no involvement of muscarinic receptors Any small increases in baseline tension observed in the presence of the H1 antagonist pyrilamine as the concentrations were relatively small and contractions were mediated by H1 receptors in both tissues As epithelial cells of the urothelium have no contractile properties the cells most likely involved in the contraction observed in organ bath experiments from U&LP tissue strips are the myofibroblasts or muscularis mucosa cells found in the lamina propria Given the size and distribution of these cells within the connective tissue Histamine influences the contractile activity of both U&LP and detrusor of the porcine urinary bladder It was determined that stimulation of the H1 receptor results in contractions in both urothelium with lamina propria and detrusor tissue samples Activation of the H2 receptor inhibits the H1-mediated contractions in the urothelium with lamina propria but not the detrusor with H3 and H4 receptors having no functional role in bladder contractility This study presents the H1 and H2 receptors as potential targets for future treatments for overactive bladder and other bladder contractile disorders Moro, C., Uchiyama, J. & Chess-Williams, R. Urothelial/lamina propria spontaneous activity and the role of M3 muscarinic receptors in mediating rate responses to stretch and carbachol. Urology 78(1442), e1449–1415, https://doi.org/10.1016/j.urology.2011.08.039 (2011) Moro, C., Tajouri, L. & Chess-Williams, R. Adrenoceptor function and expression in bladder urothelium and lamina propria. Urology 81(211), e211–217, https://doi.org/10.1016/j.urology.2012.09.011 (2013) Moro, C., Leeds, C. & Chess-Williams, R. Contractile activity of the bladder urothelium/lamina propria and its regulation by nitric oxide. European journal of pharmacology 674, 445–449, https://doi.org/10.1016/j.ejphar.2011.11.020 (2012) Andersson, K. E., Fry, C., Panicker, J. & Rademakers, K. Which molecular targets do we need to focus on to improve lower urinary tract dysfunction? ICI-RS 2017. Neurourology and urodynamics 37, S117–s126, https://doi.org/10.1002/nau.23516 (2018) Organization of the sacral parasympathetic reflex pathways to the urinary bladder and large intestine Muscarinic receptors of the urinary bladder: detrusor Chapple, C. R. et al. The effects of antimuscarinic treatments in overactive bladder: an update of a systematic review and meta-analysis. Eur Urol 54, 543–562, https://doi.org/10.1016/j.eururo.2008.06.047 (2008) Basra, R. K. et al. A review of adherence to drug therapy in patients with overactive bladder. BJU international 102, 774–779, https://doi.org/10.1111/j.1464-410X.2008.07769.x (2008) Signaling and regulation of G protein-coupled receptors in airway smooth muscle Amin, K. The role of mast cells in allergic inflammation. Respiratory Medicine 106, 9–14, https://doi.org/10.1016/j.rmed.2011.09.007 (2012) Subtypes of bladder mast cells in interstitial cystitis International journal of urology: official journal of the Japanese Urological Association 7 Christmas, T. J. & Rode, J. Characteristics of Mast Cells in Normal Bladder, Bacterial Cystitis and Interstitial Cystitis. British Journal of Urology 68, 473–478, https://doi.org/10.1111/j.1464-410X.1991.tb15388.x (1991) Gamper, M., Regauer, S., Welter, J., Eberhard, J. & Viereck, V. Are Mast Cells Still Good Biomarkers for Bladder Pain Syndrome/Interstitial Cystitis? The Journal of urology 193, 1994–2000, https://doi.org/10.1016/j.juro.2015.01.036 (2015) The role of the mast cell in interstitial cystitis Tyagi, P. et al. Urine cytokines suggest an inflammatory response in the overactive bladder: a pilot study. International urology and nephrology 42, 629–635, https://doi.org/10.1007/s11255-009-9647-5 (2010) Parsons, M. E. & Ganellin, C. R. Histamine and its receptors. British journal of pharmacology 147(Suppl 1), S127–135, https://doi.org/10.1038/sj.bjp.0706440 (2006) Neuhaus, J., Oberbach, A., Schwalenberg, T. & Stolzenburg, J. U. Cultured smooth muscle cells of the human vesical sphincter are more sensitive to histamine than are detrusor smooth muscle cells. Urology 67, 1086–1092, https://doi.org/10.1016/j.urology.2005.11.031 (2006) Histamine receptors in urethrovesical smooth muscle Kondo, M., Taniyama, K. & Tanaka, C. Histamine H1-receptors in the guinea-pig urinary bladder. European Journal of Pharmacology 114, 89–92, https://doi.org/10.1016/0014-2999(85)90526-6 (1985) Poli, E., Coruzzi, G. & Bertaccini, G. Pre- and postjunctional effects of histamine on the guinea pig urinary bladder: Evidence for heterogeneity in the H1-receptor population? Agents and Actions 23, 241–243, https://doi.org/10.1007/BF02142552 (1988) Characterization of the rabbit detrusor response to histamine through pharmacologic antagonism Acetylcholine mediation of the contractile response to histamine in human bladder detrusor muscle Potentiation of purinergic neurotransmission in guinea pig urinary bladder by histamine Andersson, K. E. & Wein, A. J. Pharmacology of the lower urinary tract: basis for current and future treatments of urinary incontinence. Pharmacological reviews 56, 581–631, https://doi.org/10.1124/pr.56.4.4 (2004) Palea, S., Artibani, W., Ostardo, E., Trist, D. G. & Pietra, C. Evidence for Purinergic Neurotransmission in Human Urinary Bladder Affected by Interstitial Cystitis. The Journal of urology 150, 2007–2012, https://doi.org/10.1016/S0022-5347(17)35955-4 (1993) Comparison of the urinary bladder base and detrusor to cholinergic and histaminergic receptor activation in the rabbit Download references This research was supported by the Australian Bladder Foundation managed by the Continence Foundation of Australia ZS was supported by an Australian Government Research Training Program Scholarship were all equally responsible for the study design Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-019-40384-1 Metrics details Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa) This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations After different steps of digestion of the tissue using collagenase the resulting cells are purified using Percoll gradient The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosis the isolated cells can be used for further investigations like comparative studies of mRNA expression cell-proliferation assay or protein analysis This protocol can be completed within 6–7 h T cell differentiation antigens on lymphocytes in the human intestinal lamina propria Interleukin-2- and interferon-gamma-secreting T cells in normal and diseased human intestinal mucosa Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages Isolation and functional characterization of human intestinal mucosal lymphoid cells Gut mucosal lymphocytes in inflammatory bowel disease: isolation and preliminary functional characterization Preparation and purification of lymphocytes from the epithelium and lamina propria of murine small intestine Improved procedure for the isolation of functionally active lymphoid cells from the murine intestine Antibodies to interleukin 12 abrogate established experimental colitis in mice Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice Treatment of T cell-dependent experimental colitis in SCID mice by local administration of an adenovirus expressing IL-18 antisense mRNA Establishment of long-term primary cultures of human small and large intestinal epithelial cells Analysis of apoptosis by propidium iodide staining and flow cytometry Download references Download citation DOI: https://doi.org/10.1038/nprot.2007.315 Sorry, a shareable link is not currently available for this article. 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Volume 11 - 2017 | https://doi.org/10.3389/fnsys.2017.00087 This article is part of the Research TopicVisceral Pain: Recent Knowledge and AdvancementView all 13 articles The lamina propria contains a dense network of cells that may play a role in bladder function by modulating communication between urothelium nerve fibers and smooth muscle or acting as pacemakers Transient receptor potential vanilloid 4 (TRPV4) channels allow cation influx and may be involved in sensing stretch or chemical irritation in urinary bladder Urothelium was removed from rats (P0-Adult) and loaded with a Ca2+ fluorescent dye (Fluo-2 AM leak resistant or Cal 520) for 90 min (35–37°C) to measure Ca2+ events Ca2+ events were recorded for a period of 60 seconds (s) in control and after drug treatment A heterogeneous network of cells was identified at the interface of the urothelium and lamina propria of postnatal rat pups stellate with numerous projections) and expressing platelet-derived growth factor receptor alpha (PDGFRα)- and TRPV4-immunoreactivity (IR) Ca2+ transients occurred at a slow frequency with an average interval of 30 ± 8.6 s Waveform analyses of Ca2+ transients in cells in the lamina propria network revealed long duration Ca2+ events with slow upstrokes We observed slow propagating waves of activity in the lamina propria network that displayed varying degrees of coupling the number of cells with Ca2+ events and the integrated Ca2+ activity corresponding to propagation of activity among cells in the lamina propria network ATP (1 μM) perfusion increased the number of cells in the lamina propria exhibiting Ca2+ events and produced tightly coupled network activity These findings indicate that ATP and TRPV4 can activate cells in the laminar propria network leading to the appearance of organized propagating wavefronts Although the lamina propria is made up of a heterogeneous population of cells types certain cells may be important modulators of neural activity and form a communication link between the urothelium and detrusor; the importance of which may depend upon age or presence of pathology we identified a heterogeneous network of cells at the urothelial-lamina propria interface of rat pups with diverse morphology and numerous processes that exhibit PDGFRα- and TRPV4-immunoreactivity Cells in this lamina propria network express spontaneous Ca2+ events mediated through the release of Ca2+ from internal stores and also respond to TRPV4 agonists with changes in Ca2+ signaling Application of exogenous ATP evoked Ca2+ waves that propagate through the lamina propria cell network demonstrating a functional syncytium that may provide a critical communication link between the urothelium and the detrusor smooth muscle to convey sensory information or to affect detrusor contractility Quebec) of both sexes and various postnatal (P) ages (P0-Adult) were used in these studies The University of Vermont Institutional Animal Care and Use Committee approved all experimental protocols involving animal use Animal care was under the supervision of the University of Vermont's Office of Animal Care Management in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and National Institutes of Health guidelines All efforts were made to minimize the potential for animal pain Tissues were harvested after euthanasia by decapitation or isoflurane overdose followed by thoracotomy Urinary bladders were removed from adult rats (n = 13) and rat pups (n = 40 P ≤ 21) of both sexes and placed in cold HEPES solution consisting of (mM): 134 NaCl 10 glucose and adjusted to pH 7.4 with NaOH The urothelium was removed from the detrusor and carefully cleaned of lamina propria with sharpened forceps until all visible lamina propria was removed The cleaned urothelial sheet was placed in buffered 4% paraformaldehyde (pH 7.4) for immunohistochemistry or placed in a special chamber (PSS) for Ca2+ imaging studies Urothelial tissue sheets were examined under an Olympus fluorescence microscope Cy3 was visualized with a filter with an excitation range of 560–596 nm and an emission range of 610–655 nm Digital images were obtained using a charge-coupled device camera (MagnaFire SP NH) and LG-3 frame grabber (Optical Analysis) Additional images were collected with a Nikon C2 confocal system with a Plan Apo 40X DIC H oil objective with 1.0 NA Samples were scanned at 1024X1024 resolution using the 488 or 561 nm laser lines with green and red detectors collecting signal at 525/36 or 605/52 nm the urothelial-lamina propria sheets were loaded for 90 min (37°C) with a Ca2+ sensitive fluorescent dye Texas) (10 μM) or Cal 520 (AAT Bioquest California) + pluronic acid (2.5 mg/ml) in HEPES solution All experiments were conducted in physiological saline solution (PSS) (35–37°C) consisting of (mM): 119 NaCl 7 glucose and constantly bubbled with Biological Gas (5% CO2) to maintain pH at 7.4 The urothelium was visualized with a 60X water immersion (NA 1.2) fluorescent objective Images were collected with a Noran Oz laser scanning confocal microscope or with a Yokogawa CSU-W1 spinning disk confocal microscope housed in the Imaging/Physiology Core (Larner College of Medicine at The University of Vermont) and the emitted fluorescence collected at >500 nm Imaging fields were 133 × 133 μm (512 × 512 pixel) Ca2+ events were initially visualized offline using software developed in our laboratory by Dr For some experiments Tetramethylrhodamine (TRITC 1 mg/ml; ThermoFischer Scientific) was incubated with fluo-2 AM leak resistant Ca2+ sensitive dye for 90 min (35–37°C) TRITC was taken up by urothelial cells and provided contrast to Fluo-2 loaded cells The tissue was visualized using a Yokogawa CSU-W1 spinning disk confocal microscope housed in the Imaging/Physiology Core TRITC was excited at 561 nm and emitted fluorescence was collected using a 525/50 filter This equates to an imaging signal to noise ratio of >14 dB Particles less than 15 pixels (1 μm2 @ 60x: 9 μm2 @ 20x) in total area were filtered out and calcium transient particle (PTCL) files were created the spatial overlap between particles on successive frames was tabulated then an existence filter was applied that preserved particles that had a continuous spatial overlap for more than 0.25 s Ca2+ transient particles that met or exceeded the above conditions were considered to be resolved Ca2+ transients Initiation sites of Ca2+ transients were flagged by choosing the first appearance of a resolved Ca2+ transient The refined Ca2+ transient particles were summed throughout the movie to create a prevalence map of activity (as % of recording time a resolvable Ca2+ event was present) Ca2+ transient prevalence maps were thresholded (>0.5% activity during recording) and bitMasks created from which intensity traces were calculated in the original recording The interval, duration, amplitude, and rise/decay times of lamina propria cells Ca2+ transients were calculated from intensity traces calculated within cell bitMasks. To measure the overall effect of drugs on cells in the lamina propria network, we integrated the area of resolvable Ca2+ transient PTCLs over the course of the recording period (see Figures 4–6) The pattern of Ca2+ transients in lamina propria cells ranged from apparently random to well demarcated wavefronts To quantify the degree of coordination of Ca2+ activity in lamina propria cells the temporal order of every resolvable Ca2+ transient in cells within the FOV was determined (ii) distance of separation and (iii) angle of between sites was calculated Any bias in the number of “next-to-fire” angles in a particular direction demonstrate a non-random pattern of activation and was used to gauge relationships between Ca2+ transients within cells in the lamina propria syncytium Random patterns of activation have a little bias (equal representation of all angles between cells in the activation sequence) where as a greater degree of bias (maximum number of events at a particular angle range divided by the minimum number of events at a particular angle range: 360° range; 20° bin size) are indicative of a well-formed wave front Images were imported into Photoshop 7.0 (Adobe Systems CA) or Powerpoint (Microsoft PowerPoint for Mac 2011 Microsoft Corporation) where groups of mages were assembled and labeled Values are expressed as mean ± S.E.M Statistical comparisons between groups were made using one-way analysis of variance (ANOVA) When F ratios exceeded the critical value (p ≤ 0.05) the Dunnett's post-hoc test was used to compare groups GSK1016790 [N-((1S)-1-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide] and GSK2193874 [3-([1,4′-Bipiperidin]-1′-ylmethyl)-7-bromo-N-(1-phenylcyclopropyl)-2-[3-(trifluoromethyl)phenyl]-4-quinolinecarboxamide] were purchased from Sigma (St and superfused over the urothelial preparation Tissue preparations (92.3%) isolated from rat pups aged >P25 exhibited few cells in the urothelial-lamina propria junction Because cells in the urothelial-lamina junction that loaded with the calcium reporter dyes were most numerous from rat pups aged ≤ P21 recordings and analyses of spontaneous and evoked calcium events were restricted to this postnatal period Whole mounts of urinary bladder tissue were prepared at the urothelial-lamina junction and contained a dense network of capillaries small blood vessels and cells from rat pups aged ≤ P21 blood vessels and cells were loaded with the fluorescent dye (Fluo-2 Tetramethylrhodamine (TRITC; red) was picked up by urothelial cells and provided contrast to Fluo-2 loaded cells (A,B) The cellular network in the lamina propria was heterogeneous with numerous small round stellate or spindle shaped cells (10–20 μm) with multiple processes projecting from the soma in rat pups aged P ≤ 21 Numerous cells in the lamina propria exhibited PDGFRα-immunoreactivity (IR) whereas fewer cells exhibited TRPV4-IR (C,D) a dense capillary network was in close proximity to the network of cells expressing PDGFRα- and TRPV4-IR (E) Capillaries were isolated using their signature long tubular shape in the PTCL analysis software and recolored red to distinguish them from lamina propria cells 3-D images of the capillary network and the lamina propria cell network at different angles of rotation (E,i,ii) Calibration bar in (B) represents 10 μm (A,B) Calibration bar in (E) represents 5 μm (A) Prevalence maps of Ca2+ transient PTCLs were thresholded (B) to create ROI bitMasks (C) to measure PTCL area (D) and Ca2+ induced fluorescence (E) from the original recording (F) Interval histogram shows slow frequency of Ca2+ transients in lamina propria cells (average interval = 30 ± 8.6 s; n = 4 (G) Duration of Ca2+ transients in lamina propria cells was 15.6 ± 2.4 s (n = 56 cells from n = 7 different experiments) (H) Examples of Ca2+ transients in 15 lamina propria cells Ca2+ transients had long durations (5–10 s) and a near symmetrical upstroke and downstroke phases Ca2+ transient waveform characteristics The propagating Ca2+ waves illustrate the ability of the lamina propria network to act as a functional syncytium Example of tightly and loosely coupled Ca2+ network activity in a continuous recording from the lamina propria cellular syncytium (A) Plot of the firing characteristics of lamina propria cells showing (i) the interval (blue dots) (ii) the distance of separation (green triangles) and (iii) the angle (orange squares) between lamina propria cells in the forward firing sequence the lowest values are zero; for separation distance (B) Spatio-temporal map of lamina propria cells firing showing 4 network firing events denoted by red (C) Histograms of the frequency of angles between next-to-fire cells (forward sequence) during the 4 network firing events The first and last network firing events show a high degree organization (B: red and purple overlays) with a cluster of small delays (A: blue dots) between firing of cells and a strong bias for next-to-fire cells to occur at specific angles (C: red & purple lines; 160 & 340°) corresponding to the direction of the wavefront (90° to the propagation direction) The second network event (B: green overlay) does not show a tightly organized network firing sequence and has variable delays (A: blue dots) with little bias in the angle between next-to-fire cells (C: green line) The third event (B: blue overlay) in which the wavefront propagates in the opposite direction shows partially coupled network activity with some bias in the angle between next-to-fire cells but does not involve all of the activateable cells in the field of view (FOV) Composite prevalence maps of (A) lamina propria cell control activity (n = 2 @ 20x; inset n = 2 @ 60x) and (B) lamina propria cell activity after the addition of GSK2193874 (1 μM) The addition of GSK2193874 did not appreciably alter the number of cells displaying Ca2+ transients (C) or the duration (B) of Ca2+ transients (D) Traces from individual experiments showing integrated Ca2+ activity in control conditions (green lines) and after the addition of GSK2193874 (1 μM: red lines) To identify the potential sources of Ca2+ underlying Ca2+ transients we used the sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitor cyclopiazonic acid (CPA; 30 μM). CPA significantly (p ≤ 0.01) reduced the overall duration of cell activation and the number of cells exhibiting Ca2+ events in the lamina propria network suggesting that Ca2+ transients are dependent on release of Ca2+ from internal stores (Figures 6A–D) Composite prevalence maps of (A) lamina propria cell control activity (n = 3 @ 60x) and (B) lamina propria cell activity after the addition of CPA 30 μM) The addition of CPA significantly reduced the overall time lamina propria cells were active and significantly reduced the number of cells in the FOV that had active Ca2+ events (C: * (D) Traces from individual experiments showing integrated Ca2+ activity in control conditions (green lines) and after the addition of CPA (30 μM: red lines) Since the lamina propria cells in this study are located immediately beneath the urothelium and are likely exposed to factors produced and released by the urothelium such as ATP, we applied ATP to the lamina propria network. Gradual perfusion of ATP (1 mM) increased the number of cells in the lamina propria exhibiting Ca2+ events (Figures 7A–C) that eventually assembled into a propagating wavefront (Figure 7B) This finding indicates that ATP facilitates a behavior transition from apparently random spontaneous Ca2+ events in lamina propria cells into well-defined propagating wavefronts Response of lamina propria cell network to the addition of ATP (1 mM) (A) Prevalence map of lamina propria syncytium during the recording (4 min) (B) Gradual perfusion of ATP 1 mM (green bar) resulted in increasing number of cells firing eventually leading to the development of propagating wavefronts as evidenced by the clustering of intervals (blue dots) separation distances (green triangles) and separation angles (orange squares) between next-to-fire cells and coherent propagating wavefronts in the ST Map (lower panel) (C) Angle histograms showing the progression from unorganized activity to well-defined wavefronts based on angle bias In preparations where propagating network Ca2+ events were present the velocity of propagation was consistent at 60–70 μm/s even though the direction of propagation was often variable To determine the overall degree of coupling between active lamina propria cells the bias in angle averaged 10.65 ± 1.63 (n = 8) indicating lamina propria cell activity was not occurring randomly and had a defined wavefront angle and direction of propagation The current studies confirm the presence of a heterogeneous cellular network in the lamina propria that exhibits spontaneous Ca2+ transients that can be loosely or tightly coupled (wavefronts) between cells these studies demonstrate several novel findings including: (i) the predominance PDGFRα- and TRPV4-immunoreactivity in the lamina propria layer from early postnatal rat pups (P ≤ 21) (ii) ATP and a TRPV4 agonist activated and increased the number of lamina propria cells that exhibited active Ca2+ events (iii) lamina propria cell activity was not random with spatio-temporal maps and PTCL analysis demonstrating varying degrees of coupling (e.g. partial or loose organization) and (iv) the coupling of Ca2+ activity of cells in laminar propria network could be modified to generate organized propagating bands of activity (wave fronts) with ATP These findings are consistent with the hypothesis that lamina propria Ca2+ signaling facilitates communication through this syncytial network to other cell types or tissue layers of the urinary bladder; our study shows the spatio-temporal patterning of this potential communication that may affect sensory processing and/or detrusor contractility we suggest that the lamina propria network is a primary source or originator of coordinated activation of the urinary bladder where it senses and responds to stimuli and communicates this information to the whole urinary bladder Such a signaling network would be important when neural circuits are not fully mature (i.e. postnatal development) or when compromised by neural injury or disease the present studies demonstrate functional expression of TRPV4 in lamina propria cells in postnatal rat pups In wholemount tissue preparations isolated from rat pups aged ≤ P21 TRPV4-IR was observed in cells in the lamina propria that exhibited similar morphology to those expressing PDGFRα-IR increased the time lamina propria cells were active and increased the number of cells that exhibited active Ca2+ events as evidenced by the rate of integrated Ca2+ activity to alter the number of lamina propria cells displaying Ca2+ transients or the duration of Ca2+ transients may indicate that Ca2+ influx through TRPV4 channels does not contribute to basal Ca2+ signaling in the lamina propria We suggest that the bladder sensory roles of TRPV4 in the normal micturition reflex or following injury or pathology may also be related to TRPV4 expression and function in lamina propria cells the lamina propria network may have an active role in sensing (e.g. between bladder layers and cell types to achieve coordinated bladder function The importance of the lamina propria network may depend upon the integrity and maturity of neural pathways that coordinate micturition reflex events it would be of interest and potential importance to continue to examine the lamina propria network at the urothelial-lamina propria junction following neural injury disease or urinary bladder dysfunction to fully understand its functional significance The studies described from the Vizzard laboratory were performed in accordance with institutional and national guidelines and regulations discussed and outlined the experimental design: TH Research described herein was funded by the National Institutes of Health (NIH) grants to DK051369 (MV) This publication was also made possible by NIH Grants: 5 P30 RR032135 from the COBRE Program of the National Center for Research Resources and 8 P30 GM103498 from the National Institute of General Medical Sciences The authors declare that the research described from the Vizzard laboratory were conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest had no role in the studies described including: design data collection and analysis of studies performed in the Vizzard laboratory decision to publish or preparation of the review The contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH Todd Clason for assistance with immunohistochemistry and imaging The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnsys.2017.00087/full#supplementary-material adenosine 5′-triphosphate; AUC_ZERO_START area under the curve using the start of the transient as the zero point; BPS platelet-derived growth factor receptor alpha; PNS transient receptor potential cation channel subfamily vanilloid member 4; μm Effects of TRPV4 cation channel 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rat as revealed by the injection of pseudorabies virus into the urinary bladder Gap junctions and connexin expression in human suburothelial interstitial cells Electrical characteristics of suburothelial cells isolated from the human bladder Characterization of the purinergic receptor subtype on guinea-pig suburothelial myofibroblasts Up-regulation of P2X3 receptor during stretch of bladder urothelial cells from patients with interstitial cystitis Augmented extracellular ATP signaling in bladder urothelial cells from patients with interstitial cystitis Augmented stretch activated adenosine triphosphate release from bladder uroepithelial cells in patients with interstitial cystitis Stretch-activated release of adenosine triphosphate by bladder uroepithelia is augmented in interstitial cystitis N-((1S)-1-{[4-((2S)-2-{[(2,4-Dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A) a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves The role of the human bladder lamina propria myofibroblast Purinergic regulation of guinea pig suburothelial myofibroblasts Functional TRPV4 channels and an absence of capsaicin-evoked currents in freshly-isolated Differential localizations of the transient receptor potential channels TRPV4 and TRPV1 in the mouse urinary bladder Non-neuronal cholinergic system in human bladder urothelium Urinary bladder function in conscious rat pups: a developmental study Nelson MT and Vizzard MA (2017) Rhythmic Calcium Events in the Lamina Propria Network of the Urinary Bladder of Rat Pups Received: 15 September 2017; Accepted: 14 November 2017; Published: 11 December 2017 Copyright © 2017 Heppner, Hennig, Nelson and Vizzard. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) or licensor are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Margaret A. Vizzard, bWFyZ2FyZXQudml6emFyZEB1dm0uZWR1 Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish A problem has occurred and we're working to fix it as fast as we can Metrics details Intestinal resident macrophages (Mϕs) regulate gastrointestinal homeostasis via production of an anti-inflammatory cytokine interleukin (IL)-10 Although a constant replenishment by circulating monocytes is required to maintain the pool of resident Mϕs in the colonic mucosa the homeostatic regulation of Mϕ in the small intestine (SI) remains unclear we demonstrate that direct stimulation by dietary amino acids regulates the homeostasis of intestinal Mϕs in the SI Mice that received total parenteral nutrition (TPN) which deprives the animals of enteral nutrients displayed a significant decrease of IL-10-producing Mϕs in the SI whereas the IL-10-producing CD4+ T cells remained intact enteral nutrient deprivation selectively affected the monocyte-derived F4/80+ Mϕ population but not non-monocytic precursor-derived CD103+ dendritic cells the replenishment of SI Mϕs and their IL-10 production were not regulated by the gut microbiota SI Mϕs were directly regulated by dietary amino acids microbiota-independent regulation of IL-10-producing resident Mϕs in the SI little is known about the regulation of resident Mϕs in the small intestine (SI) Since the density of bacteria in the lumen is markedly lower in the SI compared to the colon it is possible that other factors contribute to the replenishment of resident Mϕs in the SI blunted mucosal IL-10 production is a key driving factor of the adverse phenotypes seen in TPN recipients at present the precise mechanisms by which the lack of enteral nutrients due to TPN causes diminished mucosal IL-10 production remain unclear we show that the deprivation of enteral nutrients associated with TPN leads to a decrease in the number of IL-10-producing F4/80+CD11b+ Mϕs in the SI LP the replenishment of SI Mϕ is not regulated by the gut microbiota our study highlights the role of dietary stimulation through the enteral route in the regulation of resident Mϕ homeostasis in the SI Enteral nutrient deprivation due to TPN leads to a disruption of SI homeostasis through the impairment of IL-10 production by resident Mϕs Deprivation of enteral nutrition leads to a decline in IL-10-producing F4/80+CD11b+ macrophages in the small intestine (a) LPMCs were isolated from the SI of TPN- or sham-treated mice Isolated LPMCs (5 × 106 cells/ml) were then cultured for 13 hrs with or without LPS stimulation (100 ng ml−1) (b) IL-10Venus reporter mice received TPN or sham-treatment Cell subpopulations of IL-10-expressing cells (Venus+ cells) within the CD45+7-AAD− population were analyzed by flow cytometry Frequencies of IL-10-producing MHC-II+F4/80+CD11b+ macrophages (Mϕs) and CD3+CD4+ T cells are shown Data are representative of 3 independent experiments (c) Frequencies of total IL-10-producing leukocytes (CD45+7-AAD−) F4/80+CD11b+ Mϕs and CD3+CD4+ T cells are shown Dietary nutrients regulate replenishment of resident macrophages and their IL-10 production in the small intestine (a) LPMCs were isolated from the SI of TPN- or sham-treated mice and then analyzed by flow cytometry CD45+MHC-II+ antigen presenting cells were further gated either as F4/80+CD11b+ Mϕs or CD103+CD11c+ DCs The numbers adjacent to the outlined area indicate the frequencies of cells above CD3+CD4+ T cells and CD3+CD8α+ T cells are shown (c) Representative histograms of (% of Venus+ cells) in CD45+MHC-II+F4/80+CD11b+ Mϕs and CD45+CD3+CD4+ T cells The numbers adjacent to the lines indicate the percentages of IL-10-Venus+ cells above At least 3 independent experiments produced similar results CCL2/CCR2-dependent recruitment of monocytes is not impaired by depletion of dietary antigens Frequencies of Ly6ChiCD11b+ monocytes in the spleen (a,b) and bone-marrow (BM) (c,d) of TPN- and sham-treated mice Representative FACS plots (a,c) and pooled results (b,d) are shown (e) Expression of Ccl2 mRNA in the SI mucosa of TPN and Sham Gut microbiota is not involved in the impairment of SI macrophage homeostasis caused by enteral nutrient deprivation CD3+CD4+ T cells and CD3+CD8α+ T cells in the SI LP of TPN and sham mice with or without antibiotic treatment (4Abx) (b) Frequencies of Ly6ChiCD11b+ monocytes in the spleen and BM isolated from TPN and sham mice with or without antibiotic treatment (4Abx) Dietary amino acids regulate SI macrophage homeostasis (a) SI LPMCs were isolated from control (Ctrl) diet- or protein-free (ΔAA) diet-fed mice and analyzed by flow cytometry F4/80+ and CD103+ cells within CD45+7-AAD− cells are shown CD11b and CD11c on either F4/80+ cells (Mϕs) or CD103+ cells (DCs) is shown in histogram (b) Frequencies of F4/80+CD11b+ Mϕs and CD103+CD11c+ DCs are shown ΔAA; N = 3) **P < 0.01; ***P < 0.001 by Student’s t-test (c,d) IL-10 expression (% of Venus+ cells) in CD45+MHC-II+F4/80+CD11b+ Mϕs and CD45+CD3+CD4+ T cells in the SI LPMCs isolated from Ctrl diet- and ΔAA diet-fed mice (c) or isolated from rapamycin-treated (Rap) or untreated control (Ctrl) mice (d) Further studies are needed to clarify the mechanism by which dietary amino acids regulate Mϕ function and to elucidate which amino acids control this function in vitro and in vivo it is conceivable that the impact of dietary antigen deprivation on total CD103+ DCs (including both CD11b+ and CD11b−) becomes negligible It is noteworthy that dietary antigen deprivation brought about by TPN and low antigenicity or ΔAA diets may influence the number as well as function of other immune and non-immune cells (e.g epithelial cells) in the SI in addition to the above mentioned cell types Further studies are required to better understand the role of dietary antigens in the development and regulation of the mucosal immune system enteral nutrient deprivation due to TPN leads to a profound decline in the number of IL-10-producing Mϕ in the SI Mϕ in the SI do not require stimulation from the resident microbes in order to be constantly replenished direct stimulation from dietary amino acids contribute to the regulation of homeostasis of IL-10-producing Mϕ in the SI Supplementation of certain key nutrients through the enteral route may prevent disruption of SI homeostasis in patients receiving TPN since intestinal Mϕs and Mϕ-derived IL-10 production play crucial roles in limiting mucosal immune responses against food antigens as well as commensal microbial antigens a better understanding of the impact dietary amino acids have on the regulation of intestinal Mϕs will result in novel treatments for food allergies as well as inflammatory bowel disease All animal studies were performed in accordance with protocols approved by the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan we used an antibiotic cocktail (4Abx) consisting of ampicillin (1 g l−1) metronidazole (1 g l−1) and vancomycin (500 mg l−1) The antibiotics were administered in drinking water when the mice reached the age of 9 weeks starting 4 days before surgery for a total of 7 days After 7 days the animals received sterilized drinking water until sacrificed (5 days later) All mice were housed in individually vented cage racks All mice were sacrificed on day 7 by CO2 asphyxiation the dissected mucosal tissue was incubated in calcium and magnesium-free Hank’s balanced salt solution (HBSS) (Life technologies CA) containing 2.5% heat-inactivated fetal bovine serum (FBS) (Life technologies) and 1 mM dithiothreitol (Sigma-Aldrich) to remove mucus The mucosa was then incubated with agitation in HBSS containing 2 mM and 1 mM EDTA (Quality Biological The tissues were then collected and incubated with agitation in HBSS containing 400 U ml−1 of type 3 collagenase and 0.01 mg ml−1 of DNase I (Worthington Biochemical re-suspended in a 40% Percoll solution (GE Healthcare Life Sciences layered on top of a 75% Percoll solution and centrifuged at 2,000 r.p.m Viable LPMCs were recovered from the discontinuous gradient interface mashed and digested with 200 U ml−1 of type 3 collagenase and 0.01 mg ml−1 DNase I (Worthington Biochemical) for 30 min at 37 °C The BM was collected from mouse femurs and tibias both spleen and BM cell pellets were lysed with 1× RBC lysis buffer (eBioscience CA) to remove residual red blood cells and the remaining cells were analyzed by flow cytometry followed by stimulation with 100 ng ml−1 LPS for 24 hours Culture supernatants were harvested and cytokine levels were measured by ELISA Isotype-matched antibodies (eBioscience) were used for control staining All antibodies were used at 1:200 dilution except CD11b (used in 1:100 dilution) and Ly6C The concentration of cell suspension was adjusted to 1 × 106 cells per 100 μl Statistical analyses were performed using GraphPad Prism software version 6.0 (GraphPad Software) Student’s t-test (parametric) was used to assess significance between two populations Differences at P < 0.05 were considered significant microbiota-independent regulation of IL-10-producing lamina propria macrophages in the small intestine Intestinal macrophages and dendritic cells: what’s the difference Regulation of the immune system by the resident intestinal bacteria The regulation of IL-10 production by immune cells Macrophages in intestinal homeostasis and inflammation Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2 Monocyte recruitment during infection and inflammation Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice Overview of pediatric short bowel syndrome ESPEN Guidelines on Parenteral Nutrition: surgery Early versus late parenteral nutrition in critically ill adults Decreased phospho-Akt signaling in a mouse model of total parenteral nutrition: a potential mechanism for the development of intestinal mucosal atrophy Dissociation of E-cadherin and beta-catenin in a mouse model of total parenteral nutrition: a mechanism for the loss of epithelial cell proliferation and villus atrophy Enteral versus parenteral nutrition: effect on intestinal barrier function Annals of the New York Academy of Sciences 1165 Decline in intestinal mucosal IL-10 expression and decreased intestinal barrier function in a mouse model of total parenteral nutrition Am J Physiol 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infection microbiology 3 Total parenteral nutrition promotes bacterial translocation from the gut Impact of molecular mimicry on the clinical course and outcome of sepsis syndrome Changes to the Intestinal Microbiome with Parenteral Nutrition: Review of a Murine Model and Potential Clinical Implications Blood monocytes consist of two principal subsets with distinct migratory properties Gr1(+) inflammatory monocytes are required for mucosal resistance to the pathogen Toxoplasma gondii Cutting edge: LPS-induced emergency myelopoiesis depends on TLR4-expressing nonhematopoietic cells TLRs control hematopoiesis during infection Commensal microbiota induce LPS hyporesponsiveness in colonic macrophages via the production of IL-10 A single strain of Clostridium butyricum induces intestinal IL-10-producing macrophages to suppress acute experimental colitis in mice receptor for niacin and the commensal metabolite butyrate suppresses colonic inflammation and carcinogenesis The transcription factor E4BP4 regulates the production of IL-10 and IL-13 in CD4+ T cells Inflammatory T cell responses rely on amino acid transporter ASCT2 facilitation of glutamine uptake and mTORC1 kinase activation Dietary folic acid promotes survival of Foxp3+ regulatory T cells in the colon Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine Stimulation by food proteins plays a critical role in the maturation of the immune system Immunological activities are modulated by enteral administration of an elemental diet in mice Dietary glutamine affects mucosal functions in rats with mild DSS-induced colitis Dietary histidine ameliorates murine colitis by inhibition of proinflammatory cytokine production from macrophages Cutting edge: mTORC1 in intestinal CD11c+ CD11b+ dendritic cells regulates intestinal homeostasis by promoting IL-10 production Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition shapes intestinal function TPN-associated intestinal epithelial cell atrophy is modulated by TLR4/EGF signaling pathways Parenteral nutrition decreases paneth cell function and intestinal bactericidal activity while increasing susceptibility to bacterial enteroinvasion Journal of parenteral and enteral nutrition 38 Alteration of the intestinal intraepithelial lymphocytes during total parenteral nutrition Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria Intestinal specific overexpression of interleukin-7 attenuates the alternation of intestinal intraepithelial lymphocytes after total parenteral nutrition administration Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans Download references We thank Kei Sakamoto (University of Michigan Department of Pathology) for advice on quantification of bacteria Immunology Core) for performing ELISAs and Aaron Robida Mark Savary and Michael Pihalja (University of Michigan Flow Cytometry Core) for assistance with the use of LSR Fortessa and FACS Canto II This work was supported by JSPS Postdoctoral Fellowship for Research Abroad (to S.K the Crohn’s and Colitis Foundation of America Michigan Gastrointestinal Peptide Research Center NIDDK P30DK034933 (to N.K.) and NIH AI44076 (to D.H.T.) conducted the experiments with the help from S.K. Download citation Metrics details The herbal medicine berberine (BBR) has been recently shown to be an AMP-activated protein kinase (AMPK) productive activator with various properties that induce anti-inflammatory responses We investigated the effects of BBR on the mechanisms of mucosal CD4+T cell activation in vitro and on the inflammatory responses in T cell transfer mouse models of inflammatory bowel disease (IBD) We examined the favorable effects of BBR in vitro using lamina propria (LP) CD4+ T cells in T cell transfer IBD models in which SCID mice had been injected with CD4+CD45RBhigh T cells BBR suppressed the frequency of IFN-γ- and Il-17A-producing LP CD4+ T cells This effect was found to be regulated by AMPK activation possibly induced by oxidative phosphorylation inhibition We then examined the effects of BBR on the same IBD models in vivo BBR-fed mice showed AMPK activation in the LPCD4+ T cells and an improvement of colitis Our study newly showed that the BBR-induced AMPK activation of mucosal CD4+ T cells resulted in an improvement of IBD and underscored the importance of AMPK activity in colonic inflammation Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract BBR may be of potential therapeutic utility in the treatment of immune-mediated diseases the widely used models of colitis are chemically induced which is not appropriate for representing the chronic inflammatory features of IBD we investigated the effect and inhibitory mechanisms of BBR on LP CD4+ T cells of IBD—in terms of the cellular energy metabolism related to AMPK activity—using a T cell transfer colitis model involving the transfer of naïve (CD4+CD45RBhigh) T cells into congenic immunodeficiency mice to induce CD4+ T cell-specific colitis Our results provide new insight into how BBR regulates the LP CD4+ T cell function as well as underscore the importance of AMPK activity in colonic inflammation the involvement of AMPK activity in colonic inflammation may lead to the development of new therapeutic targets that are safe and useful for treating IBD BBR suppressed inflammatory cytokines of LP CD4+ T cells from colitis SCID mice in vitro Colitis LP CD4+ T cells were stimulated with PMA plus ionomycin mixed with BBR (BBR) or not (Control) The bar graphs show number of LP CD4+ T cells (B) Colitis LP CD4+ T cells were stimulated with PMA plus ionomycin mixed with BBR (BBR) or not (Control) for 8 h and intracellular staining was performed to analyze the CD3+CD4+IFN-γ+- or IL-17A+-producing cells by flow cytometry Representative flow cytometry images are shown Flow cytometry showed the percentage of CD3+CD4+IFN-γ+-or IL-17A+-producing cells in colitis LP CD4+ T cells (C) The bar graphs show the percentage of IFN-γ- and IL-17A-producing cells in LP CD4+ T cells As shown in Fig. 1B,C the frequency of IFN-γ-producing CD4+ T cells in BBR-treated colitis LP CD4+ T cells was significantly lower than in non-BBR-treated colitis LP CD4+ T cells The frequency of IL-17A-producing CD4+ T cells was also lower in BBR-treated cells than in untreated cells These results indicated that BBR directly affected the colitis LP CD4+ T cells All gels were run in the same experimental conditions (see material and methods for details) The bar graphs show the percentage of each protein expression These data strongly indicated that the production of IFN-γ and IL-17A by colitis LP CD4+ T cells was regulated through AMPK activities BBR affected the oxidative phosphorylation and decreased the total adenosine triphosphate production Colitis LP CD4+ T cells were stimulated with PMA plus ionomycin mixed with BBR (BBR) or not (Control) for 8 h (B) Bioenergetic profile of colitis LP CD4+ T cells The OCR profile following the addition of mitochondrial inhibitors (oligomycin rotenone/antimycin A) was determined by a Flux analyzer xXF 96 S mitochondrial ATP and ECAR profiles were separately analyzed This was due to the effect of increased AMPK activation compensating for the loss of ATP production in OXPHOS according to the total ATP production in our experiment glycolysis did not sufficiently compensate for ATP production These experiments suggest that BBR suppressed OXPHOS and total ATP production All gels were run in the same experimental conditions (see Materials and methods for details) The numerical values represent the mean values of 6 samples per group Pictures and dot plots of flow cytometry show representative samples from each group All data are shown as the mean ± SEM for 6 mice per group These data indicated that BBR reduced the T cell inflammatory responses and ameliorated experimental colitis (A,B) A comparison of the fecal microbial community at the phylum and genus level between colitis and BBR (D,E) Beta diversity_UniFrac_unweighted and UniFrac unweighted distance Each point represents the gut microbiota community of each mouse with black points and gray points indicating the colitis and BBR groups The alpha diversity was evaluated to assess the species richness within the gut microbiota community diversity of the two groups (Fig. 6C) There were no considerable differences in the number of observed OTU Faith’s phylogenetic diversity whole tree and the diversity of the species estimated by Chao1 the species richness and evenness calculated by the Shannon index were significantly reduced with BBR supplementation These data suggested that BBR affected the gut microbiota composition and this change might be one of the factors ameliorating colitis we showed that BBR ameliorated CD4+ T cell-related chronic colitis in a mouse model and revealed the relationship between BBR and AMPK Our message based on the present data can be summarized in the following three points: 1) We adapted the CD4+ T cell-transfer model a chronic colitis model that clearly differs from the widely used chemically induced acute colitis models; 2) BBR affected the AMPK-related metabolic pathway and reduced the inflammatory responses; and 3) BBR reduced the microbiota diversity Such chemically induced models showed acute colitis that did not accurately reflect the chronic inflammatory process observed in IBD patients CD4+T cell-transfer colitis models show chronic colitis with T cell infiltration and may be superior to chemically induced models The use of CD4+T cell-transfer colitis models revealed a new inhibitory mechanism of BBR particularly in relation to colitis LP CD4+ T cells We therefore focused on AMPK and attempted to reveal its immunological effects Our results showed that BBR increased the AMPK activity in colitic LP CD4+ T cells we showed that the AMPK activity regulated IFN-γ- and IL-17A-producing LP CD4+ T cells These data suggested that the regulation of AMPK in colitis LPCD4+ T cells was important to control mucosal inflammation We further investigated the relationship between AMPK and metabolic pathways It is well-established that AMPK is activated under conditions of ATP shortage in order to restore the decreased intracellular energy and thereby ensure the cell survival and maintenance of the cell function ATP-consuming anabolic pathways are turned off and ATP-producing catabolic processes are stimulated Our results showed BBR decreased the total ATP content suggesting that the increase in the AMPK activity was induced by BBR through a reduction in the ATP content our present results indicated the strong effect of BBR on AMPK activation and an AMPK agonist such as BBR may be expected to reduce inflammatory cell activation We therefore emphasize the importance of AMPK in our immune-mediated colitis model as well as the effects of BBR on AMPK activation Further studies are needed to gain more insight into the glycolysis of colitis LPCD4+T cells We also did not examine the detailed relationships between the effects of BBR and the characteristics of these cells but the effects of BBR on these cells might offer further insight into the regulation of mucosal inflammation Our data revealed that BBR reduced the severity of colitis and that the change in the gut microbiota was due to the anti-inflammatory effects of BBR We therefore considered the change in the gut microbiota in our experiment to be reasonable we showed for the first time that BBR ameliorated CD4+ T cell-related chronic colitis in a mouse model with changes in gut microbiota via AMPK activity possibly induced by the inhibition of OXPHOS according to in vitro and in vivo experiments The AMPK activity of colonic inflammation may represent a new therapeutic target for IBD may be recognized as new candidates for use in IBD therapy Balb/c and CB17-icr SCID mice were purchased from Japan CLEA (Tokyo Mice were maintained under specific-pathogen-free conditions in the Animal Care Facility of Okayama University The Balb/c donors and recipients were used at 5 to 7 weeks of age donors and CB17-icr SCID recipients were used at 5 weeks of age All protocols and procedures conformed to the guidelines of the Okayama University Committee for Care and Use of Laboratory Animals and were approved by the Animal Experiments Ethics Committee of Okayama University BBR (chloride form) was obtained from Sigma-Aldrich (St The resulting compound used in this study had a purity of 98% BBR was added at a concentration of 100 µM we mixed normal diet with BBR at a concentration of 0.35% To detect the expression of a variety of molecules on LP mononuclear cells they were incubated with antibodies for 20 min after their surface molecules were stained cells were fixed using a Cytofix/Cytoperm Kit (BD Pharmingen) and incubated with antibodies for 20 min Multi-color flow cytometric analyses were performed using MACS (Miltenyi Biotec a Quant flow cytometer and Flowjo (FlowJo LLC The following monoclonal antibodies (mAbs) were obtained from Biolegend (San Diego FITC anti-mouse IFN-γ (XMG1.2) and Purified anti-mouse CD16/32 (93) The following mAbs were obtained from BD Pharmigen (San Diego USA): FITC anti-mouse CD45RB (16 A); PE anti-mouse Bcl-2 (3F11) The following mAbs were obtained from eBioscience (San Diego consisting of the sum of three parameters: crypt elongation Single-cell suspensions were prepared from the LP as described2 the entire length of the colon was opened longitudinally and cut into small pieces The dissected mucosae were incubated for 45 min with Ca2+- Mg2+- free Hanks’ balanced salt solution containing 1 mM dithiothreitol (Sigma-Aldrich) to remove mucus and then treated with 3.0 mg/ml collagenase (Roche Diagnostics GmbH The cells were pelleted twice through a Ca2+- Mg2+- free Hanks’ balanced salt solution and then subjected to Ficoll-Paque density gradient centrifugation Enriched LP CD4+ T cells were obtained by positive selection using anti-CD4 (L3T4) MACS magnetic beads Each experiment contained a group with equal concentrations of DMSO as a control LP CD4+ T cells enriched from colitis SCID mice were cultured by the method described previously cells were collected and plated at 5 × 105 cells in 96-well plates The cells were lysed with 100 µl lysis buffer and placed directly into the chamber of a luminometer (Flex Station3 Light emission was recorded after the addition of 100 µl of luciferin-luciferase solution (TOYO B-NET The ATP values were averaged among at least two technical replicates per sample Total proteins were extracted from cultured LP CD4+ T cells using a protein extraction kit (Minute Protein Extraction Kit; Invent Biotechnologies Extracted proteins were quantitated by the BCA method (BCA protein Assay Kit; Takara Bio Japan) and diluted to the same protein concentrations Equivalent amounts of each group were run on SDS polyacrylamide gels Proteins were transferred electrically to a PVDF membrane (Bio-Rad The membranes were blocked using polyvinylidene fluoride Blocking Reagent (Toyobo the membranes were incubated with the following rabbit monoclonal antibodies overnight at 4 °C: anti-AMPK(Thr172) anti-JAK2 (Tyr221) (Cell Signaling Technology After three washes with Tris-buffered saline with Tween-20 (Sigma) the membranes were incubated with secondary antibodies for 1 h at room temperature The rabbit monoclonal antibodies were used at a 1:1000 dilution and the anti-rabbit secondary antibodies (Cell Signaling Technology) at a 1:2000 dilution Bands were detected by using a LAS 4000 imager (GE Healthcare Life Sciences Contrast was adjusted with Adobe Photoshop Elements 14 The quantity in each sample was analyzed using an Image J64 (NIH The fecal DNA was sequenced using Illumina Miseq platforms according to the manufacturer’s recommendation (San Diego all sequences were normalized to the smallest number of reads (5324 reads) before being assigned operation taxonomic units (OTUs) using the Greengenes reference database with a 97% similarity threshold and the alpha and beta diversity were compared The relative abundance of OTUs in each group was evaluated at the phylum and genus levels Faith’s phylogenetic diversity whole tree and the Shannon diversity index were determined to estimate the alpha diversity The weighted and unweighted UniFrac distance in beta diversity were evaluated to compare the microbial diversity between the BBR diet group and non-BBR diet group All DNA sequences are available on the Sequence Read Archive under the relevant project identification number Results are expressed as means ± standard error of the means for parametric data and medians for non-parametric data All parametric data were compared using the Student’s t test and non-parametric data were compared by the Mann Whitney U-test Data were considered to be statistically significant at P < 0.05 Japan) was used for all statistical analyses Contact the authors or provide an SRA number Immunosenescent colitogenic CD4(+) T cells convert to regulatory cells and suppress colitis Pharmacological and therapeutic effects of Berberis vulgaris and its active constituent A comparative study on the anti-inflammatory antinociceptive and antipyretic effects of isoquinoline alkaloids from the roots of Turkish Berberis species Berberine reduces insulin resistance through protein kinase C-dependent up-regulation of insulin receptor expression Berberine sulfate inhibits tumor-promoting activity of teleocidin in two-stage carcinogenesis on mouse skin Berberine differentially modulates the activities of ERK and JNK to suppress Th17 and Th1 T cell differentiation in type 1 diabetic mice Regulation of Th1 and Th17 cell differentiation and amelioration of experimental autoimmune encephalomyelitis by natural product compound berberine Berberine enhances the AMPK activation and autophagy and mitigates high glucose-induced apoptosis of mouse podocytes activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic switch Preexisting high frequencies of memory CD8+ T cells favor rapid memory differentiation and preservation of proliferative potential upon boosting Reducing CD73 expression by IL1beta-Programmed Th17 cells improves immunotherapeutic control of tumors Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages AMPK-dependent and independent effects of AICAR and compound C on T-cell responses The energy sensor AMPK regulates T cell metabolic adaptation and effector responses in vivo Berberine ameliorates TNBS induced colitis by inhibiting inflammatory responses and Th1/Th17 differentiation Berberine promotes recovery of colitis and inhibits inflammatory responses in colonic macrophages and epithelial cells in DSS-treated mice Berberrubine attenuates mucosal lesions and inflammation in dextran sodium sulfate-induced colitis in mice Berberine inhibits macrophage M1 polarization via AKT1/SOCS1/NF-kappaB signaling pathway to protect against DSS-induced colitis Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases Stat3 and Stat4 direct development of IL-17-secreting Th cells Inhibition of the AMP-activated protein kinase-alpha2 accentuates agonist-induced vascular smooth muscle contraction and high blood pressure in mice The role of AMPK in T cell metabolism and function AMPK: a nutrient and energy sensor that maintains energy homeostasis induces AMP activated protein kinase activity leading to autophagic cell death in breast cancer cells Berberine Regulates Treg/Th17 Balance to Treat Ulcerative Colitis Through Modulating the Gut Microbiota in the Colon Effects of berberine on human rheumatoid arthritis fibroblast-like synoviocytes Experimental biology and medicine (Maywood Immunometabolism: Cellular Metabolism Turns Immune Regulator Mitochondrially targeted effects of berberine [Natural Yellow 18 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] on K1735-M2 mouse melanoma cells: comparison with direct effects on isolated mitochondrial fractions Berberine and its more biologically available derivative inhibit mitochondrial respiratory complex I: a mechanism for the action of berberine to activate AMP-activated protein kinase and improve insulin action Antioxidant and anti-inflammatory activities of berberine in the treatment of diabetes mellitus Antioxidant Effect of Berberine and its Phenolic Derivatives Against Human Fibrosarcoma Cells Asian Pacific journal of cancer prevention: APJCP 16 IL-7 promotes long-term in vitro survival of unique long-lived memory subset generated from mucosal effector memory CD4+ T cells in chronic colitis mice Role of regulatory T cell in the pathogenesis of inflammatory bowel disease A central role for induced regulatory T cells in tolerance induction in experimental colitis Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel disease Antibacterial Mechanisms of Berberine and Reasons for Little Resistance of Bacteria Chinese Herbal Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5′-methoxyhydnocarpin Proceedings of the National Academy of Sciences of the United States of America 97 Structural changes of gut microbiota during berberine-mediated prevention of obesity and insulin resistance in high-fat diet-fed rats Berberine and inflammatory bowel disease: A concise review Immune-mediated antitumor effect by type 2 diabetes drug Proceedings of the National Academy of Sciences of the United States of America 112 predominant lactobacilli in the intestinal flora of healthy thoroughbreds Animal science journal = Nihon chikusan Gakkaiho 80 QIIME allows analysis of high-throughput community sequencing data Download references No potential conflicts of interest relevant to this article are reported Department of Gastroenterology and Hepatology Okayama University Graduate School of Medicine Okayama University Graduate School of Environmental and Life Science Tien Thi Thuy Nguyen & Hidetoshi Morita conceived and designed the study and performed the microbiome analysis; S.E. conceived and designed the study and performed experiments; Y.S Berberine improved experimental chronic colitis by regulating interferon-γ- and IL-17A-producing lamina propria CD4⁺ T cells through AMPK activation Download citation DOI: https://doi.org/10.1038/s41598-019-48331-w Metrics details The intestinal immune system is chronically challenged by a huge plethora of antigens derived from the lumen B-cell responses in organized gut-associated lymphoid tissues and regional lymph nodes that are driven chronically by gut antigens generate the largest population of antibody-producing cells in the body: the gut lamina propria plasma cells Although animal studies have provided insights into mechanisms that underpin this dynamic process some very fundamental differences in this system appear to exist between species this prevents extrapolation from mice to humans to inform translational research questions in this review we will describe the structures and mechanisms involved in the propagation and regulation of this immense plasma cell population in man we will seek our evidence exclusively from studies of human cells and tissues Immune responses in the intestine are initiated in gut-associated lymphoid tissue (GALT) and in gut regional lymph nodes Whereas food and ingested antigens are present in the small bowel microbial antigens represent a major immune challenge in the colon Antigens at both gut locations are sampled by follicle-associated epithelium (FAE) Dendritic cells in the subepithelial area are key regulators of the fate of responding B cells They express the enzyme retinaldehyde dehydrogenase that catalyzes the production of retinoic acid that subsequently imprints homing via the induction of α4β7 and C-C motif receptor 9 (CCR9) They also secrete immunoglobulin A (IgA)-inducing protein (IGIP) and APRIL (a proliferation-inducing ligand) that together with stromally derived transforming growth factor-β (TGFβ) support IgA class switch recombination hypermutation and selection in the germinal centers of Peyer’s patches or regional lymph nodes Plasmablasts subsequently home back to the lamina propria where they differentiate into plasma cells The chemokines C-C motif chemokine ligand 25 (CCL25) and CCL28 may contribute to the differential homing of plasmablasts primed in the small intestine or colon though how and where the lymphocyte receptor of CCL28 The precursors of lamina propria IgA plasma cells are generated in microanatomically discrete zones of organized lymphoid inductive sites, such as the gut-associated lymphoid tissue (GALT) and gut regional lymph nodes (Figure 1) First we will consider the structure and function of human GALT and then the derivation and unique properties of intestinal plasma cells themselves Paraffin sections of gut-associated lymphoid tissue (GALT) in human colon stained for (a) CD20 (brown) (b) IgD (brown) and (c) CD3 (pink) and CD8 (brown) The germinal center (GC) is surrounded by a dashed circle Highly immunoglobulin D (IgD) expressing naive B cells in b surround the GC The more extensive marginal zone B-cell population extends between the mantle zone and the follicle-associated epithelium (FAE) T follicular helper cells defined by their location in the GC are rarely CD8+ and are located predominantly in the light zone (LZ) of the GC where they are involved in repertoire selection T cells are rarely present in the dark zone (DZ) Paraffin section of human ileum stained with CD20 (brown). Nuclei are counterstained in blue. CD20 expressing B cells are abundant in gut-associated lymphoid tissue (GALT). The lamina propria adjacent to GALT may contain B cells that tend to be large cells resembling plasmablasts or plasma cells (identified with a box in (a) and magnified in panel (b)). Distant lamina propria, however, contains few, if any, CD20+ B cells (box (C)). Illustration of factors that support plasma cells that can bind antigen through their B-cell receptor in the lamina propria niche Stromal cell–derived interleukin (IL)-6 may bind to plasma cell IL-6 receptor and APRIL (a proliferation-inducing ligand) produced by epithelial cells or eosinophils may bind to BCMA on plasma cells to support plasma cell survival C-X-C motif chemokine ligand 12 (CXCL12) may promote plasma cell retention and survival through binding C-X-C motif chemokine receptor 4 (CXCR4) Plasma cell frequencies in the intestine may also be regulated by T cells through the presentation of cognate antigen to local T-cell populations despite selective pressure to maintain structural viability It may be a consequence of drive from the high antigenic load or lower selective pressure so that a greater range of variants is tolerated (a) Paraffin section of gut-associated lymphoid tissue (GALT) from the ileum stained for a proliferation-inducing ligand (APRIL; brown) and activation-induced cytidine deaminase (AID; pink) Nuclei are counterstained with hematoxylin (blue) The germinal center (GC) is identified with a dashed line (b) AID staining is confined to the GC with the exception of occasional large interfollicular B cells that have cytoplasmic AID APRIL is produced adjacent to B cells expressing AID in the GC (c) APRIL is abundant in cells with dendritic and macrophage morphology beneath the follicle-associated epithelium (FAE) (d) An area where the crypt epithelium expressing APRIL abuts the lymphoid tissue is indicated with a dotted line Areas magnified in B–D are identified with labeled boxes in a (e) The receptors for soluble APRIL and B-cell activating factor (BAFF) are illustrated schematically for reference the maintenance of plasma cells in the gut lamina propria could also be explained by some gut plasma cells having long lifespans (see below) may suggest that the VH usage of B cells that later home to and operate in the gut as plasma cells is influenced not only by antigen but possibly also by the mucosal priming The significance of this and any possible link to the development of marginal zone B cells remains to be determined and afterwards the plasma cells are seeded into the lamina propria in wide areas of the gut it hence seems likely that long-lived B-cell responses initiated in GCs do exist in humans but the relative contribution of long-lived circulating memory B cells and long-lived plasma cells in the lamina propria to maintain a persistent gut humoral response awaits clarification Paraffin sections of human (a and b) ileum and (c–e) colon stained for immunoglobulin A (IgA) (a) and a proliferation-inducing ligand (APRIL; b–e) IgA plasma cells (arrow heads) secrete IgA that is transported into the gut lumen following binding to the polymeric immunoglobulin receptor (pIgR) on the basolateral surface of epithelial cells IgA in transit through epithelial cells is indicated with an arrow is expressed mostly by 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immunoglobulin-A-expressing plasma cells and contribute to gut immune homeostasis Human intestinal epithelial and smooth muscle cells are potent producers of IL-6 CXCL12 is a constitutive and inflammatory chemokine in the intestinal immune system Identification of tissue transglutaminase as the autoantigen of celiac disease Screening detects a high proportion of celiac disease in young HLA-genotyped children Decrease by 50% of plasma IgA tissue transglutaminase antibody concentrations within 2 months after start of gluten-free diet in children with celiac disease used as a confirming diagnostic test Immunoglobulins in jejunal mucosa and serum from patients with adult coeliac disease Celiac disease: autoimmunity in response to food antigen Responsive population dynamics and wide seeding into the duodenal lamina propria of transglutaminase-2-specific plasma cells in celiac disease Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association Download references This work was supported by grants from the European Commission (grant ERC-2010-Ad-268541) the Research Council of Norway (partially through its Centres of Excellence funding scheme and the South-East Norway Regional Health Authority (to L.M.S.) and Medical Research Council of Great Britain grant MR/L009382/1 (to J.S.) We thank Jorunn Stamnaes and Ramus Iversen for commenting on the manuscript J Spencer and L M Sollid: These authors contributed equally to this work Centre for Immune Regulation and Department of Immunology University of Oslo and Oslo University Hospital-Rikshospitalet The author declared no conflict of interest Reprints and permissions Download citation Metrics details The intestinal immune system must elicit robust immunity against harmful pathogens but must also restrain immune responses directed against commensal microbes and dietary antigens The mechanisms that maintain this dichotomy are poorly understood Here we describe a population of CD11b+F4/80+CD11c− macrophages in the lamina propria that expressed several anti-inflammatory molecules but little or no proinflammatory cytokines even after stimulation with Toll-like receptor ligands retinoic acid and exogenous transforming growth factor-β the differentiation of Foxp3+ regulatory T cells lamina propria CD11b+ dendritic cells 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devoid of Stat3 in macrophages and neutrophils CD11b facilitates the development of peripheral tolerance by suppressing Th17 differentiation Chemokines and the tissue-specific migration of lymphocytes Human G protein-coupled receptor GPR-9–6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse Interleukin-10-deficient mice develop chronic enterocolitis Abrogation of TGFβ signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease Localization of transforming growth factor β isoforms in the normal murine small intestine and colon Essential role for CD103 in the T cell-mediated regulation of experimental colitis Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo Interleukin-10 and the interleukin-10 receptor The role of IL-10 in crossregulation of TH1 and TH2 responses IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells CD70+ antigen-presenting cells control the proliferation and differentiation of T cells in the intestinal mucosa Download references Aguilar Mertens for assistance with cell sorting; T Querec for assistance with gene array data analysis; R Mittler (Emory University School of Medicine) for recombinant Flt3 ligand; A Murthy for synthesis of clodronate liposomes; W Ahmed (Emory University School of Medicine) and A Sharpe (Harvard Medical School) for PD-L1 deficient mice Supported by the National Institutes of Health (AI0564499 AI057157 and AI-50019 to B.P.; and DK007771-06A1 (Pathobiology of Mucosal/Epithelial Disease) to T.L.D.) and the Crohn's and Colitis Foundation of America (T.L.D.) Vaccine Research Center and Yerkes Regional Primate Research Center Department of Pathology and Laboratory Medicine Download citation Metrics details Intramucosal lipomas are rare and easily overlooked by pathologists despite their diagnostic significance for Cowden syndrome (PTEN hamartoma tumor syndrome) Only 25–35% of patients harbor identifiable PTEN mutations The significance and diagnostic approach to intramucosal lipomas have not been thoroughly addressed in the literature Intramucosal lipomas are mimicked by pseudolipomatosis coli an artifactual mucosal gas infiltration from endoscopic insufflation This differential was investigated by morphology and S-100 immunohistochemistry Twenty-five colonic intramucosal lipomas were identified from 176 archival gastrointestinal lipomas from 1998 to 2017 Controls included 40 submucosal lipomas and 30 pseudolipomatoses S-100 immunohistochemistry on all 95 lesions confirmed delicate fat vacuole membranous and nuclear S-100 staining in lipomas absent from pseudolipomatoses Differentiating morphology between intramucosal lipoma and pseudolipomatosis and mucosal lymphoid aggregate involvement (12% vs 80%) five patients (20%) had confirmed Cowden syndrome (four with PTEN mutations) the intramucosal lipoma was the index diagnostic lesion Three (12%) intramucosal lipoma patients had additional clinical features associated with Cowden syndrome Sporadic-type intramucosal lipomas were identified in 17 patients (68%) without evidence of Cowden syndrome including three with normal PTEN genetic testing No distinguishing endoscopic or pathologic polyp features were identified between sporadic and syndromic intramucosal lipomas These data provide evidence that intramucosal lipomas are important harbingers of Cowden syndrome making up approximately one-third of this series We also show for the first time that two-thirds of intramucosal lipomas are sporadic and geneticists should increase their awareness of this subtle but diagnosable lesion strongly associated with Cowden syndrome The aim of this study was to assess the sensitivity and specificity and diagnostic approaches to gastrointestinal intramucosal lipomas for Cowden syndrome in comparison with pertinent controls in this the largest reported series in the literature We seek to raise the awareness of gastrointestinal pathologists and thereby improve the diagnosis of Cowden syndrome an important inherited and high-risk multicancer syndrome Germline PTEN mutation testing was performed with informed patient consent and detailed family and past medical histories and physical examinations were conducted for Cowden syndrome evaluation by the Genetics Counseling Department Two control groups consisted of 40 patients with submucosal lipomas and 30 with pseudolipomatosis coli The archival histologic slides of all cases and controls were obtained and reviewed systematically by experienced gastrointestinal pathologists To distinguish true intramucosal lipomas from pseudolipomatosis morphologic features were compared and routine S-100 immunohistochemical staining was performed The morphologic features evaluated included the (1) size and regularity of the lesional open spaces/holes were they gas in pseudolipomatosis or lipid in lipomas (2) the location of the lesions within the mucosa (3) their relationship to colonic crypts and mucosal-associated lymphoid aggregates (4) whether submucosal lipomas ever breached the muscularis mucosae to enter the overlying lamina propria and (5) associated lamina propria spindle cell mesenchymal proliferations S-100 immunohistochemical staining patterns were assessed for either (1) total non-staining of lesional holes in pseudolipomatosis (2) staining of scattered non-lesional lamina propria dentritic cells with definable delicate dentritic processes or (3) delicate membranous cytoplasmic rim staining around true lipid vacuoles in lipomas plus/minus nuclear staining of the small adipocyte nuclei within plane of sectioning Genetic germline PTEN testing was performed through sequencing of all exons and adjacent introns additional large rearrangement testing was performed In the case of a known mutation in one family site-specific PTEN testing for the familial mutation was undertaken Finally, 17 patients (68%) had no additional features associated with Cowden syndrome. PTEN genetic testing was pursued in three of these patients that did not disclose pathogenic variants. These 68% of intramucosal lipomas in this series then document the first reported sporadic-type intramucosal lipomas in the literature, unrelated to Cowden syndrome (Table 2) Endoscopic image of a small 3 mm colonic and syndromic intramucosal lipoma from a Cowden patient No specific distinguishing endoscopic features are present Spindle cell proliferations characteristic of 80% of true intramucosal lipomas and absent from all pseudolipomatosis coli in this series (a) Prominent paucicellular lamina propria spindle cell proliferation associated with a sporadic-type intramucosal lipoma (× 200) (b) Intramucosal lipoma with a more typical subtle spindle cell proliferation infiltrating the lamina propria from a Cowden syndrome intramucosal lipoma (× 200) Table 2 summarizes and compares the histologic and other phenotypic clinical features across both sporadic-type and Cowden syndromic intramucosal lipoma patients in this series there were no pathologic features that distinguished sporadic and Cowden syndromic intramucosal lipomas this distinction was accomplished through PTEN germline genetic testing and careful evaluation by trained geneticists for the other phenotypic manifestations of Cowden syndrome One serrated sessile polyp with an accompanying submucosal lipoma was included in the submucosal lipoma control group It behaved as all of the other submucosal lesions by not crossing the muscularis mucosae into the mucosa Spindle cell proliferations were not seen in association with the submucosal lipoma controls S-100 delicately highlights the adipocyte outer membrane (arrows) and occasionally a nucleus within plane of section S-100 does not highlight the insufflation artifact (arrow) but rather stains only lamina propria dendritic cells (arrowheads) for which care should be taken not to over interpret as a false-positive S-100 staining in pseudolipomatosis This series of 25 rare intramucosal lipomas revealed five patients (20%) with the important inherited multicancer disorder of Cowden syndrome it was the gastrointestinal pathologists’ diagnosis of intramucosal lipoma with commentary about the possibility of Cowden syndrome that first lead to the diagnosis Three patients (12%) had additional features for Cowden syndrome but did not meet full clinical diagnostic criteria and 17 patients (68%) were found to have incidental or sporadic intramucosal lipomas without clinical or genetic features Identification of an intramucosal lipoma in the context of other hamartomatous polyps or a major criterion cancer (breast endometrial) is highly suggestive of Cowden syndrome clinical information regarding many features of Cowden syndrome will not be readily available at the time of polyp pathology review and genetic counseling referral for further assessment can help determine if PTEN gene testing or enhanced cancer screening are needed Intramucosal lipomas are therefore an important hallmark feature of possible Cowden syndrome for which gastrointestinal pathologists should be aware Clues that clear vacuoles resembling fat in the mucosa actually represent artifactual gas bubbles are their variable and generally small to tiny size and their common association with mucosal-associated lymphoid tissue True lipomas tend to have relatively large or macrovesicular fat vacuoles and more uniform vacuoles that are usually located at the base of the lamina propria so that it is fortunate that S-100 staining offers an additional means to distinguish these mimics Given the serious consequences of the diagnosis of a true intramucosal lipoma confirmatory and simple S-100 staining is highly useful The S-100 staining pattern of true adipocytes ringing around the cytoplasmic fat vacuoles in a thin they are frequently out of the plane of section of the S-100 stain Pathologists are therefore cautioned to examine S-100 staining of possible intramucosal lipomas under high magnification before excluding this subtle staining pattern The other caveat with S-100 staining is that normal and often abundant lamina propria dentritic cells stain strongly for S-100 If admixed with the gas bubbles of pseudolipomatosis dendritic cell positivity can lead to false-positive interpretation of the gas bubbles Recognition of the branching dendrites of the dentritic cells helps prevent false-positive interpretation Some degree of lamina propria spindle cell proliferation was seen in 80% of intramucosal lipoma cases while none of the submucosal lipomas or pseudolipomatosis controls revealed this feature This spindle cell change was present in sporadic and Cowden syndromic intramucosal lipomas alike It did however serve to make the intramucosal lipoma more obvious on H&E morphology and to exclude the pseudolipomatosis mimics intramucosal lipomas appear also to exist incidentally or sporadically occurring in about two-thirds of patients in this series The true prevalence of intramucosal lipomas is almost certainly under-recognized by this study as these lesions are relatively small and unimpressive morphologically and are easily overlooked by gastrointestinal pathologists or dismissed as pseudolipomatosis Intramucosal adipose pathology has not previously been of much interest or clinical significance and this combined with its unimpressive appearance microscopically leads the authors of this study to the strong suspicion that they have been commonly overlooked in the past overlooked cases would not have been included in this study which by necessity was based on a search of diagnosed archival lipomas The true prevalence of this colonic polyp remains unknown but it is certainly possible that the two-thirds prevalence of sporadic intramucosal lipomas reported in this series is an underestimate the strong association with Cowden syndrome in this series indicates its potential importance as a diagnostic finding this series further demonstrates that the intramucosal lipomas are an important diagnostic feature of Cowden syndrome Proper pathologic and clinical genetic diagnosis of this syndrome enables improved cancer prevention through targeted cancer surveillance for these patients and their families Intramucosal lipomas are subtle and easily missed lesions that gastrointestinal pathologist have not routinely and specifically sought diagnostically but can be recognized readily by pathologists through heightened awareness that fat is never normal within the mucosa and stains positively by S-100 in comparison with pseudolipomatosis gas bubbles which do not The described morphologic features are also highly useful including the large and regular size of the fat vacuoles the lack of involvement of mucosal lymphoid tissue and the associated benign lamina propria spindle cell proliferations Increased awareness of this lesion by gastrointestinal pathologists and geneticists should improve the diagnosis of the important and enigmatic condition of Cowden syndrome Pneumatosis intestinalis versus pseudo-pneumatosis: review of CT findings and differentiation Morphologic characterization of hamartomatous gastrointestinal polyps in Cowden syndrome Best Pract Res Clin Gastroenterol 2009;23:219–231 Cowden syndrome and Bannayan–Riley–Ruvalcaba syndrome represent one condition with variable expression and age-related penetrance: results of a clinical study of PTEN mutation carriers Predicting PTEN mutations: an evaluation of Cowden syndrome and Bannayan–Riley–Ruvalcaba syndrome clinical features A clinical scoring system for selection of patients for PTEN mutation testing is proposed on the basis of a prospective study of 3042 probands Cowden syndrome and the PTEN hamartoma tumor syndrome: Systematic review and revised diagnostic criteria Germline PTEN mutation analysis for PTEN hamartoma tumor syndrome Clinical implications for germline PTEN spectrum disorders Endocrinol Metab Clin N Am 2017;46:503–517 Colonic polyposis and neoplasia in Cowden syndrome Gastrointestinal polyposis in Cowden syndrome Colonic manifestations of PTEN hamartoma tumor syndrome: case series and systematic review Download references This study was conducted with support from the Genetic Counseling and the Biorepository and Molecular Pathology Shared Resources supported by the Cancer Center Support Grant awarded to the Huntsman Cancer Institute by the National Cancer Institute of the National Institutes of Health Department of Pathology and ARUP Laboratories Division of Gastroenterology University of Utah Health Sciences Center Download citation DOI: https://doi.org/10.1038/modpathol.2017.161 Metrics details The intestinal cell types responsible for defense against pathogenic organisms remain incompletely characterized Here we identify a subset of CD11chiCD11bhi lamina propria dendritic cells (LPDCs) that expressed Toll-like receptor 5 (TLR5) in the small intestine When stimulated by the TLR5 ligand flagellin TLR5+ LPDCs induced the differentiation of naive B cells into immunoglobulin A–producing plasma cells by a mechanism independent of gut-associated lymphoid tissue by a mechanism dependent on TLR5 stimulation these LPDCs promoted the differentiation of antigen-specific interleukin 17–producing T helper cells and type 1 T helper cells the LPDCs specifically produced retinoic acid supported the generation and retention of immunoglobulin A–producing cells in the lamina propria and positively regulated the differentiation interleukin 17–producing T helper cells Our findings demonstrate unique properties of LPDCs and the importance of TLR5 for adaptive immunity in the intestine induce differentiation and support function of T cells with regulatory properties Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody Control of intestinal homeostasis by regulatory T cells and dendritic cells Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid Endotoxin-mediated dendritic cell release from the intestine Characterization of released dendritic cells and TNF dependence CCR7 is critically important for migration of dendritic cells in intestinal lamina propria to mesenteric lymph nodes Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17–producing T cell responses TLR signaling in the gut in health and disease Detection of pathogenic intestinal bacteria by Toll-like receptor 5 on intestinal CD11c+ lamina propria cells Intestinal IgA synthesis: a primitive form of adaptive immunity that regulates microbial communities in the gut Transfer of lymphocytes of Peyer's patches between immunoglobulin allotype congenic mice: repopulation of the IgA plasma cells in the gut lamina propria A population of resting IgM-IgD double-bearing lymphocytes in Peyer's patches: the major precursor cells for IgA plasma cells in the gut lamina propria In situ class switching and differentiation to IgA-producing cells in the gut lamina propria Two distinctive pathways for recruitment of naive and primed IgM+ B cells to the gut lamina propria Sphingosine 1-phosphate regulates peritoneal B-cell trafficking for subsequent intestinal IgA production A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria Regulation of IgA production by naturally occurring TNF/iNOS-producing dendritic cells Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID) Signaling via LTβR on the lamina propria stromal cells of the gut is required for IgA production CC chemokine ligands 25 and 28 play essential roles in intestinal extravasation of IgA antibody-secreting cells Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway Activation of retinoic acid receptor-α favours regulatory T cell induction at the expense of IL-17-secreting T helper cell differentiation Expanding dendritic cells in vivo enhances the induction of oral tolerance Modulating dendritic cells to optimize mucosal immunization protocols Interactions between commensal intestinal bacteria and the immune system Interleukin-17 family members and inflammation Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2 Impaired immune and acute-phase responses in interleukin-6-deficient mice Defective TCR expression in transgenic mice constructed using cDNA-based α- and β-chain genes under the control of heterologous regulatory elements Download references Matsumoto for the distribution of OT-II-transgenic mice Kopf (Swiss Federal Institute of Technology) and OT-II transgenic mice (C57BL/6) were provided by W.R Heath (The Walter and Eliza Hall Institute of Medical Research) the 21st Century Center of Excellence Program of Japan (S.A.) the World Premier International Research Center (S.A.) and the National Institutes of Health (AI070167 to S.A.) Satoshi Uematsu and Kosuke Fujimoto: These authors contributed equally to this work Osaka University Graduate School of Medicine Nihon University School of Dentistry at Matsudo Exploratory Research for Advanced Technology helped with the immunohistochemical analysis; Y.-J.J provided advice for the experiments and manuscript; S.U Supplementary Figures 1–9 and Supplementary Table 1 (PDF 3389 kb) Download citation The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission All content on this site: Copyright © 2025 Elsevier B.V., its licensors, and contributors. All rights are reserved, including those for text and data mining, AI training, and similar technologies. For all open access content, the relevant licensing terms apply. Volume 14 - 2023 | https://doi.org/10.3389/fmicb.2023.1258796 Salmonella enterica serovar Typhimurium (STm) is a major foodborne pathogen and poultry are a key reservoir of human infections To understand the host responses to early stages of Salmonella infection in poultry and mesenchymal cells that are characteristic of the lamina propria We infected these enteroids with wild-type (WT STm) a non-invasive mutant lacking the prgH gene (ΔprgH STm) or treated them with STm lipopolysaccharide (LPS) and analyzed the expression of innate immune related genes by qPCR at 4 and 8 h The localization of the tight junction protein expression was disrupted in WT STm infected enteroids but not ΔprgH STm or LPS treated enteroids suggesting a loss of epithelial barrier integrity The innate immune response to LPS was more pronounced in 2D enteroids compared to 3D enteroids and by 8 hpi the response in 3D enteroids was almost negligible when STm adhered to or invaded the enteroids both 2D and 3D enteroids exhibited an upregulation of inflammatory responses The presence of lamina propria cells in 3D enteroids resulted in the unique expression of genes associated with immune functions involved in regulating inflammation 2D and 3D enteroids showed temporal differences in response to bacterial invasion or adherence innate responses in 3D but not 2D enteroids continued to increase after infection with WT STm whereas the responses to the non-invasive strain decreased at 8 hpi in both 2D and 3D enteroids STm infection of chicken enteroids recapitulated several observations from in vivo studies of Salmonella-infected chickens including altered epithelial barrier integrity based on ZO-1 expression and inflammatory responses Our findings provide evidence that Salmonella-infected enteroids serve as effective models for investigating host-pathogen interactions and exploring the molecular mechanisms of microbial virulence although the 3D model mimics the host more accurately due to the presence of a lamina propria STm can colonise the gastrointestinal tract without an associated clinical disease The aim of this study was to disentangle the innate immune response between a system with (3D) and without (2D) lamina propria cells to STm infection by comparing the gene expression profiles between uninfected and infected enteroids we analyzed the effects of an invasion deficient strain on the innate immune response in each enteroid model Our study reveals marked differences in the response to STm infection in both models these models provide valuable insights into deciphering the distinct responses in systems where lamina propria cells are present or absent such that findings with simpler cell-based models should be interpreted with caution Experiments were performed using embryonic day 18 (ED18) Hy-Line Brown fertile embryos (Gallus gallus) obtained from the National Avian Research Facility Embryos were humanely culled under the authority of a UK Home Office Project License (PE263A4FA) in accordance with the guidelines and regulations of the Animals (Scientific Procedures) Act 1986 cells were resuspended in FOM media supplemented with 1X N2 supplement (TFS) 100 ng/mL human (hu) epidermal growth factor (huEGF 10 μM CHIR 99021 (Stratech Scientific) 10 μM Y27632 (Stem Cell Technologies) and 100 nM LDN193189 (Cambridge Bioscience) Cells were incubated for 3 h at 37°C counted and seeded at 2 × 105 cells in uncoated 24 well plates with 350 μl of FOM media supplemented with 1X N2 supplement 10 μM CHIR 99021 and incubated at 37°C which was maintained in the presence of 50 μg/mL of ampicillin (Merck) Bacteria were incubated for 18 h at 37°C with shaking at 180 rpm to an optical density of 1 at 600 nm and pelleted at 2,000 g for 10 min Bacteria were washed twice with PBS and resuspended in 10 mL of PBS Ten-fold serial dilutions were plated in duplicate on LB agar containing 20 μg/mL of naladixic acid incubated at 37°C overnight to determine viable counts retrospectively 3D enteroids were pelleted at 100 g for 4 min and reseeded at 200 enteroids per well on 24 well plates (Corning) in 400 μL of FOM media without antibiotics 2D enteroids were washed twice with PBS and cultured for a further 24 h in FOM without antibiotics the 3D and 2D enteroids were treated with WT or ΔprgH STm strains (2 × 105) LPS (1 ug/mL) derived from STm (product code L6143 At 4 and 8 h post-infection (hpi) the supernatant was removed and cells washed with PBS and lysed in RLT Plus buffer (Qiagen) containing 10 μg/mL 2-mercaptoethanol (Merck) 3D enteroids were infected with 1 × 103 The 3D enteroids were further homogenized using QIAshredder columns (Qiagen) Samples were stored at −20°C until use For immuno-fluorescent staining, chicken 2D enteroids were grown on 2% Matrigel (Corning) coated transwell inserts (VWR, 0.33 cm2) in 24 well plates (Orr et al., 2021) while 3D enteroids were grown as outlined above Chicken 2D and 3D enteroids were treated with WT or ΔprgH STm cells were gently washed with PBS and fixed with 4% w/v paraformaldehyde (TFS) for 15 min at room temperature and blocked with 5% v/v goat serum in permeabilization buffer (0.5% w/v bovine serum albumin and 0.1% w/v Saponin in PBS; Sigma-Aldrich) Permeabilization buffer was used to dilute all antibodies Cells were stained with mouse anti-human ZO-1 (Abcam clone A12) overnight at 4°C followed by the secondary antibody goat anti-mouse IgG1 Alexa Fluor®594 (TFS) for 2 h on ice Cells were counterstained with Hoechst 33,258 and Phalloidin Alexa Fluor®647 (TFS) to stain for nuclei and F-actin Slides were mounted using ProLong™ Diamond Antifade medium (TFS) Controls comprising of secondary antibody alone were prepared for each sample Images and Z-stacks were captured using an inverted LSM880 (Zeiss) with 40X and 63X oil lenses using ZEN 2012 (Black Edition) software and were analyzed using ZEN 2012 (Blue Edition) Z-stack modeling was performed using IMARIS software (V9) Total RNA from the enteroids was extracted using an RNeasy Plus Mini Kit (Qiagen) consisting of a genomic DNA column eliminator according to manufacturer’s instructions and quantified spectrophotometrically Five independent 3D enteroids samples and three independent 2D enteroids samples that were of high quality were used for qPCR analysis (RNA concentration of > 100 ng and a 260/230 ratio of 2) Reverse transcription was performed using the High Capacity Reverse Transcription Kit (Applied Biosystems) according to manufacturer’s instructions with random hexamers and oligo (dT)18 The cDNA samples were stored in −20°C until use The fluorescence emission was recorded after each cycling step Raw qPCR data quality threshold was set to 0.65-baseline correction to linear (derivative) and quantitation cycle (Cq) threshold method to auto (global) using the Real-Time PCR Analysis software 3.1.3 (Fluidigm) Principal component analysis was performed using prcomp function and plots were generated using ggplot in RStudio (V1.1.442) Statistical analysis of the differentially expressed genes (DEGs) between control (untreated) and LPS treated or between control and STm infected 2D or 3D enteroids was performed using GENEx5 and GenEx Enterprise (MultiD Analyses AB) Correction for multiple testing was performed with the estimation of false discovery rate using Dunn-Bonferroni correction threshold of 0.00054 DEG with a significant difference (P < 0.05) and a fold change of ≥ 1.5 were identified the fold change of untreated and treated samples at their respective timepoint was calculated Graphs were prepared using GraphPad Prism 9 This is consistent with the known role of T3SS-1 in promoting membrane ruffling and invasion Untreated and LPS-treated 2D and 3D enteroids exhibit unaltered organization of F-actin Confocal micrographs of F-actin organization in (A) untreated or (B) LPS-treated 2D and 3D enteroids Chicken 2D enteroids were cultured on Matrigel coated transwell inserts and images were retrieved using 20X objective both epithelial cells and the underlying mesenchymal cells with long F-actin filaments can be observed (white arrow) Images are representative of 3 independent experiments Scale bars = 200 μm (2D) and 100 μm (3D) WT STm infection remodels F-actin in chicken enteroids Confocal micrographs showing F-actin remodeling in (A) WT STm infected 2D and 3D enteroids dense F-actin staining can be observed surrounding the invading bacteria consistent with reorganization of subcortical actin stimulating membrane ruffling (white arrows) numerous bacteria can be observed within epithelial cells At 8 hpi the number of internalized bacteria was markedly higher in 3D enteroids inoculated with the same bacterial dose (B) No F-actin remodeling was observed in ΔprgH STm infected 2D and 3D enteroids at 8 hpi although bacteria were observed in close association with the apical surface of epithelial cells Scale bars = 100 μm and 50 μm Distribution of the tight junction protein ZO-1 at cell-cell junctions in untreated and LPS treated enteroids Confocal micrographs of ZO-1 distribution in (A) untreated or (B) LPS treated 2D and 3D enteroids show typical ZO-1 distribution at 8 h WT STm but not ΔprgH STm infection alters the distribution of the tight junction protein ZO-1 in chicken enteroids Confocal micrographs showing the distribution of ZO-1 in (A) WT STm infected 2D and 3D enteroids which leads to reduced ZO-1 expression in 2D enteroids and altered ZO-1 distribution in 3D enteroids at 8 hpi (yellow/magenta dash insert images) (B) ZO-1 distribution were unaltered in ΔprgH STm infected 2D and 3D enteroids at 8 hpi corresponding to the presence of different cell types such as immune cell in 3D enteroids while the effect of treatment (PC1) was preserved Principal component analysis (PCA) of gene expression profiles and WT and ΔprgH STm infected (A) 2D enteroids (n = 3) and (B) 3D enteroids (n = 5) at 4 hpi and 8 hpi alone and (C) with both datasets There was no core set of common DEGs between 2D and 3D enteroids at 4 and 8 h post-LPS treatment Of the 11 DEGs regulated by 3D enteroids at 4 h post-LPS treatment DEGs specifically regulated in 2D enteroids were related to TLR signaling (TOLLIP LYG2) and regulation of immune responses (BATF3 Chicken 2D enteroids exhibit more pronounced responses to LPS treatment (A) Pie charts indicating the number of statistically significant (P < 0.05) and non-significant (P > 0.05) DEGs in LPS treated 2D enteroids (n = 3) and 3D enteroids (n = 5) at 4 h and 8 h compared to time-matched DEGs with a significant difference (P < 0.05) and a fold change of ≥ 1.5 were identified (B) Venn diagram showing common and unique DEGs across each enteroid model and timepoint The mRNA expression levels of a majority of the DEGs increased by 2-7-fold at P-values of less than 0.05 in LPS treated enteroids compared to untreated enteroids except for SELE and TIMD4, which decreased in expression in 2D enteroids (Supplementary Material 2) Salmonella-derived LPS treatment of chicken 2D and 3D enteroids resulted in differential innate immune responses with 2D enteroids having a more pronounced response compared to 3D enteroids This core set of genes are involved in the regulation of immune responses The second largest set of common genes (16 DEGs) was shared between 2D enteroids at 4 hpi and 3D enteroids at 4 and 8 hpi These genes are involved in regulating immune responses (JUN were differentially expressed at 4 hpi in both models Innate immune response increase with time in WT STm infected 3D enteroids (A) Pie charts indicating number of statistically significant (P < 0.05) and non-significant (P > 0.05) DEGs in WT STm infected 2D enteroids (n = 3) and 3D enteroids (n = 5) at 4 hpi and 8 hpi compared to their time-matched The number of DEGs that were uniquely expressed in one or the other model differed substantially with the 2D enteroids expressing 5 unique genes; specifically those encoding a regulator of Notch signaling (DTX2) the 3D enteroids uniquely expressed 21 genes upon STm infection with a majority associated with immune cell functions The seven genes shared between 4 and 8 hpi are involved in regulating inflammation (IL10RA genes involved in the regulation of interferon (IFN) and IFN-inducible genes (IRF1 NLRC5) and macrophage and DC activities (CCL19 TIMD4) were found to be differentially expressed the data suggests that 2D and 3D enteroids have differing temporal responses to STm infection Lamina propria cells temporally govern the response to WT STm infection (A) A comparison of the fold change levels of the common DEGs between the enteroid models demonstrates increased expression levels in 2D enteroids compared to 3D enteroids at 4 hpi the expression levels of the common DEG are higher in 3D enteroids compared to 2D enteroids (B) The expression levels of a majority of the common DEGs decrease with time in WT STm infected 2D enteroids and increase with time in 3D enteroids Fold change values represent the mean of 3 (2D) or 5 (3D) independent experiments relative to their time-matched respective controls An additional four genes were upregulated in ΔprgH STm infection and were involved in the regulation of glycolysis (PFKFB3) and cytokine signaling (SOCS3) When disentangling the innate responses between the models 8 and 20 genes were differentially expressed only in 2D and 3D enteroids Similar to the response to WT STm infection ΔprgH STm infected 2D enteroids regulated CCL5 and DTX2 at 4 hpi and genes involved in the regulation of antigen presentation (CD83) and inhibition of NFκB activation (NFAIP2) a gene involved in cell-cell junctions (STEAP1) and a serine protease (PRSS23) were upregulated and by 8 hpi encoding a phosphatidylserine receptor for apoptotic cells was specifically regulated in 2D enteroids The shared genes in 3D enteroids were involved in nitric oxide synthesis (GCH1 negative regulation of inflammation (NFκBIZ and membrane trafficking in endosomes (SNX10) Chicken enteroids mount an innate immune response to non-invasive STm (A) Pie charts indicating the number of statistically significant (P < 0.05) and non-significant (P > 0.05) DEGs in STm infected 2D enteroids (n = 3) and 3D enteroids (n = 5) at 4 hpi and 8 hpi The data indicates that bacterial invasion is not required for 2D and 3D enteroids to mount an early response to STm but invasion is required in 3D enteroids to increase the responses over-time ΔprgH STm induces a rapid but brief innate immune response in chicken enteroids (A) Fold change difference in the common DEGs in ΔprgH STm infected 2D and 3D enteroids at 4 hpi and 8 hpi (B) The innate immune responses decrease with time in ΔprgH STm infected 2D and 3D enteroids Key KEGG pathways such as Toll-like and NOD-like receptor signaling pathways Cytosolic DNA-sensing pathway and Cytokine-cytokine receptor interaction were regulated in 3D enteroids Transcriptional regulation of innate immune responses does not require bacterial invasion in chicken enteroids (A) Venn diagram showing the DEGs that are altered by WT or ΔprgH STm infected 2D and 3D enteroids at 4 hpi and 8 hpi (B) Heat maps of the fold change difference in the DEGs (Fold change ≥ 1.5 and 250 CFU of WT STm infected 3D enteroids demonstrates that the transcriptional regulation of innate immune genes is not significantly affected by bacterial burden Fold change values represent the mean of 3 independent experiments To determine the effects of bacterial burden on the innate immune responses of lamina propria cells, we infected 3D enteroids with 1,000, 500 and 250 CFU of WT STm for 3 h and analyzed the mRNA expression levels using Fluidigm Biomark high-throughput qPCR. Few genes were significantly differentially regulated, with a fold change of ≥ 1.5 and P-values < 0.05, between the different doses of WT STm (Figure 11B) This demonstrates that the burden of bacteria does not significantly alter the transcriptional regulation of innate immune genes in chicken 3D enteroids and further supports the role lamina propria cells have on the regulation of responses to Salmonella intestinal enteroids represent the gold standard in vitro models for studying host-pathogen interactions the inner core of chicken 3D enteroids is solid and contains all cells that are present in the lamina propria because the isolated intact villi become enclosed to develop into 3D structures our 2D model contains all subsets of epithelial cells few IELs and a layer of mesenchymal cells representing the lamina basalis Chicken 2D and 3D enteroids offer valuable model systems in which to disentangle the interplay between epithelial/mesenchymal cells (2D) and epithelial/mesenchymal/lamina propria cells (3D) to reveal the cellular interplay that highly affects the overall response to danger signals including STm The 2D conformation may not be completely tight and some leakage of LPS to the basolateral sides of the cells may have occurred the chicken 3D enteroid resembles the natural conformation of the epithelium and prevents access of LPS to the basolateral sides of the cells similar to the in vivo situation we would recommend to either grow the 2D enteroids on a transwell insert and measure epithelial barrier integrity prior to LPS exposure or use the 3D conformation intracellular bacterial replication was not quantified we demonstrated that varying the size of the bacterial inoculum induced no significant changes in gene expression levels and therefore the differences in temporal gene expression may be related to the presence of lamina propria cells in 3D enteroids and the structural differences between the models Future studies involving use of fluorescence dilution may allow replication of Salmonella to be measured at the single cell level including to understand the fate of STm in different types of infected cells the differences in temporal gene expression may be related to the presence of lamina propria cells in 3D enteroids and the structural differences between the models we utilized the small intestines from ED18 embryos therefore taken together both these attributes may be driving an immune response independent of bacterial invasion Further research is necessary to examine the differences in the immune response between enteroids-derived from older chickens and how different STm virulence factors affect immune responses most key features of the immune response against STm in vivo could be replicated in 2D and 3D enteroids Future single-cell RNA sequencing studies will help to fully resolve the contributions and cross-talk between the different epithelial mesenchymal and immune cells to STm in 2D and 3D enteroids Our data indicates that chicken 3D enteroids provide a suitable ex vivo intestinal model to examine the relationship between macrophage polarization and immuno-metabolism during infection with different Salmonella strains the chicken 2D and 3D enteroids allowed for the first time a description of the distinct innate immune responses exhibited by epithelial cells and lamina propria cells The enteroid models replicated several observations demonstrated after in vivo infection of chickens with Salmonella including the alteration of tight junctions and the induction of inflammatory responses The chicken enteroid models offers many advantages over other models to reduce animal use in the study of host-pathogen interactions The original contributions presented in this study are included in the article/Supplementary material further inquiries can be directed to the corresponding authors The animal study was approved by The Roslin Institute’s Animal Welfare and Ethical Review Board The study was conducted in accordance with the local legislation and institutional requirements LV secured funding to undertake the work and supervised the project and JM performed the experiments and analyzed the data with support of PV The author(s) declare financial support was received for the research This work was supported by the Biotechnology and Biological Sciences Research Council Institute Strategic Program Grant funding (BB/X010937/1 and BBS/E/D/20002174) and an iCase doctoral studentship to TN (BB/MO14819) We wish to thank the animal caretakers of the National Avian Research Facility for the supply of eggs and for helpful advice from staff at the Roslin Institute’s Bio-imaging Facility the author has applied a Creative Commons Attribution (CC BY) license to any Authors Accepted Manuscript version arising from this submission The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1258796/full#supplementary-material Identification of key transcription factors and their functional role involved in Salmonella typhimurium infection in chicken using integrated transcriptome analysis and bioinformatics approach Antigen sampling CSF1R-expressing epithelial cells are the functional equivalents of mammalian m cells in the avian follicle-associated epithelium Observations on the pathogenesis of experimental Salmonella typhimurium infection in chickens Infection by Salmonella enterica serovar typhimurium DT104 modulates immune responses and the function of 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited *Correspondence: Kate Sutton, a2F0ZS5zdXR0b25Acm9zbGluLmVkLmFjLnVr; Lonneke Vervelde, bG9ubmVrZS52ZXJ2ZWxkZUByb3NsaW4uZWQuYWMudWs= Metrics details Mononuclear phagocytes are essential for the maintenance of intestinal homeostasis and have crucial roles in inflammatory bowel diseases (IBDs) The maintenance of robust gut homeostasis requires the presence of two mononuclear phagocyte populations of distinct origin and function CD11b+CD103+ dendritic cells (DCs) are poised to migrate to the draining mesenteric lymph nodes to initiate and modulate primary T cell immune responses CX3C-chemokine receptor 1 (CX3CR1)+ mononuclear phagocytes (which have also been termed DCs) do not migrate to the mesenteric lymph nodes but function locally in the mucosa and may maintain effector and regulatory T cells Mouse research requires a better understanding of mononuclear phagocytes during disease but in humans we lack fundamental insights into the steady state functions of these cells For research into IBDs to capitalize on the progress made with small animal models it will be important to align human and mouse mononuclear phagocyte populations The intestinal landscape comprises the host's own tissue and immune cells as well as a diverse intestinal microbiota Intricate regulatory mechanisms have evolved to maintain peaceful coexistence at this site the breakdown of which can result in devastating inflammatory bowel diseases (IBDs) Mononuclear phagocytes promote both innate and adaptive immune responses in the gut and are essential for the maintenance of intestinal homeostasis we review the origins and functions of the mononuclear phagocytes found in the intestinal lamina propria highlighting the problems that have arisen from their classification Understanding these cells in their physiological context will be important for developing new therapies for IBDs Microbial ecology of the gastrointestinal tract The genetics and immunopathogenesis of inflammatory bowel disease Bacterial invasion: the paradigms of enteroinvasive pathogens Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut Dendritic cells in intestinal homeostasis and disease Recent progress in understanding the phenotype and function of intestinal dendritic cells and macrophages Dendritic cells in intestinal immune regulation Intestinal lamina propria dendritic cell subsets have different origin and functions Origin of the lamina propria dendritic cell network These two articles established that CD103+ and CX 3 CR1+ lamina propria DCs originate from classic DC precursors and Ly6C+ monocytes antigen sampling cells migrate in lymph and serve classical dendritic cell functions This elegant study used two-photon microscopy and the cannulation of intestinal lymphatics to show that CX 3 CR1+ lamina propria DCs do not migrate from the lamina propria to the mesenteric lymph nodes Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans In vivo analysis of dendritic cell development and homeostasis The origin and development of nonlymphoid tissue CD103+ DCs Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8α+ conventional dendritic cells Transepithelial pathogen uptake into the small intestinal lamina propria Microbe sampling by mucosal dendritic cells is a discrete Intestinal villous M cells: an antigen entry site in the mucosal epithelium Regulation of intestinal dendritic cell migration and activation by plasmacytoid dendritic cells TNF-α and type 1 IFNs after feeding a TLR7/8 ligand Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells A nice study that established the crucial role of CCR7-dependent DC migration for oral tolerance establishment Retinoic acid receptor signaling levels and antigen dose regulate gut homing receptor expression on CD8+ T cells Stromal mesenteric lymph node cells are essential for the generation of gut-homing T cells in vivo Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis The first paper to show that intestinal mononuclear phagocytes are in involved in the maintenance of FOXP3+ T Reg cells in the lamina propria in vivo ATP drives lamina propria TH17 cell differentiation The immune geography of IgA induction and function The biology of intestinal immunoglobulin A responses Adaptive immune regulation in the gut: T cell-dependent and T cell-independent IgA synthesis Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury Efficient colonic mucosal wound repair requires Trem2 signaling Regulatory mechanisms of immune responses to intestinal bacteria A human gut microbial gene catalogue established by metagenomic sequencing Immune adaptations that maintain homeostasis with the intestinal microbiota Symbiotic bacteria direct expression of an intestinal bactericidal lectin The nuclear IκB protein IκBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria Intestinal macrophages: unique effector cells of the innate immune system IκBNS inhibits induction of a subset of Toll-like receptor-dependent genes and limits inflammation a negative regulator of Toll-like receptor-interleukin 1 receptor signaling Intestinal inflammation in mice deficient in Tir8 an inhibitory member of the IL-1 receptor family Increased susceptibility to colitis-associated cancer of mice lacking TIR8 an inhibitory member of the interleukin-1 receptor family TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection Requirement for lymphoid tissue-inducer cells in isolated follicle formation and T cell-independent immunoglobulin A generation in the gut Enteric flora expands gut lamina propria CX3CR1+ dendritic cells supporting inflammatory immune Responses under Normal and Inflammatory Conditions Salmonella induces flagellin- and MyD88-dependent migration of bacteria-capturing dendritic cells into the gut lumen Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria References 60 and 61 established the effects of a specific bacterial strain in determining the composition of the T helper cell compartment in the mouse gut TLR11 activation of dendritic cells by a protozoan profilin-like protein Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response Gut commensal bacteria direct a protective immune response against Toxoplasma gondii Gr1+ inflammatory monocytes are required for mucosal resistance to the pathogen Toxoplasma gondii Distribution of human colonic dendritic cells and macrophages Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-γ axis Cytokine profiles differ in newly recruited and resident subsets of mucosal macrophages from inflammatory bowel disease TREM-1-expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases Intestinal epithelial cells promote colitis-protective regulatory T-cell differentiation through dendritic cell conditioning Human intestinal epithelial cells promote the differentiation of tolerogenic dendritic cells A subset of human dendritic cells in the T cell area of mucosa-associated lymphoid tissue with a high potential to produce TNF-α Mouse models of intestinal inflammation as tools to understand the pathogenesis of inflammatory bowel disease Differential activity of IL-12 and IL-23 in mucosal and systemic innate immune pathology Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system This article described a new colitis model and showed the effects of the gut immune system on the microbiota Colitis-associated colorectal cancer driven by T-bet deficiency in dendritic cells Ulcerative colitis and autoimmunity induced by loss of myeloid αv integrins Loss of integrin αvbeta8 on dendritic cells causes autoimmunity and colitis in mice References 79 and 80 establish the role of DC-derived αVβ8 integrin-mediated activation of latent TGFβ in the maintenance of gut homeostasis The role of dendritic cells in the development of acute dextran sulfate sodium colitis Conventional dendritic cells regulate the outcome of colonic inflammation independently of T cells IL-10-dependent partial refractoriness to Toll-like receptor stimulation modulates gut mucosal dendritic cell function A role for CD47 in the development of experimental colitis mediated by SIRPα+CD103− dendritic cells Elimination of local macrophages in intestine prevents chronic colitis in interleukin-10-deficient mice Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis Intestinal macrophages: differentiation and involvement in intestinal immunopathologies Increased expression of DC-SIGN+IL-12+IL-18+ and CD83+IL-12−IL-18− dendritic cell populations in the colonic mucosa of patients with Crohn's disease CD83+CCR7− dendritic cells accumulate in the subepithelial dome and internalize translocated Escherichia coli HB101 in the Peyer's patches of ileal Crohn's disease Macrophage migration inhibitory factor activates antigen-presenting dendritic cells and induces inflammatory cytokines in ulcerative colitis Characteristics of intestinal dendritic cells in inflammatory bowel diseases Tumour necrosis factor-α production stimulated by heat shock protein 70 and its inhibition in circulating dendritic cells and cells eluted from mucosal tissues in Crohn's disease Cutting edge: inflammatory responses can be triggered by TREM-1 a novel receptor expressed on neutrophils and monocytes Th1/Th17 immune response is induced by mesenteric lymph node dendritic cells in Crohn's disease Increased expression of interleukin 17 in inflammatory bowel disease Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis Interleukin-23 drives innate and T cell-mediated intestinal inflammation IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6 Dependence of intestinal granuloma formation on unique myeloid DC-like cells A genome-wide association study identifies IL23R as an inflammatory bowel disease gene Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to promote interleukin-17 production in human memory T cells Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn's disease susceptibility A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1 A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1β production NOD2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation This study provides intriguing evidence for a surprising potential link of two IBD candidate genes ( NOD2 and ATG16L1 ) involved in pathogen sensing and autophagy Distinct differentiation potential of blood monocyte subsets in the lung Collaboration of epithelial cells with organized mucosal lymphoid tissues and double-negative Peyer's patch dendritic cells Download references This work was supported by the Israel Science Foundation The Thyssen Foundation and a research grant from the estate of Edith F Groups of lymphoid nodules present in the small intestine They occur massed together on the intestinal wall opposite the line of attachment of the mesentery B cell follicles and interfollicular T cell areas Dynamic aggregates of lymphoid cells found in the mouse small intestinal lamina propria; similar lymphoid aggregates have also been identified in the mouse colon Isolated lymphoid follicles have germinal centres and an overlying follicle-associated epithelium containing microfold (M) cells specialized for antigen uptake They are formed de novo in adult animals in response to commensal organism-derived stimuli and although their function is not completely clear they may be inductive sites of immunity in the intestinal lamina propria A thymidine analogue that can be incorporated into DNA during DNA replication Treatment with bromodeoxyuridine enables the detection of cells that are dividing or have recently divided A subset of blood monocytes that is derived from MDPs in the bone marrow and emigrates in a CCR2-dependent way to the blood Ly6Chi monocytes can home back to the bone marrow in the steady state and differentiate into Ly6Clow monocytes Ly6Chi monocytes can give rise to splenic TIP-DCs Furthermore they are the precursors of CX3CR1+ mononuclear phagocytes in the intestinal lamina propria Ly6Chi monocytes can contribute to tissue remodelling and healing processes Tubular invaginations of the intestinal epithelium Paneth cells are found at the base of the crypts along with continuously dividing stem cells which are the source of all intestinal epithelial cells The genomic content of a sample of organisms obtained from a common habitat separated from the mother by Caesarean section These animals can be monocolonized with defined microbial species in order to investigate specific relationships between the host and the microbiota An approach that involves rapidly scanning single nucleotide polymorphism markers across the complete genomes of many individuals to find genetic variations associated with a particular disease A carbohydrate modification of the P-selectin glycoprotein 1 (PSGL1) SLAN is expressed by a subset of DCs found in human blood and is recognized by the monoclonal antibody MDC8 Any process involving degradative delivery of a portion of the cytoplasm to the lysosome that does not involve direct transport through the endocytic or vacuolar protein sorting pathways Paneth cells are found at the base of intestinal crypts and produce antimicrobial proteins and peptides Download citation Metrics details Telocytes have recently emerged as unique interstitial cells defined by their extremely long thin and moniliform prolongations termed telopodes Despite growing evidence that these cells consistently reside in the stromal compartment of various organs from human beings studies dealing with telocytes in structures of the oral cavity are scarce the present morphologic study was undertaken to explore for the first time the presence and specific localization of telocytes within tissues of the normal human tongue a complex muscular organ whose main functions include taste Telocytes were initially identified by CD34 immunostaining and confirmed by CD34/PDGFRα double immunofluorescence and transmission electron microscopy CD34+/PDGFRα+ telocytes were organized in interstitial meshworks either in the tongue lamina propria or in the underlying striated muscle Lingual telocytes were immunonegative for CD31 Telopodes were finely distributed throughout the stromal space and concentrated beneath the lingual epithelium and around CD31+ vessels They also enveloped salivary gland units outside the α-SMA+ myoepithelial cells and delimited lymphoid aggregates These findings establish telocytes as a previously overlooked interstitial cell population worth investigating further in the setting of human tongue pathophysiology we undertook this morphologic study to provide a first proof of the existence and microanatomic localization of TCs in tissues of the normal human tongue a peculiar striated muscular organ covered in oral mucosa with functions as various as taste food manipulation and swallowing initiation Immunohistochemical localization of telocytes (TCs)/CD34+ stromal cells in the interstitium of the human tongue striated muscle (A) Hematoxylin and eosin staining demonstrating the normal appearance of the tongue muscle (B–H) CD34 immunohistochemistry with hematoxylin counterstain (B,C) A diffuse CD34+ reticular network is evident in the perimysium encasing skeletal muscle bundles (B) and around intramuscular vessels (dashed arrows) and nerves (arrows) (C) (D–F) The endomysium is populated by a dense meshwork of CD34+ TCs projecting long and moniliform telopodes in close relationship with skeletal muscle fibers (G) CD34+ TCs intimately encircle intramuscular arterioles (BV (H) TCs form an outer sheath (arrows) for intramuscular nerves (NE) and ganglia (asterisk) Double immunofluorescence staining of human tongue tissue sections (A–F) CD34 (green) and platelet-derived growth factor receptor α (PDGFRα; red) immunofluorescence with 4′,6-diamidino-2-phenylindole (DAPI; blue) counterstain for nuclei Single green and red images are shown in (A,D,B,E) All telocytes (TCs)/CD34+ stromal cells within the tongue stromal compartment are PDGFRα+ Colocalization of CD34 and PDGFRα on the TC surface gives rise to yellow staining Ultrastructural identification of telocytes (TCs) in human tongue muscle interstitium (A) Semithin sections stained with toluidine blue and observed by light microscopy Spindle-shaped cells with very long and thin moniliform cytoplasmic processes (arrows) are observed in the stroma surrounding skeletal muscle fibers (B–D) Tongue muscle ultrathin sections stained with UranyLess and bismuth subnitrate solutions and observed by transmission electron microscopy TCs (digitally colored in blue) are ultrastructurally identifiable as interstitial cells with telopodes (Tp) namely cytoplasmic prolongations with a moniliform silhouette due to the alternation of thin segments (podomers) and expanded portions (podoms) oval or piriform cell body mostly occupied by the nucleus dashed arrow) and in close relationship with skeletal muscle fibers (C,D) we revealed that spindle-shaped TCs/CD34+ stromal cells displaying distinctive moniliform/varicose prolongations (i.e telopodes) constitute a previously unrecognized cell meshwork spanning in the whole stromal compartment of the normal human tongue from the basement membrane underneath the stratified squamous epithelium to the underlying lamina propria and the interstitium of the deeper striated muscle Even though we frankly acknowledge that a comprehensive ultrastructural analysis of human tongue stromal space was not possible because of the unavailability of specimens of the lamina propria it is remarkable that we could confirm the presence of cells ultrastructurally identifiable as TCs within the tongue muscle interstitium in the same locations previously disclosed by light microscopy the arrangement of a subset of lingual TCs to precisely delimit and separate subepithelial lymphoid aggregates from the surrounding tissue may be in favor of a participation of those cells in local immunosurveillance the present findings on the human tongue add to a growing body of data published over the past ten years and strengthen the notion that it is time to reconsider our knowledge of the human microscopic anatomy by recognizing TCs as a distinctive stromal cell identity further studies should be devoted to definitely prove the multiple proposed TC functions and to decipher the mechanisms mediating the cross-talk between TCs and other cell types the ‘strategic’ TC locations that we herein highlighted within the stromal space of the normal human tongue represent the essential groundwork for upcoming functional studies as well as for future investigations of TCs in different tongue pathologies One paraffin-embedded normal tongue tissue block per case was selected with informed consent to prepare tissue slides for histochemical and immunohistochemical analyses The study was carried out in accordance with the Declaration of Helsinki and the approval of the institutional review board of Careggi University Hospital Tongue sections (3 µm thick) from each paraffin-embedded tissue block were deparaffinized and routinely stained with hematoxylin and eosin to confirm the normal tissue appearance (i.e absence of any obvious histopathologic feature) Negative controls were performed by overnight incubation of serial sections with isotype-matched and concentration-matched irrelevant mouse IgG (Sigma-Aldrich Antigen-antibody complexes were revealed by sequentially applying to tissue sections biotinylated secondary antibodies streptavidin peroxidase reagent and 3-amino-9-ethylcarbazole (AEC TA-125-SA; Lab Vision) chromogenic solution tissue slides were mounted and observed under a Leica DM4000 B microscope equipped with a Leica DFC310 FX 1.4-megapixel digital color camera and the Leica software application suite LAS V3.8 (Leica Microsystems TCs and telopodes detected in electron microscopy images were digitally colored in blue using Adobe Photoshop CS6 software (Adobe Systems Telocytes heterogeneity: From cellular morphology to functional evidence TELOCYTES - a case of serendipity: the winding way from Interstitial Cells of Cajal (ICC) via Interstitial Cajal-Like Cells (ICLC) to TELOCYTES The history of telocyte discovery and understanding cardiac and smooth muscle interstitium: morphological and functional aspects a distinct type of cell among the stromal cells present in the lamina propria of jejunum Identification of telocytes in skeletal muscle interstitium: implication for muscle regeneration Reappraising the microscopic anatomy of human testis: identification of telocyte networks in the peritubular and intertubular stromal space Morphological evidence of telocytes in human synovium miR-193 expression differentiates telocytes from other stromal cells Protein profiling of human lung telocytes and microvascular endothelial cells using iTRAQ quantitative proteomics Comparative proteomic analysis of human lung telocytes with fibroblasts Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells CD34+ stromal cells/fibroblasts/fibrocytes/telocytes as a tissue reserve and a principal source of mesenchymal cells Telocytes in human fetal skeletal muscle interstitium during early myogenesis Telocytes of the human adult trigeminal ganglion Telocytes express PDGFRα in the human gastrointestinal tract Cardiac telocytes are double positive for CD34/PDGFR-α Telocytes in minor salivary glands of primary Sjögren’s syndrome: association with the extent of inflammation and ectopic lymphoid neogenesis Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium Human myometrium - the ultrastructural 3D network of telocytes Cardiac telocytes – their junctions and functional implications Telocytes and their extracellular vesicles—Evidence and Hypotheses Telocytes transfer extracellular vesicles loaded with microRNAs to stem cells Cardiac telocyte-derived exosomes and their possible implications in cardiovascular pathophysiology Extracellular vesicles release by cardiac telocytes: electron microscopy and electron tomography Telocytes in their context with other intercellular communication agents Telocytes as supporting cells for myocardial tissue organization in developing and adult heart Telocytes play a key role in prostate tissue organisation during the gland morphogenesis The secretome of myocardial telocytes modulates the activity of cardiac stem cells Human resident CD34+ stromal cells/telocytes have progenitor capacity and are a source of αSMA + cells during repair Telocyte implications in human pathology: an overview The failing human heart is characterized by decreased numbers of telocytes as result of apoptosis and altered extracellular matrix composition Evidence for progressive reduction and loss of telocytes in the dermal cellular network of systemic sclerosis The potential role of telocytes in tissue engineering and regenerative medicine Transplantation of telocytes attenuates unilateral ureter obstruction-induced renal fibrosis in rats Telocytes are the physiological counterpart of inflammatory fibroid polyps and PDGFRA-mutant GISTs Telocytes in normal and keratoconic human cornea: an immunohistochemical and transmission electron microscopy study Molecular phenotypes of the human kidney: Myoid stromal cells/telocytes and myoepithelial cells Telocytes subtypes in human urinary bladder Telocytes in female reproductive system (human and animal) Temporospatial localization of telocytes during esophageal morphogenesis in rabbit Evolution of the structure and function of the vertebrate tongue A three-dimensional atlas of human tongue muscles Critical review: Cardiac telocytes vs cardiac lymphatic endothelial cells Telocytes in human skin–are they involved in skin regeneration Myofibroblasts: paracrine cells important in health and disease Subepithelial telocytes are an important source of Wnts that supports intestinal crypts Exosomes derived from cardiac telocytes exert positive effects on endothelial cells An in vitro investigation of telocytes-educated macrophages: morphology Phenotypical and ultrastructural features of Oct4-positive cells in the adult mouse lung Telocytes within human skeletal muscle stem cell niche Molecular and cellular regulatory mechanisms of tongue myogenesis A Wnt/Notch/Pax7 signaling network supports tissue integrity in tongue development Telocytes in the interstitium of human exocrine pancreas: ultrastructural evidence Surgical margins in head and neck squamous cell carcinoma: what is ‘close’ Postoperative pathologic assessment of surgical margins in oral cancer: A contemporary review Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis Download references This work was supported by research funds granted to Dr dell’Università e della Ricerca (FFABR 2017) and the University of Florence Department of Experimental and Clinical Medicine Institute of Histopathology and Molecular Diagnosis Manetti conceived and designed the research study Manetti contributed to the acquisition of data Manetti contributed to the analysis and interpretation of data Manetti prepared the figures and wrote the paper All authors have read and approved the final version of the manuscript Download citation DOI: https://doi.org/10.1038/s41598-019-42415-3 Metrics details Interpretation of gastrointestinal tract mesenchymal lesions is simplified merely by knowing in which anatomic layer they are usually found Kaposi sarcoma is detected on mucosal biopsies whereas inflammatory fibroid polyp is nearly always in the submucosa Gastrointestinal stromal tumors (GISTs) are generally centered in the muscularis propria Schwannomas are essentially always in the muscularis propria Mesenteric lesions are usually found in the small bowel mesentery Knowledge of the favored layer is even most important in interpreting colon biopsies as many mesenschymal polyps are encountered in the colon Although GISTs are among the most common mesenchymal lesions we will concentrate our discussion on other mesenchymal lesions some of which are in the differential diagnosis of GIST Glomus tumor. (a) These generally arise in the muscularis propria of the stomach. The cells are perfectly round. There is hemangiopericytoma-like vascular pattern. (b) Note the round nuclei and the well-developed cell borders. (c) Smooth muscle actin immunolabeling. Glomus cells are modified smooth muscle cells. They express actin and calponin but not desmin. (d) Collagen type 4 immunolabeling. Each cell is invested in a network of collagen type 4. Malignant-appearing gastrointestinal glomus tumor. (a) This lesion arose in the muscularis propria of the small bowel. It has areas similar to those depicted in Figure 1 but with transitions to solid and spindle zones. (b) Note the spindling and mitotic activity. (c) Calponin stain. (d) Collagen type 4 stain. Gastric Kaposi sarcoma. (a) This example is classic and features spindle cells, hemosiderin deposition, and hyaline globule formation. (b) This more subtle example merges with the existing lamina propria but appears more spindled and sclerotic. Such examples are easily mistaken for fibrosis. (c) HHV8 immunolabeling. (d) CD117 immunolabeling. This expression has no correlation with KIT mutations but can lead to a diagnostic pitfall. (a) There is a proliferation of Schwann cells and peculiar appearing ganglion cells in colonic lamina propria (b) Benign fibroblastic polyp of the colon/perineurioma This example is associated with a serrated polyp These lamina propria lesions are brightly eosinophilic and have a suggestion of palisading They are incidental sporadic (as opposed to syndromic) lesions detected at screening colonoscopy Tumor nodules are generally composed of solid sheets of cells that surround gaping capillary vessels showing a hemangiopericytoma-like pattern Tumor cells also tend to be present in the muscular walls of larger vessels The individual tumor cells are round with sharply defined cell membranes Some tumors have brightly eosinophilic cytoplasm Although most glomus tumors behave in a benign fashion, rare examples metastasize and are lethal (Figure 2) we mention several commonly encountered lesions of each layer If the spindle cell proliferation is noted it typically appears more spindled than normal lamina propria and often contains hemosiderin The proliferation is composed of CD34-reactive spindle cells Antibodies to HHV8 are also available to confirm this impression Interest in the condition remained relatively dormant in the literature for the next 100 years until the disease began manifesting in immunosuppressed individuals first in the setting of iatrogenic immunosuppression associated with solid organ transplantation and later as a feature of AIDS females comprise a higher percentage of those affected; the male-to-female ratio ranges from 2 to 3:1 Although Kaposi’s sarcoma may assume a chronic or aggressive course in these patients regression frequently follows discontinuation of the immunosuppressive treatment Patients receiving immunosuppressive agents for various other collagen vascular and skin disorders are also included in this category KS frequently assumes an aggressive course Areas prone to KS in the adult AIDS population include: the oral cavity Lesions more often involve the upper half of the body than in classic KS Postmortem studies have demonstrated a high incidence of concurrent disease in lymph nodes Pediatric cases of AIDS-KS have a proclivity for lymphadenopathic disease and lesions have been detected in adults from ages 20 to 90 years with a peak incidence between the ages of 40 and 60 years (mean age about 50 years) and the lesions are detected during routine colonoscopy Solitary lesions are not associated with genetic syndromes ganglioneuromatous polyposis is associated with familial adenomatous polyposis (FAP) and diffuse ganglioneuromatosis is associated with multiple endocrine neoplasia (MEN) type IIB and with NF1 Diffuse ganglioneuromatosis is found in virtually all patients with MEN IIB and often antedates the development of the endocrine neoplasms Patients with MEN IIB and ganglioneuromatosis present with diverse GI symptoms Most syndromic GI tract ganglioneuromas are found in the colorectum and in patients younger (mean age of about 35 years) than those who have sporadic isolated ganglioneuromas At low magnification, polypoid sporadic ganglioneuromas often resemble juvenile or inflammatory polyps, in that they have disturbed crypt architecture and expanded lamina propria. At higher magnification, the lamina propria is expanded by collections of spindle cells within a fibrillary matrix and irregular nests and groups of ganglion cells (Figure 4a) Sporadic examples may also have submucosal extension and a plexiform arrangement involving the submucosal nerve plexus such that they superficially resemble neurofibromas (differing by the presence of many ganglion cells) The ganglioneuromas in ganglioneuromatous polyposis show overlapping features with sporadic ganglioneuromas but tend to be more variable and have more numerous ganglion cells and filiform architecture the process is centered in the myenteric plexus is either diffusely intramural or transmural and consists of fusiform expansions or confluent transmural ganglioneuromatous proliferations These lesions are easily diagnosed without immunohistochemistry but the spindle cells react with S100 protein and the ganglion cells mark with neuron specific enolase (NSE) The primary distinction is from neurofibroma which is based on the presence of ganglion cells in ganglioneuromas and their lack in neurofibromas NSE or synaptophysin staining may help detect them Ganglioneuromas are distinguished from gangliocytic paraganglioma (PGL)by the presence of epithelioid cells in gangliocytic PGL; these latter cells may be keratin positive gangliocytic PGLs arise primarily in the duodenum Sporadic ganglioneuromas are treated by polypectomy and seldom recur Patients with syndromic ganglioneuromas must be carefully followed Those with NF1 may develop other neural lesions including malignant peripheral sheath tumors; those with MEN IIB may develop endocrine neoplasms Ganglioneuromatous polyposis may herald Cowden disease whereas the diffuse type is most likely associated with NF1 and MEN IIB This latter type may cause strictures requiring resections Benign fibroblastic polyps are managed by simple polypectomy and require no endoscopic follow-up The lesional cells are diffusely immunoreactive with S100 protein rare associated axons may be highlighted with a NFP immunostain The main differential diagnosis is with colonic neurofibroma an important distinction given its clinical association with NF1 colonic neurofibromas display more cellular heterogeneity with more nuclear variability and varying amount of cytoplasm the spindle cells in neurofibromas are only focally immunoreactive with S100 protein which are highlighted with a NFP immunostain mucosal Schwann cell hamartomas are unassociated with syndromic states Endoscopic follow-up is not necessary after diagnosis (a) The tumor is seen here occupying the mucosa and submucosa and is composed of bland spindle to polygonal plump bland cells with moderate amount of pink cytoplasm and visible nucleoli Note scattered cells with intracytoplasmic brown pigment (b) Higher magnification shows better nuclear detail they are unlike the macronucleoli typical of melanoma (c) This high magnification image shows a psammoma body These were rare in this particular example Note the scattered intracytoplasmic pigment (d) A Ki67 immunostain shows a low proliferation index (e) The lesional cells are immunoreactive with HMB45 (f) A S100 immunostain highlights the lesional cells Benign epithelioid nerve sheath tumors are identified at colonoscopy as incidental polyps37 (the mean age in our series was 58.6 years) although we have also rarely encountered larger examples None of the patients that we have encountered with these tumors has had a known history of neurofibromatosis or MEN IIB Benign epithelioid nerve sheath tumor (epithelioid schwannoma) (a) The lesion in fact involved the full thickness of the bowel and they are present in a background of eosinophils but it seldom arises in the GIT and lacks an inflammatory backdrop GIST is the key entity in the muscularis propria and is well studied and will be addressed only briefly in this discussion The stomach is the most common site for GISTs and they are occasionally diagnosed on mucosal biopsies those diagnosed on biopsies are aggressive lesions that have invaded the mucosa GISTs are mesenchymal tumors arising in the GIT and within the abdomen with no demonstrable GI connection and clarification of their immunohistochemical profile careful morphological examination and clinicopathological correlation remain essential for excluding mimics and for assessing likely behavior in this heterogeneous group of neoplasms This neoplasm was shown to have SDHB loss on immunolabeling Note the monotonous appearance of the neoplastic cells About a third of gastric adenocarcinomas express DOG1 a potential pitfall in the differential diagnosis of epithelioid gastrointestinal stromal tumor (f) Epithelioid gastrointestinal stromal tumor Some examples appear similar to gastric adenocarcinomas although there tends to be less conspicuous nuclear pleomophism (and usually no mucosal component) (a) The lymphoid cuff is a diagnostic clue on frozen sections even though gastric schwannoma almost always arises in the muscularis propria (b) The mild nuclear pleomorphism and the scattered background lymphocytes differ from the appearance of GIST which typically has essentially no inflammatory cells and very monomorphic nuclei The features are those of a nerve sheath tumor Retroperitoneal schwannomas are the source for diagnostic issues on needle biopsies This example shows strong diffuse keratin expression GIT schwannomas are typically not encapsulated, a feature that distinguishes them from schwannomas in the peripheral nervous system. They can also be plexiform. Diffuse intralesional lymphocytes are seen in all cases (Figure 10b) They are composed of interlacing bundles of spindle cells that are only weakly palisaded Occasional cases show epithelioid morphology Most cases present scattered cells with nuclear atypia and vascular hyalinization can be encountered in some examples Although schwannomas appear quite similar to GISTs the lymphoid cuff is a tip-off that they are These tumors are all strongly S100 protein-positive and lack muscle markers and CD117 gastric schwannomas lack KIT and PDGFRA mutations (0/9 cases studied) Few studied cases show multiple copies of chromosomes 22 Most mesenteric lesions are found in the small bowel mesentery, but all of them can affect the gastric mesentery. These include fibromatosis, inflammatory myofibroblastic tumor, sclerosing mesenteritis (which has overlapping features with retroperitoneal fibrosis/IgG4-related sclerosing diseases), and calcifying fibrous pseudotumor. Their features are summarized in Table 2 Mesenteric fibromatosis is probably the commonest among the intra-abdominal fibromatosis group It usually presents as a slowly growing mass that involves small bowel mesentery or retroperitoneum where distinction may become extremely difficult from retroperitoneal fibrosis There are cases associated with pregnancy and Crohn’s disease even though the majority is considered to be secondary to trauma in individuals with the appropriate predisposition Mesentric fibromatosis in patients with Gardner’s syndrome appears to have a substantially higher recurrence rate than in patients without this syndrome Gardner’s syndrome is an autosomal dominant familial disease with a female predilection and consists of numerous colorectal adenomatous polyps a disorder caused by germline adenomatous polyposis coli (APC) gene mutations It is associated with an 8–12% incidence of developing fibromatosis intestinal and extra-intestinal neoplasms typically arise through bi-allelic (germline then somatic) inactivation of the APC gene whereas the corresponding tumors in non-FAP patients occur either through somatic bi-allelic APC inactivation or somatic mutation of a single beta-catenin allele As the various FAP-associated tumors have been studied somatic alterations of the APC/beta-catenin pathway have been initially detected in familial examples and then subsequently demonstrated in the sporadic counterparts The first tumors studied were gastrointestinal adenomas all of which occur more frequently in FAP patients than in controls It has been estimated that FAP patients in general have an 852-fold increased risk of developing desmoids The pale collagen contrasts with the eosinophilic vascular walls a feature that is chatracteristic of mesenteric examples Cytoplasmic staining is not considered positive for diagnosis of fibromatosis (a) Inflammatory myofibroblastic tumor The lesional myofibroblastic cells display prominent nucleoli This shows the stellate contours of the myofibroblastic cells to advantage It may display IgG4 immunolabeling in plasma cells but lacks the storiform process of classic IgG4-related fibrosclerosis but may be in a spectrum with it Sclerosing mesenteritis (also known as mesenteric panniculitis and mesenteric lipodystrophy) most commonly affects the small bowel mesentery presenting as an isolated large mass although about 20% of patients have multiple lesions The etiology remains unknown though it is assumed to reflect a reparative response although the stimulus is not clear; prior trauma/surgery is usually not reported This process is benign but a minority or affected patients die of complications Disease does not typically progress or recur and the patients’ symptoms are relieved by resection These tumors were originally described as ‘childhood fibrous tumor with psammoma bodies’ Calcifying fibrous tumor/pseudotumor is a rare benign fibrous lesion Most soft tissue examples affect children and young adults without gender predilection whereas visceral examples usually occur in adults These tumors were originally described in the subcutaneous and deep soft tissues (extremities and scrotum) but have subsequently been reported all over the body notably in the mesentery and peritoneum and pleura (sometimes multiple) Visceral examples may produce site-specific symptoms Calcifications are apparent on CT and may be thick and band-like or punctuate with a mottled appearance and a signal closer to that of muscle than fat Although examples have followed trauma and occurred in association with Castleman’s disease and inflammatory myofibroblastic tumors The lesion is hypocellular and has lymphoid aggregates and calcifications These tumors can be multiple but are always benign (b) Note the paucicellularity and calcification In addition to the aforementioned entities occasionally lesions analogous to myositis ossificans are present in the mesentery (heterotopic myositis ossificans) the diagnosis of mesenchymal lesions of the GIT can be simplified if one is familiar with the layer in which they arise (mucosa and familiarity with these traps is crucial to avoid diagnostic errors Gastrointestinal glomus tumors: a clinicopathologic Glomus tumor of the stomach: a clinicopathologic analysis of 10 cases and review of the literature Gastric glomus tumor: analysis of endosonographic characteristics and computed tomographic findings Gastroduodenal glomus tumors: differentiation from other subepithelial lesions based on dynamic contrast-enhanced CT findings Novel MIR143-NOTCH fusions in benign and malignant glomus tumors Genes Chromosomes Cancer 2013;52:1075–1087 Opportunistic disorders of the gastrointestinal tract in the age of highly active antiretroviral therapy Idiopathic multiple pigmented sarcoma of the skin by Kaposi Kaposi's sarcoma: a clinicopathologic study with particular reference to its relationship to the reticuloendothelial system Association of Kaposi's sarcoma with second primary malignancies: possible etiopathogenic implications Kaposi's sarcoma in sub-Saharan Africa: a current perspective HIV—associated and non—HIV associated types of Kaposi's sarcoma in an African population in Tanzania Status of immune suppression and HHV-8 seroprevalence AIDS-related Kaposi's sarcoma: epidemiological treatment and control aspects in sub-Saharan Africa Kaposi's sarcoma in immunosuppressed patients Kaposi's sarcoma among persons with AIDS: a sexually transmitted infection Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma The pathobiology of Kaposi's sarcoma: advances since the onset of the AIDS epidemic The nature of hyaline (eosinophilic) globules and vascular slits of Kaposi's sarcoma distinguishes vascular neoplasms from potential mimics Vascular endothelial growth factor receptor-3 (VEGFR-3): a marker of vascular tumors with presumed lymphatic differentiation kaposiform and Dabska-type hemangioendotheliomas Expression of D2-40 in lymphatic endothelium of normal tissues and in vascular tumours Immunocytochemistry in the diagnosis of Kaposi's sarcoma and angiosarcoma Latency-associated nuclear antigen expression and human herpesvirus-8 polymerase chain reaction in the evaluation of Kaposi sarcoma and other vascular tumors in HIV-positive patients including cardiac myxoma and the Cushing syndrome Ganglioneuromas of the gastrointestinal tract Relation to Von Recklinghausen disease and other multiple tumor syndromes Vasoactive intestinal polypeptide-producing ganglioneuromatosis involving the entire colon and rectum Vasoactive intestinal polypeptide-secreting ganglioneuromatosis affecting the entire colon and rectum Benign fibroblastic polyps of the colon: a histologic Benign serrated colorectal fibroblastic polyps/intramucosal perineuriomas are true mixed epithelial-stromal polyps (hybrid hyperplastic polyp/mucosal perineurioma) with frequent BRAF mutations Early colonic perineurioma: a report of 11 cases Fibroblastic polyp of the colon and colonic perineurioma: 2 names for a single entity Intestinal perineuriomas: clinicopathologic definition of a new anatomic subset in a series of 10 cases Mucosal Schwann cell ‘hamartoma’: clinicopathologic study of 26 neural colorectal polyps distinct from neurofibromas and mucosal neuromas Mucosal benign epithelioid nerve sheath tumors Gastric submucosal granuloma with eosinophilic infiltration Inflammatory fibroid polyps of the stomach Inflammatory fibroid polyps of the gastrointestinal tract: spectrum of clinical Multiple and recurrent inflammatory fibroid polyps in a Devon family ('Devon polyposis syndrome'): an update Multiple and recurrent inflammatory fibroid polyps in three generations of a Devon family: a new syndrome Inflammatory fibroid polyps harbour mutations in the platelet-derived growth factor receptor alpha (PDGFRA) gene CD34 expression by inflammatory fibroid polyps of the stomach Inflammatory fibroid polyps of the gastrointestinal tract: evidence for a dendritic cell origin Its morphologic spectrum and immunohistochemical profile Prognosis of gastrointestinal smooth-muscle (stromal) tumors: dependence on anatomic site Gastrointestinal stromal tumors in patients with neurofibromatosis 1: a clinicopathologic and molecular genetic study of 45 cases SDHB immunohistochemistry: a useful tool in the diagnosis of Carney-Stratakis and Carney triad gastrointestinal stromal tumors gastric stromal tumours and pulmonary chondromas (Carney triad) and the dyad of paragangliomas and gastric stromal sarcomas (Carney-Stratakis syndrome): molecular genetics and clinical implications Immunohistochemistry for SDHB divides gastrointestinal stromal tumors (GISTs) into 2 distinct types Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor PDGFRA activating mutations in gastrointestinal stromal tumors c-kit and PDGFRA mutations in extragastrointestinal stromal tumor (gastrointestinal stromal tumor of the soft tissue) DOG1 antibody in the differential diagnosis of gastrointestinal stromal tumors: a study of 1840 cases Monoclonal antibody DOG1.1 shows higher sensitivity than KIT in the diagnosis of gastrointestinal stromal tumors A novel monoclonal antibody against DOG1 is a sensitive and specific marker for gastrointestinal stromal tumors Expression of melanoma antigens in epithelioid gastrointestinal stromal tumors: a potential diagnostic pitfall Gastric schwannoma: a clinicopathologic study of 51 cases and critical review of the literature Cytologic findings of gastric schwannoma: a case report Keratin expression in schwannoma; a study of 115 retroperitoneal and 22 peripheral schwannomas Nuclear beta-catenin expression distinguishes deep fibromatosis from other benign and malignant fibroblastic and myofibroblastic lesions The diagnostic value of beta-catenin immunohistochemistry World Health Organization Classification of Tumours In: Sohin L (ed) Pathology and Genetics of Tumours of Soft Tissue and Bone Hogendoorn PC et al WHO Classification of Tumours of Soft Tissue and Bone Bosman F Are tumefactive lesions classified as sclerosing mesenteritis a subset of IgG4-related sclerosing disorders Consensus statement on the pathology of IgG4-related disease Download references Lysandra Voltaggio & Elizabeth A Montgomery Download citation DOI: https://doi.org/10.1038/modpathol.2014.126 Nuclear Medicine and Molecular Imaging (2021) Metrics details To identify in vivo the intestinal cell type that has the capacity to educate migratory DCs and to elucidate the mechanisms that lead to RA production by CD103+ DCs we dissected and characterized various cell subsets from the intestinal LP and identified a stromal cell (SC) population capable of imprinting DCs with RALDH activity These SCs are an abundant component of the intestinal LP and might represent a direct source of RA we show that these SCs are in close contact with CD103-expressing DCs and that this interaction conversely promotes granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion by the SCs is absolutely required for effective RALDH induction in the DC compartment the constitutive RALDH expression by LP SCs is independent of RA whereas it did require the presence of the microbiota Our findings therefore identified an RA-producing LP SC as a direct sensor of the gut environment and an important regulator of the functional maturation of mucosal DCs The results also demonstrate an unexpected two-way cross-talk between these SCs and the DCs that might have an important role in controlling the tolerogenic or inflammatory nature of the mucosal immune response A subset of lamina propria (LP) stromal cells (SCs) have the capacity to produce retinoic acid (RA) (a) Analysis of RALDH activity using aldefluor in single-cell suspensions prepared from small intestinal (SI) LP and assessed by flow cytommetry for the indicated markers Left lower panel displays CD45−aldefluor+ cells (gate R1 black color) within the whole SI LP SC population (gray color) Data show a representative experiment of >5 independent experiments (b) messenger RNA (mRNA) levels of the RA-producing enzymes RALDH1 (aldh1a1) RALDH2 (aldh1a2) and RALDH3 (aldh1a3) were measured by quantitative real-time PCR in sorted CD45−Epcam−CD31−Pdpn+ cells (SCs black bars) and CD45−Epcamhi cells (intestinal epithelial cell Data shown correspond with RNA from three independent experiments that were quantified at the same time (c) Supernatants from sorted SCs were used to stimulate F9-RARE-lacZ cells and β galactosidase production by these cells was measured using a colorimetric reaction Left panel compares the absorbances obtained when F9-RARE-lacZ cells were left untreated (black bar) or supernatants were added to the culture (white bar); right panel shows an estimation of the RA cc present in the supernatants from three independent experiments pooled together (*P<0.05 t-test non paired) (d) Aldefluor staining of CD45− Pdpn+ SCs was compared between specific pathogen-free (SPF) and germ-free mice (GF) or (e) between vitamin A-sufficient (VAS) and vitamin A-deficient (VAD) mice Shown is a representative experiment out of three independent experiments (P=0.18 Aldefluor-positive stromal cells (SCs) are abundant within the intestinal lamina propria and locate closely to intestinal dendritic cells (DCs) (a–c) Confocal imaging of aldefluor-stained live small intestinal explants after intestinal epithelial cell removal Aldefluor staining was followed by immunofluorescence with the indicated antibodies central panel) explants were stained with aldefluor in the presence of the RALDH inhibitor DEAB White arrowheads point out aldefluor+ DCs in close contact with aldefluor+ SCs yellow arrowheads indicate podoplanin+ aldefluor+ cells Quantification shows the percentage of CD11c+ CD103+ cells in direct contact with aldefluor bright lamina propria cells from >10 different images Data shown correspond to RNA from three independent experiments that were quantified at the same time (d) Aldefluor staining of splenic DCs (gated CD45+MHCIIhiCD11c+) after 24 h culture alone or together with sorted SCs (SCs cond. left panel) or their supernatants (SCs sups The global RAR global antagonist BMS204493 (RAR inh.) was added to the culture at a concentration of 20 nM or otherwise indicated Data shown are from one representative experiment of at least three experiments suggesting that the LP SCs also enhanced the functional priming capacity of the DCs they interacted with To investigate whether soluble factors released by Pdpn+ CD31− SCs were able to educate DCs, we conditioned splenic DCs for 24 h with media harvested from Pdpn+ CD31− SCs overnight cultures. Splenic DCs stimulated with these supernatants specifically upregulated aldh1a2 expression (Figure 3c) which is the RALDH isoform typically expressed by the mucosal migratory DCs Furthermore, a global RA receptor antagonist (BMS 204493) blocked RALDH expression in DCs induced either by direct contact with Pdpn+ CD31− SCs (Figure 3d, left panel) or by SC-derived supernatants (Figure 3d indicating that RALDH induction in DCs depended on RA signaling these data demonstrate that LP Pdpn+ CD31− SCs can induce RA-dependent transcriptional activation of aldh1a2 in DCs which leads to the expression of RALDH2 and the production of RA a hallmark feature of the migratory mucosal CD103+ DC Granulocyte-macrophage colony-stimulating factor (GM-CSF) production by stromal cells is enhanced by dendritic cells (DCs) and required for the imprinting of DCs with high RALDH activity (a) Aldefluor staining of splenic DCs after 24 h treatment with SI LP CD45−Epcam−Pdpn+CD31− stromal cells (SCs) or with media supplemented with increasing concentrations of retinoic acid (RA) Data from one representative experiment of three experiments with similar results (b) Left panel shows aldefluor staining of a cell line established from sorted primary CD45−Pdpnhi splenic cells (Sp SCs) vs SI LP CD45−Epcam−Pdpn+CD31− stromal cells (SCs) Middle panel shows aldefluor staining of splenic DCs (CD45+CD11c+MHCIIhi gate) cultured for 24 h with the aforementioned cell line in the presence or not of RA (100 nM) Right panel shows aldefluor staining of splenic DCs treated with RA plus concentrated suspension (sups) from the aforementioned cell line Sups were digested with proteinase K when indicated Shown is the data from one representative experiment of two (c) Left panel shows aldefluor staining of splenic DCs after 24 h treatment with RA plus sequential fractions of the sups from Sp SCs (fractionation details within the Methods section) Right panel shows an estimate of the size of the proteins contained within the fraction that gave us the most activity (d) Left panel shows aldefluor staining of splenic DCs cultured for 24 h alone or together with SCs from wild-type (WT) or GM-CSF knockout (KO) mice Anti-GM-CSF blocking antibody was added where indicated Right panel shows the fold increase on aldefluor staining (mean fluorescence intensity MFI) on splenic DCs cocultured with SI LP CD45−Epcam−Pdpn+CD31−SCs from WT vs Data show the results from three independent experiments *P<0.05 (unpaired t-test) (e) Aldefluor staining of splenic DCs after 24 h culture with SI LP CD45-Epcam-Pdpn+CD31− SCs in the same well or separated by a permeable membrane (transwell) Bar graphs shows data from three replicate wells within one experiment Data show one representative experiment out of two (f) Shows GM-CSF by intracellular staining (left panel) or enzyme-linked immunosorbent assay (right) on SI LP CD45−Epcam−Pdpn+CD31−SCs cultured for 24 h alone or together with splenic DCs Left panel shows one representative experiment out of three Right panel shows data analyzed corresponding to five independent experiments the data show that RA and GM-CSF produced by LP SCs synergize to imprint DCs with the ability to produce RA and to functionally mature the in vivo observation indicating a direct contact between RALDH+ LP SCs and CD103+ DC together with the finding that this interaction regulates GM-CSF production by the SCs suggest that a direct cross-talk between these two cell types is required to ensure local imprinting of mucosal migratory DCs which direct tolerogenic and inflammatory immune responses in the intestine CD11b+CD103+ dendritic cell (DC) numbers and retinoic acid (RA)-producing capacity are diminished in granulocyte-macrophage colony-stimulating factor (GM-CSF)−/−mice CD103 staining of small intestinal (SI) lamina propria (LP) DCs (gated as CD45+ CD11chi MHCII high) in wild-type (WT) vs Lower row shows the percentage of the indicated populations among the total number of SI LP cells in WT vs (b) Aldefluor staining of the CD11b+ (upper row) and CD11b− (lower row) SI LP CD103+ DC population in WT vs GM-CSF KO mice indicating the percentage of aldefluor high cells (middle panel) as well as the aldefluor mean fluorescence intensity (MFI) for the whole population (right panel) Data show one representative experiment of >3 performed (NS it is still possible that MLNs SCs imprint local resident DCs in the LN or provide RA directly to T cells during priming Further investigation is needed to fully understand the role of these RA-producing MLN SCs suggesting that these SCs might serve as a primary source of RA for the initial education and imprinting of the RA-processing machinery in migratory DC precursor cells splenic DCs cultured overnight with Pdpn+CD31− intestinal SCs upregulated aldh1a2 expression and gained other features associated with intestinal mucosal DCs SCs depended on the microbiota for their educational activity and failed to produce RA in germ-free mice the location of CD103+ DCs within the LP separated by the basal membrane from the epithelium makes it unlikely that imprinting by intestinal epithelial cells contributes significantly to the education of migratory DCs in vivo might be key factors in directing and controlling the immune response at the mucosal forefront The fact that the SCs depend on the presence of the microflora for their educational role further suggests that they are important sentinels that have the capacity to report on the condition of the intestinal lumen and accordingly regulate the tolerogenic or inflammatory nature of the immune response C57BL/6 and OT-II TCR-transgenic were purchased from the Jackson Laboratories (Bar Harbor GM-CSF knockout mice were generously provided by Dr Whitsett (Cincinnati Children’s Research Foundation) Mice were maintained under specific pathogen-free conditions at the La Jolla Institute for Allergy and Immunology vivarium Animal care and experimentation were consistent with the National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at the La Jolla Institute for Allergy and Immunology Swiss Webster germ-free mice and age and gender-matched controls were purchased from Taconic Farms (Germantown pregnant females received either a chemically defined diet that lacked vitamin A (AIN-93M) or control diet containing retinyl acetate (25,000 IU kg−1) (Dyets Inc VAD diet started at 7–10 days of gestation and the pups were weaned at 3 weeks of age and maintained on the same diet at least until 11 weeks of age before analysis was performed Epithelial cell removal and single-cell preparation from intestinal LP these pieces were placed in Hank’s balanced salt solution 5% fetal bovine serum 0.5 M EDTA and shaken at 250 r.p.m and vortex for 1 min at room temperature; the tissue pieces were recovered using a stainless steel sieve The remaining intestinal tissue was minced and digested using collagenase type IV (Sigma Aldrich Released cells were pelleted by centrifugation washed with Hank’s balanced salt solution 5% fetal bovine serum and passed through a 70-μm in order to obtain a single cell suspension These cells were stained for the markers Epcam Analysis of RALDH activity by aldefluor staining RALDH activity in individual cells was analyzed using the aldefluor staining kit (StemCell Technologies 1 × 106 cells were resuspended in the kit Assay Buffer containing activated aldefluor substrate (150 nM) and incubated for 30 min at 37 °C in the presence or absence of the RALDH inhibitor DEAB (100 μM) Total RNA was extracted using TriZol reagent (Qiagen and complementary DNA obtained with the iScript cDNA synthesis kit (Bio-Rad Target messenger RNA was quantified using SYBR green (Roche IN)) and gene expression normalized relative to glyceraldehyde 3-phosphate dehydrogenase Data were collected and analyzed on a LightCycler 380 (Roche) Primers used were GAPDH fwd 5′-ATGGCCTTCCGTGTTCCTAC-3′ Rarb rev 5′-CTCTGTGCATTCCTGCTTTG-3′ Rarg fwd 5′-AAGTACACCACGAACTCCAGT-3′ and expression of surface markers and cytokines was assessed by flow cytometry cells were incubated for 4–5 h with 50 ng ml−1 paraformaldehyde CA) in a tissue culture incubator at 37 °C cells were resuspended in fixation/permeabilization solution (BD Cytofix/Cytoperm kit and intracellular cytokine staining performed according to the protocol in this kit intestinal explants were washed three times in Hank’s balanced salt solution 5% fetal bovine serum 0.5 M EDTA for 10 min at 37 °C and shaking at 250 r.p.m tissues were incubated in aldefluor assay buffer together with aldefluor reagent (1.5 μM) in the presence or absence of the RALDH inhibitor DEAB (100 μM) F9-RARE-lacZ reporter cell line cells were grown in gelatinized flasks in Dulbecco’s modified Eagle’s media supplemented with 15% fetal bovine serum and 0.8 mg ml−1 geneticin (G418) media Once cells were near confluency they were detached from the flask using trypsin/EDTA (Gibco) Cells were washed and resuspended at 2 million cells ml−1 density and plated in a flat-bottom 96-well tissue culture plate at 2 × 105 cells per well (100 μl from initial dilution) plus 100 μl media different concentrations of RA or cell supernatants supernatants were removed and cells were washed extensively with ice cold phosphate-buffered saline cells were lysed and β-galactosidase activity was assayed using the β-galactosidase enzyme assay system with reporter lysis buffer from Promega (Madison Values obtained from known RA concentrations were used to draw a standard curve to quantify RA levels on the cell supernatants Supernatants were subjected to anion exchange chromatography (AEC cat #17-5166-01) and 1-ml fractions were collected along an increasing gradient of sodium chloride (0–1 M) Fractions were used to treat DCs (50 μl per well in a 96-well plate total volume per well 200 μl) and the fraction that stimulated the highest RALDH2 upregulation was subjected to size exclusion chromatography 0.5 ml fractions were collected and used as before to stimulate DCs Function of retinoid nuclear receptors: lessons from genetic and pharmacological dissections of the retinoic acid signaling pathway during mouse embryogenesis Interactions of retinoid binding proteins and enzymes in retinoid metabolism Regulation of gene expression by vitamin A: the role of nuclear retinoic acid receptors Retinoic acid imprints gut-homing specificity on T Cells A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism All-trans retinoic acid mediates enhanced T reg cell growth Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens How vitamin A metabolizing dendritic cells are generated in the gut mucosa Vitamin A and immune regulation: role of retinoic acid in gut-associated dendritic cell education Expression of retinaldehyde dehydrogenase enzymes in mucosal dendritic cells and gut-draining lymph node stromal cells is controlled by dietary vitamin A GM-CSF and IL-4 synergistically trigger dendritic cells to acquire retinoic acid-producing capacity Retinoic acid induces homing of protective T and B cells to the gut after subcutaneous immunization in mice MyD88 and retinoic acid signaling pathways interact to modulate gastrointestinal activities of dendritic cells Retinal dehydrogenase gene expression in stomach and small intestine of rats during postnatal development and in vitamin A deficiency Localization of retinal dehydrogenase type 1 in the stomach and intestine Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells Stromal cell contributions to the homeostasis and functionality of the immune system Lymph node stromal cells support dendritic cell-induced gut-homing of T Cells MyD88-dependent TLR1/2 signals educate dendritic cells with gut-specific imprinting properties IRF4 transcription-factor-dependent CD103+CD11b+ dendritic cells drive mucosal T helper 17 cell differentiation Regulation of dendritic cell development by GM-CSF: molecular control and implications for immune homeostasis and therapy Use of reporter cells to study endogenous retinoid sources in embryonic tissues Download references We thank Dr Noelle for providing us with the F9-RARE-lacZ cell line and Dr Buckley for the Pdpnhi splenic SC line This work was supported by NIH R01 grants: R01AI050265 and DP1OD006433 La Jolla Institute for Allergy and Immunology Division of Integrative Biosciences and Biotechnology Pohang University of Science and Technology International Research and Development Center for Mucosal Vaccines The authors declared no conflict of interest and contributed in the writing of the manuscript performed experiments and analyzed results discussed the results and their implications SUPPLEMENTARY MATERIAL is linked to the online version of the paper Download citation Metrics details Significant progress has been made in the standardization of bladder neoplasm classification and reporting Accurate staging using the American Joint Committee on Cancer/International Union Against Cancer (AJCC/UICC) TNM system is essential for patient management and has been reinforced by clinical evidence in recent years It is now recognized that ‘superficial’ bladder carcinomas are a heterogenous group of tumors with diverse biological and clinical manifestations is no longer used for bladder tumor nomenclature Recognition of diagnostic pitfalls associated with lamina propria invasion is critical for the evaluation of bladder tumor specimens Neither the 1973 nor the 2004 WHO grading system appears to be useful for predicting the clinical outcome of invasive urothelial carcinoma This review will discuss recent progress and controversial issues on the staging and substaging of bladder carcinomas Essential elements for handling and reporting of bladder tumor specimens will also be discussed assess the extent of spread at the time of diagnosis and stratify patients into prognostic groups for treatment planning Adoption of a uniform staging system permits comparison of therapeutic interventions among different institutions pT1 urothelial carcinoma invading into the lamina propria. Wisps of muscularis mucosae, thin-walled vessels, and inflammatory infiltrate are present at the deepest penetration of the tumor. pT1 urothelial carcinoma with different invasive patterns. (a) The smooth contour of basement membrane is disrupted in infiltrating tumor cells. (b and c) Irregular small clusters of tumor cells invading the stroma. (d) Variably sized nests with single cells and irregular clusters of invading cells. pT1 urothelial carcinoma. (a) Invading fronts of the neoplasm show tentacular or finger-like extensions with myxoid reaction of the stroma. (b) Smooth contours of basement membrane are lost and irregular clusters or single cells are present in the lamina propria. pT1 urothelial carcinoma with retraction artifact. (a) Retraction artifact around invasive clusters is a useful feature for diagnosing stromal invasion. (b) The appearance of clusters with retraction artifact should not be mistaken for lymphovascular invasion. Paradoxical differentiation in pT1 urothelial carcinoma The invasive tumor cells often acquire abundant eosinophilic cytoplasm and appear to be more differentiated than overlying noninvasive tumor cells Urothelial carcinoma may involve von Brunn's nests The smooth regular contour of basement membrane Stromal responses in invasive urothelial carcinoma. The stromal reaction to invasive tumor may be (a) myxoid; (b) fibrous; (c) inflammatory; (d) pseudosarcomatous; or (e) desmoplastic. (f) Retraction artifact is also common adjacent to early invasive carcinoma, an important diagnostic clue for stromal invasion. (a) Prominent inflammation at the epithelial–stromal interface may obscure isolated cells or small nests of invasive carcinoma (b) Immmunostaining with anti-cytokeratin antibodies highlights the tumor cells (c and d) Myofibroblasts and smooth muscle cells may show positive cytokeratin immunoreactivity is weak and the nuclei are small with indistinct smudged chromatin The proliferating stroma associated with invasion is usually nonexpansile being limited to areas around the neoplasm and composed of cells whose nuclei have a degenerate or smudged appearance Muscularis mucosae and muscularis mucosae invasion. (a) Muscularis mucosae is composed of thin and wavy fascicles of smooth muscle in the submucosa of the bladder wall. (b) Muscularis mucosae is characterized by thin, often interrupted, bands of smooth muscle cells at high power. (c) Muscularis mucosae invasion. (d) Hyperplastic muscularis mucosae may be difficult to be distinguished from muscularis propria. (a) The lamina propria is thin in the trigone area and merges imperceptibly into the muscularis propria wall (b) Higher magnification view of the trigone area Substaging of pT1 bladder carcinoma is particularly challenging for tumors arising from the trigone Microinvasive carcinoma arising from urothelial carcinoma in situ (a) Individual single cells permeate the stroma adjacent to von Brunn's nests containing carcinoma in situ (b) Prominent stromal inflammatory response may obscure the presence of individual tumor cells Retraction artifact is a useful clue for the diagnosis of invasion (a) pT2 urothelial carcinoma invades into muscularis propria Infiltrating tumor cells permeate the thick muscularis propria wall (b) Sometimes it is difficult to ascertain the presence of muscularis propria invasion when columns and nests of tumor cells widely separate bundles of detrusor muscle (c and d) Extensive destruction of muscularis propria may result in fragmentations of detrusor muscle (e) It is not uncommon for tumor cells to abut but not penetrate bundles of mucularis propria The muscle–tumor interface may even have a smooth contour (f) Even without direct invading into muscularis propria wall tumor cells immediately adjacent to broad smooth muscle fibers should be categorized as pT2 carcinoma In 1978, Jewett87 concluded ‘it seems probable that our arbitrary dividing line drawn 30 years ago at the halfway level to separate B1 (pT2a) from B2 (pT2b) tumors was too superficial.’ Substaging of T2 bladder carcinoma based on the level of muscularis propria invasion is of limited value for stratifying patients into prognostic groups and should be eliminated from future TNM classification Tumor size may be a more pertinent parameter for the subclassification of T2 bladder cancer (a) Tumor invades into perivesical adipose tissue The prognostic significance of perineural invasion is uncertain Urothelial carcinoma invading into prostatic stroma (pT4a urothelial carcinoma) (a) Urothelial carcinoma often elicits a strong inflammatory response (b) Tumor cells invading prostatic stroma are usually highly pleomorphic (c and d) Immunostaining for high molecular weight cytokeratins is diffusely and strongly positive in urothelial carcinoma The basal cells in the adjacent prostatic glands show positive stainings for high molecular weight cytokeratin (c) Lymph node metastasis from urothelial carcinoma Extranodal extension is present at low power (a) and is confirmed on high power examination (b) Other specimens include cystectomy (partial or total) Surgical excision of an urachal carcinoma usually includes the bladder dome Bladder biopsy and/TUR specimens are to provide diagnostic and prognostic information for urologists to plan surveillance and treatment Tissues from biopsy specimens should be entirely embedded for histological examination These biopsy specimens obtained through the cystoscope often vary in size At least two levels of sectioning should be obtained on each small biopsy Proper orientation of bladder tumor biopsy specimens is difficult It may be necessary to re-embed and reorient the tissue to facilitate assessment of the depth of invasion Transurethral resection of the bladder specimens should be weighted in aggregate Papillary tumors may be grossly recognizable in these specimens The number of tissue chips with involvement and gross tumor size should be recorded At least one block per centimeter of tumor diameter should be submitted at the initial sampling Overfilling of specimen cassettes should be avoided submitting any residual specimen may be necessary to firmly rule out stromal invasion If there is invasion into the lamina propria in the initial sampling then additional sampling is recommended to rule out muscularis propria invasion We recommend that the urologist submit superficial and deep tumor-base specimens in separate containers to facilitate the detection of deep muscle invasion Processing of these specimens may be summarized in three steps: (1) orientation of the specimen and identification of relevant anatomic structures (eg (2) fixation of the specimen and (3) dissection of the specimen Peritoneum covering the surface of the bladder is a reliable anatomic landmark the peritoneum descends further along the posterior wall of the bladder than it does along the anterior wall the bladder adjoins the rectum and seminal vesicles posteriorly Location and dissection of the ureters is easier after fixation The outer dimensions of the urinary bladder as well as the length and diameter of ureters The external surface of the bladder should be inked Proper fixation of the specimen is a prerequisite for adequate histological evaluation We recommend that large bladder specimens be fixed in formalin overnight Some prefer to expand the bladder with formalin Injection of formalin into the urinary bladder cavity is accomplished either through the urethra by a Foley catheter or through the bladder dome using a large-gauge needle after the urethra has been clamped We prefer to open the bladder before formalin fixation It should be opened anteriorly from the urethra to the bladder dome Thus the bladder mucosa may be everted for close examination color and consistency of the tumor should be documented the dissection is resumed by shaving the margins from each ureter and the urethra the distal urethral margin is the distal end of the prostate at the apex The ureters are opened at their trigone orifices a full-thickness cut through the tumor and bladder wall should be made The tumor should be generously sampled for accurate staging at least one section should be taken for each centimeter of tumor diameter Sections are taken in such a way as to show the relationship of tumor to adjacent urothelium several sections are taken from the tumor base to adequately assess the extent of invasion Normal appearing mucosa is also sampled to detect occult multifocal carcinomas due to the field effect of bladder carcinogenesis The entire bladder is transversely step-sectioned at 5-mm intervals from the bladder neck to the dome Perivesical fat is carefully searched for lymph nodes The presence or absence of gross fat invasion should be documented Illustration of a cystoprostatectomy specimen ureteral orifices (including intramural portion) The prostate should be sampled using the standard protocol for radical prostatectomy specimens Preoperative treatments or repeat TUR may render residual tumors grossly invisible there is increasing incidence of pT0 carcinoma at cystectomy the bladder should be extensively sampled with particular attention to abnormal appearing mucosa and to sites of previously documented tumor resection uterus and vagina should be evaluated according to the standardized protocols for these organs Pelvic soft tissue margins should be documented As the uterus and rectum are located posterior to the bladder an anterior opening of the bladder is preferred to facilitate documentation of urothelial tumors and to evaluate the tumor's relationship to these other organs for staging Sections should be taken to confirm the presence of each pelvic organ and to show the relationship between the tumor and each of these structures These sections also document the resection margins for each organ and examine each organ for primary disease Partial cystectomy specimens (including resections of diverticula) should be fixed and dissected according to the guidelines of radical cystectomy (see earlier discussion) The edges of the specimen are inked because these represent the surgical margins of the bladder wall A variation of the partial cystectomy is performed for resections of urachal tract neoplasms These specimens consist of the dome of the bladder in continuity with the urachal tract including the umbilicus the urachal tract should be serially sectioned at right angles to the long axis from the bladder to the umbilicus Submit for histology a number of these urachal tract cross-sections Appropriate samples of the soft tissue margin surrounding the urachus and of the skin margin around the umbilicus should be submitted for histology the minimum number of lymph nodes that should be sampled has not been established We recommend that at least eight lymph nodes be sampled a clearing solution may be used to aid in detection of lymph nodes so a thorough search should be made of this area in addition to other pelvic node-bearing structures which illustrate synoptic formats for biopsy and cystectomy cancer specimen reports Micropapillary (a and b) and nested (c and d) variants of urothelial carcinoma These variants are associated with poor prognosis and aggressive treatment is warranted Retraction artifact is common in micropapillary variant (a) The tumor cells of nested variant may appear to be bland (c) yet immunostaining for cytokeratin highlights the aggressively infiltrative nature of these cancer cells (d) Substaging of T2 bladder cancer (T2a verrus T2b) is not feasible in bladder biopsy or TURB specimens as the entire thickness of the detrusor muscle is not present The term ‘superficial muscle invasion’ in the pathology report leads to confusion For biopsy and TUR of invasive bladder carcinoma some urologists prefer a statement of pathological stage (T1 or T2) in the report we recommend that tumor stage be indicated as ‘at least’ pT1 or pT2 Adipose tissue may be present in both the lamina propria and muscularis propria the presence of fat in a biopsy or TUR specimen does not necessarily indicate extravesical extension (pT3 carcinoma) Laboratories accredited by the College of American Pathologists are required to use the CAP cancer reporting guidelines Infiltrative growth pattern in invasive urothelial carcinoma The tumor is composed of highly pleomorphic and anaplastic cells in narrow cords or as individual cells permeating the stroma Lymphovascular invasion of urothelial carcinoma endothelial cells are identified on routing H&E sections eliminating the need for CD31 or CD34 staining Positive soft tumor margins. Tumor cells are present at the inked perivesical soft tissue margin. The resection margins should be individually specified in the pathology report The following margins should be reported separately: ureteral (right and left) and pelvic soft tissue margins (for pelvic exenteration specimens) In cases of urachal adenocarcinoma in which partial cystectomy with excision of the urachal tract and umbilicus is performed the soft tissue surrounding the urachus and the skin around the umbilical margin Consistent and standardized pathological evaluation is essential for comparison of treatment results between clinical trials and for translational research endeavors Predicting the survival of bladder carcinoma patients treated with radical cystectomy Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients Cystectomy for bladder cancer: a contemporary series Outcomes of radical cystectomy for transitional cell carcinoma of the bladder: a contemporary series from the Bladder Cancer Research Consortium It is time to abandon the ‘superficial’ in bladder cancer Eliminate the term ‘superficial’ bladder cancer American Joint Committee on Cancer Staging Manual Cancer specific outcomes in patients with pT0 disease following radical cystectomy P0 stage at radical cystectomy for bladder cancer is associated with improved outcome independent of traditional clinical risk factors Does of stage pT0 cystectomy specimen confer a survival advantage in patients with minimally invasive bladder cancer Effect of a pT0 cystectomy specimen without neoadjuvant therapy on survival Outcome of radical cystectomy for bladder cancer according to the disease type at presentation Histologic grading of noninvasive papillary urothelial neoplasms Stage pT1 bladder carcinoma: diagnostic criteria Value of immunohistochemistry in staging T1 urothelial bladder carcinoma pT1 Urothelial carcinoma of the bladder: criteria for diagnosis Prognostic factors in stage T1 bladder cancer: tumor pattern (solid or papillary) and vascular invasion more important than depth of invasion Detection of residual tumor cells in bladder biopsy specimens: pitfalls in the interpretation of cytokeratin stains Clinical significance of interobserver differences in the staging and grading of superficial bladder cancer Reproducibility and prognostic variability of grade and lamina propria invasion in stages Ta Management of stage T1 tumors of the bladder: International Consensus Panel Progression of T1 bladder tumors: better staging or better biology Predicting cancer progression in patients with stage T1 bladder carcinoma Grading and staging of bladder carcinoma in transurethral resection specimens Correlation with 105 matched cystectomy specimens Difficult decisions in urologic oncology: management of high-grade T1 transitional cell carcinoma of the bladder Can restaging transurethral resection of T1 bladder cancer select patients for immediate cystectomy Restaging transurethral resection of high risk superficial bladder cancer improves the initial response to bacillus Calmette-Guerin therapy Contemporary management of stage T1 transitional cell carcinoma of the bladder Muscularis mucosa differentiates two populations with different prognosis in stage T1 bladder cancer Significance of invasion to the muscularis mucosae on the progression of superficial bladder cancer The influence of the level of lamina propria invasion and the prevalence of p53 nuclear accumulation on survival in stage T1 transitional cell bladder cancer The importance of the depth of invasion in stage T1 bladder carcinoma: a prospective cohort study Is microstaging of early invasive cancer of the urinary bladder possible or useful The usefulness of the level of the muscularis mucosae in the staging of invasive transitional cell carcinoma of the urinary bladder Microstaging of pT1 transitional cell carcinoma of the bladder: does it really differentiate two populations with different prognoses Substaging of T1 bladder carcinoma based on the depth of invasion as measured by micrometer Histology and fine structure of the muscularis mucosae of the human urinary bladder Invasive carcinomas of the urinary bladder Evaluation of tunica muscularis mucosae involvement The muscularis mucosae of the human urinary bladder Outcomes after intravesical bacillus Calmette-Guerin are not affected by substaging of high grade T1 transitional cell carcinoma Further characterization of the muscle layers and lamina propria of the urinary bladder by systematic histologic mapping: implications for pathologic staging of invasive urothelial carcinoma The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder Predicting extravesical extension of bladder carcinoma: a novel method based on micrometer measurement of the depth of invasion in transurethral resection specimens Morphological and clinical observations of patients with early bladder cancer treated with total cystectomy Clinical observations on sixty-nine cases of in situ carcinoma of the urinary bladder Observation on microinvasive transitional cell carcinoma of the urinary bladder Morphologic expressions of urothelial carcinoma in situ: a detailed evaluation of its histologic patterns with emphasis on carcinoma in situ with microinvasion Preneoplastic non-papillary lesions and conditions of the urinary bladder: an update based on the Ancona International Consultation Carcinoma of the bladder: influence of depth of infiltration on the 5-year results following complete extirpation of the primary growth Staging error in the bladder tumor: the correlation between stage of TUR and cystectomy A propsed simplified staging system of invasive bladder tumors Current state of classification and staging of bladder cancer Current perspectives in the management of high grade invasive bladder cancer Total cystectomy for carcinoma of the bladder Trans Am Assoc Genitourin Surg 1968;60:22–30 Results with preoperative radiation therapy and radical cystectomy Risk of local urethral recurrence after radical cystectomy for bladder cancer The curability of invasive bladder cancer treated by radical cystectomy Simple cystectomy in the management of bladder carcinoma The relationship of local control to distant metastasis in muscle invasive bladder cancer Pathology and prognosis following total cystectomy for carcinoma of bladder The surgical management of bladder carcinoma Radical cystectomy for carcinoma of the bladder:16 years of experience Carcinoma of the bladder: treatment by radical cystectomy Long-term patient survival after cystectomy for regional metastastic transitional cell carcinoma of the bladder Management of invasive bladder cancer: a meticulous pelvic node dissection can make a difference Clinical staging and histologic grading in relation to survival A clinicopathologic evaluation of partial cystectomy for carcinoma of the urinary bladder Stage B (P 2/3A/N0) transitional cell carcinoma of bladder highly curable by radical cystectomy Radical cystectomy without radiation therapy for carcinoma of the bladder Results of contemporary radical cystectomy for invasive bladder cancer: a clinicopathological study with an emphasis on the inadequacy of the tumor Superficial (pT2a) and deep (pT2b) muscle invasion in pathological staging of bladder cancer following radical cystectomy Impact of the level of muscle invasion in organ-confined bladder cancer short course preoperative radiation therapy and immediate single stage radical cystectomy with pelvic node dissection in the management of bladder cancer Survival of patients with stage T2-T3a bladder cancer treated by radical cystectomy Tumor size predicts the survival of patients with pathologic stage T2 bladder carcinoma: a critical evaluation of the depth of muscle invasion Pathological staging of muscle invasive bladder cancer Is substaging of pT2 tumors really necessary Editorial: Comments on the staging of invasive bladder cancer two B's or not to B's: That is the question Intravesical adipose tissue: a quantitative study of its presence and location with implications for therapy and prognosis Microscopic and gross extravesical extension in pathological staging of bladder cancer perivesical fat invasion at radical cystectomy is an adverse predictor of recurrence and survival Mechanisms of prostatic stromal invasion in patients with bladder cancer: clinical significance Transitional cell carcinoma involving the prostate with a proposed staging classification for stromal invasion Is stage pT4a (D1) reliable in assessing transitional cell carcinoma involvement of the prostate in patients with a concurrent bladder cancer A necessary distinction for contiguous or noncontiguous involvement Prostatic involvement by transitional cell carcinoma in patients with bladder cancer and its prognostic significance Critical pathological evaluation of the prostate from cystoprostatectomies for bladder cancer: insights from complete sampling with the whole mount technique Prognosis of seminal vesicle involvement by transitional cell carcinoma of the bladder Outcome in patients with seminal vesicle invasion after radical cystectomy Recommendations for the reporting of urinary bladder specimens that contain bladder neoplasms Superiority of ratio based lymph node staging for bladder cancer Lymph node density is superior to TNM nodal status in predicting disease-specific survival after radical cystectomy for bladder cancer: analysis of pooled data from MDACC and MSKCC Risk factors for patients with pelvic lymph node metastases following radical cystectomy with en bloc pelvic lymphadenectomy: concept of lymph node density Evaluation of the relevance of lymph node density in a contemporary series of patients undergoing radical cystectomy Extracapsular extension of pelvic lymph node metastases from urothelial carcinoma of the bladder is an independent prognostic factor Transitional cell carcinoma of the urinary bladder with regional lymph node involvement treated by cystectomy: clinicopathologic features associated with outcome Prognostic implications of extracapsular extension of pelvic lymph node metastases in urothelial carcinoma of the bladder Uroplakin II as a promising marker for molecular diagnosis of nodal metastases from bladder cancer: comparison with cytokeratin 20 Molecular determination of perivesical and lymph node metastasis after radical cystectomy for urothelial carcinoma of the bladder Prospective evaluation of the prognostic relevance of molecular staging for urothelial carcinoma Molecular lymph node staging in bladder urothelial carcinoma: impact on survival Can immunohistochemistry enhance the detection of micrometastases in pelvic lymph nodes from patients with high-grade urothelial carcinoma of the bladder Handling and pathology reporting of specimens with carcinoma of the urinary bladder Updated protocol for the examination of specimens from patients with carcinoma of the urinary bladder Recommendations for the reporting of urinary bladder specimens containing bladder neoplasms Association of Directors of Anatomic and Surgical Pathology Pathologic evaluation of radical cystectomy specimens: a cooperative group report Diagnosis and grading of bladder cancer and associated lesions A practical approach to bladder sampling and diagnostic reporting of pathological findings Pathologic prognostic parameters in bladder urothelial biopsy Practice protocol for the examination of specimens removed from patients with carcinoma of the urinary bladder ASCP survey on anatomic pathology examination of the urinary bladder Appendix E Guidelines for handling of most common and important surgical specimens Rosai and Ackerman's Surgical Pathology Histologic variants of urothelial carcinoma: differential diagnosis and clinical implications Risk and prognostic factors—a pathologist's perspective Histological typing of urinary bladder tumours World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs Grading the invasive component of urothelial carcinoma of the bladder and its relationship with progression-free survival Patterns of invasion and histological growth as prognostic indicators in urothelial carcinoma of the upper urinary tract Superficial bladder cancer: progressioin and recurrence Prognostic factors in invasive bladder carcinoma in a prospective trial of preoperative adjuvant chemotherapy and radiotherapy Mapping cancerous and precancerous bladder changes A study of the urothelium in ten surgically removed bladders Origin and dissemination of human urinary bladder carcinoma Prognostic parameters in superficial bladder cancer: an analysis of 315 cases Predictability of recurrent and progressive disease in individual patients with primary superficial bladder cancer Mapping of the urinary bladder: its impact on the concepts of bladder cancer Nonpapillary carcinoma in situ and atypical hyperplasia in cancerous bladders: further studies of surgically removed bladders by mapping Molecular evidence supporting field effect in urothelial carcinogenesis Clonality and genetic divergence in multifocal low-grade superficial urothelial carcinoma as determined by chromosome 9 and p53 deletion analysis Cytogenetic analysis of multifocal bladder cancer supports a monoclonal origin and intraepithelial spread of tumor cells Prognostic factors for recurrence and followup policies in the treatment of superficial bladder cancer: report from the British Medical Research Council Subgroup on Superficial Bladder Cancer (Urological Cancer Working Party) Lymphovascular invasion is independently associated with poor prognosis in patients with localized upper urinary tract urothelial carcinoma treated surgically Prognostic significance of vascular and perineural invasion in urothelial bladder cancer treated with radical cystectomy Lymphovascular invasion is independently associated with overall survival and local and distant recurrence in patients with negative lymph nodes at radical cystectomy Prognostic significance of lymphovascular invasion of bladder cancer treated with radical cystectomy Muscle-invasive bladder cancer: predictive factors and prognostic difference between primary and progressive tumors The prognostic significance of vascular invasion in stage T1 bladder cancer and perineural invasion have prognostic implications for bladder cancer after radical cystectomy Positive surgical margins in soft tissue following radical cystectomy for bladder cancer and cancer specific survival Download references Institute of Pathological Anatomy and Histopathology Polytechnic University of the Marche Region (Ancona) Download citation DOI: https://doi.org/10.1038/modpathol.2009.1 Metrics details Distinguishing bladder muscularis propria from muscularis mucosae can be problematic especially in transurethral resection specimens performed for bladder carcinoma bladder carcinoma can be associated with a proliferative/desmoplastic myofibroblastic response that can resemble smooth muscle and potentially lead to overdiagnosis of muscularis propria invasion The aim of this study was to investigate the potential role of immunohistochemistry in staging bladder carcinoma by evaluating the expression of different markers in myofibroblasts and nonvascular smooth muscle cells in 15 cases of invasive bladder carcinoma Reactive myofibroblasts were consistently positive for vimentin and smooth muscle actin and had variable expression of actin and CD10 Nonvascular smooth muscle cells of the bladder were consistently positive for smooth muscle actin In contrast to smooth muscle cells of the muscularis propria which displayed strong smoothelin expression in all 15 cases the smooth muscle cells of the muscularis mucosae displayed moderate smoothelin expression in only 1 (9%) of 11 cases (P=10−7) although strongly highlighting endothelial and endomysial cells the smooth muscle cells of the muscularis propria weakly expressed vimentin in only 1 (7%) of 15 cases whereas smooth muscle cells of the muscularis mucosae had moderate or strong expression in 9 (82%) of 11 cases (P=0.00016) The sensitivity and specificity of desmin or caldesmon expression for smooth muscle cells were 100% The sensitivity and specificity of strong smoothelin expression for muscularis propria were 100% whereas those of absent vimentin expression were 93 and 82% Although morphology remains the gold standard the findings suggest that immunohistochemistry may be potentially useful for staging of bladder carcinoma especially on transurethral resection specimens can have a thickened hyperplastic appearance that may resemble the thick bundles of the muscularis propria especially in transurethral resection specimens and may also cause difficulty in staging of cancers in these specimens these myofibroblasts can be readily distinguished from the smooth muscle cells of the muscularis mucosae or muscularis propria; however we have seen cases of bladder carcinoma misdiagnosed as muscularis propria-invasive disease based solely on the presence a cellular myofibroblastic response surrounding the tumor including one where immunoreactivity for smooth muscle actin was used to ‘support’ the diagnosis of muscularis propria invasion This raises several important questions: can immunohistochemistry help distinguish between reactive myofibroblasts and smooth muscle cells of the bladder between muscularis mucosae and muscularis propria; and can immunohistochemistry be used as an adjunct in staging carcinomas of the bladder especially in the circumstances discussed above in staging bladder carcinoma has not been fully evaluated the immunohistochemical expression of different potentially discriminatory markers in myofibroblasts and smooth muscle cells of the muscularis mucosae and muscularis propria from cystectomy specimens was evaluated and presented herein After approval by the University of Alabama at Birmingham Institutional Review Board 15 consecutive cystectomy specimens resected at the University Hospital for invasive bladder carcinoma were reviewed to identify representative sections of muscularis propria-invasive tumor associated with a myofibroblastic response and/or muscularis mucosae adjacent to or involved by tumor We purposefully limited this study to cystectomy specimens as in contrast to some transurethral resection specimens the depth of invasion and type of muscle can readily be determined based on hematoxylin and eosin-stained sections this morphologically determined depth of invasion/type of muscle was considered to be the gold standard Interpretation of immunohistochemical stains in each case was performed by semiquantitatively analyzing the intensity of staining separately in each compartment (myofibroblasts Vascular smooth muscle was used as an internal control for all antibodies except CD10 with absent staining categorized as negative (0) and an intensity of staining that was weaker or stronger than the intensity of staining in vascular smooth muscle categorized as weak (1+) The patterns of staining in the different compartments were compared to each other and the utility of the different antibodies in distinguishing between myofibroblasts and smooth muscle cells and between the smooth muscle of the muscularis mucosae and that of the muscularis propria was evaluated by comparing the sensitivity (true positives/true positives+false negatives) specificity (true negatives/true negatives+false positives) and accuracy (true positives+true negatives/true positives+false positives+true negatives+false negatives) rates of each immunostain in identifying the specific components The 95% confidence intervals (CI) for proportions based on a binomial probability distribution were also calculated to assess the accuracy of the estimates of the sensitivity and accuracy rates of the different immunostains in labeling the compartment in question Statistical analysis was used to compare the categorical data obtained using either the χ2-test A P-value of 0.05 was considered statistically significant There were 14 high-grade urothelial carcinomas and 1 squamous-cell carcinoma 14 showed a variably cellular desmoplastic/myofibroblastic tumor response (including 1 with a pseudosarcomatous reaction) and 11 had residual muscularis mucosae adjacent to and/or involved by tumor Reactive myofibroblasts displayed strong vimentin expression and moderate to strong smooth muscle actin expression in all cases, with no expression of caldesmon, desmin, and smoothelin in any case (Figure 1). In addition, weak actin expression was seen in 8 (53%) cases, whereas CD10 expression, of variable intensity, was seen in 11 (73%) cases. Expression of different immunostains in the smooth muscle of the muscularis propria and reactive myofibroblastic cells associated with invasive carcinoma A case of squamous-cell carcinoma that is seen to invade the muscularis propria (a A desmoplastic reaction is seen surrounding the tumor nests which is composed of paler myofibroblasts with more tapered nuclei (b) and smoothelin (f) immunostains all appear to specifically highlight the smooth muscle of the muscularis propria a smooth muscle actin immunostain also labeled the myofibroblasts and endothelial cells around the tumor (g) Although vimentin highlighted some scattered endomysial cells within the muscle fibers the smooth muscle cells themselves appeared negative (h A CD10 immunostain (not shown) weakly highlighted some of the myofibroblasts present Expression of different immunostains in the smooth muscle of the muscularis mucosa. The smooth muscle fibers of the muscularis mucosae in this bladder (a, b) were strongly immunoreactive for desmin (c), actin (d), and smooth muscle actin (e), weakly immunoreactive for caldesmon (f), and negative for smoothelin (g). The smooth muscle fibers of the muscularis mucosae were also weakly positive for vimentin (h, inset) and negative for CD10 (not shown). Smoothelin expression in the smooth muscle of the muscularis mucosae Section of bladder showing an edematous lamina propria involved by high-grade urothelial carcinoma with angiolymphatic invasion (a In addition to being immunoreactive for desmin (b) and vimentin (c the smooth muscle fibers of the muscularis mucosae in this case displayed moderate immunoreactivity for smoothelin (d) more reminiscent of the intensity of staining associated with muscularis propria The muscularis mucosae were also positive for other muscle markers and negative for CD10 (not shown) The utility of different immunostains in distinguishing the smooth muscle of the muscularis propria from a pseudosarcomatous reactive myofibroblastic response resembling smooth muscle fibers A case of high-grade urothelial carcinoma associated with a cellular spindle-cell pseudosarcomatous response that is infiltrating between the fibers of the muscularis propria (a The residual muscle fibers display more eosinophilic cytoplasm most evident in the upper right hand corner of b (arrows) Compared to myofibroblasts within the cellular response smooth muscle cells showed more intense staining for smooth muscle actin (c; arrowheads); however as only smooth muscle cells were positive with this immunostain (d) Caldesmon and smoothelin immunostains (not shown) resulted in a pattern of staining similar to that of desmin (albeit with weaker intensity) Given that vimentin was only rarely expressed in the smooth muscle cells of the muscularis propria the sensitivity of negative or weak vimentin expression for labeling such cells was 100% but the specificity was only 9%; using a negative immunoreaction as the cutoff increased the specificity to 82% whereas the sensitivity decreased to 93% Using the two immunostains in combination also improved detection of smooth muscle cells of the muscularis propria as the sensitivity of moderate or strong smoothelin expression combined with negative vimentin expression was 93% and the specificity was 100% whereas the sensitivity of moderate or strong smoothelin expression combined with negative or weak vimentin expression was 100% and the specificity was 100% In this study we have shown that differential expression of immunohistochemical markers appears potentially useful in distinguishing between myofibroblasts and smooth muscle cells of the bladder and also between the smooth muscle cells of the muscularis mucosae and those of the muscularis propria desmin or caldesmon appeared to be accurate immunostains that can help distinguish smooth muscle cells from myofibroblasts may also help distinguish muscularis mucosae from muscularis propria Evaluation of additional cases and confirmatory studies on transurethral resection specimens are warranted to further explore this as well as the overall potential utility of immunohistochemistry as a potential aid to morphology in staging of bladder carcinoma After encountering one such case in which the diagnosis was ‘supported’ by a positive smooth muscle actin immunostain in the spindle cells in question we wanted to further characterize the immunohistochemical profile of these myofibroblasts as well as those of the smooth muscle cells of muscularis mucosae and smooth muscle cells of the muscularis propria to investigate the potential role of immunohistochemistry in distinguishing these cell types Because of the high sensitivity and specificity of desmin and smoothelin immunostains for detecting smooth muscle cells the addition of less discriminatory markers was not further useful for distinguishing them from myofibroblasts the wide variation in the intensity of CD10 staining of myofibroblasts as well as the lack of a valid internal control further curtailed its potential diagnostic utility These findings appear to corroborate ours in that there is indeed a difference in vimentin expression levels between the smooth muscle cells of the muscularis mucosae and those of the muscularis propria These findings are remarkably similar to ours despite probable variations in antibody dilutions and methodologies among the different studies An algorithmic approach to the use of desmin and vimentin immunohistochemistry in the evaluation of foci of bladder carcinoma suspicious for muscularis propria invasion It can also be argued that similar developmental and functional considerations may explain the differential vimentin expression patterns of the smooth muscle in these two compartments Although the findings in this study clearly suggest that there is a potential role for immunohistochemistry in staging of bladder carcinoma on different staining intensities in distinguishing between the muscularis propria and muscularis mucosae which might create issues with reproducibility Using vascular smooth as an internal reference for comparison would help in minimizing potential variations in intensity among different laboratories due to different fixation methods Although inclusion of a relatively small number of cystectomy specimens might also be considered another limitation a preliminary exploratory study of different immunohistochemical markers in nonequivocal cases (where there was no question regarding the depth of tumor invasion) to identify those immunostains that might be useful as an aid in staging of bladder carcinoma Our findings suggest that a panel composed of desmin, smoothelin, and vimentin immunostains, possibly using an algorithmic approach such as the one displayed in Figure 6 appears to have the most potential in this regard these results need to be validated in a larger sample of transurethral resection specimens (with questionable depth of invasion and where cystectomy follow-up staging data can be used as a gold standard) before more definitive conclusions can be made we have shown that differential expression of immunohistochemical markers can potentially distinguish between myofibroblasts and smooth muscle cells of the bladder especially transurethral resection specimens that is warranted to further assess the potential role of immunohistochemistry in staging of bladder carcinoma we would caution that any number of immunohistochemically stained sections can only supplement and never replace morphological findings based on hematoxylin and eosin-stained sections Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs Correlation between biopsy and radical cystectomy in assessing grade and depth of invasion in bladder urothelial carcinoma Urology 2001;57:1063–1066; discussion 1066–1067 Muscularis mucosae of the urinary bladder revisited with emphasis on its hyperplastic patterns: a study of a large series of cystectomy specimens Vascular plexus is a differentation criterion for muscularis mucosa from muscularis propria in small biopsies and transurethral resection materials of urinary bladder Lippincott Williams & Wilkins: Philadelphia Transitional cell carcinoma of the urinary bladder with pseudosarcomatous stroma The role of immunohistochemistry in the diagnosis of urinary bladder neoplasms CD10 expression in tumour and stromal cells of bladder carcinoma: an association with bilharziasis CD10 expression in urothelial bladder carcinomas: a pilot study Stromal expression of CD10 in invasive breast carcinoma: a new predictor of clinical outcome Expression of CD10 by stromal cells during colorectal tumor development a novel cytoskeletal protein specific for smooth muscle cells Cadherin-11 is expressed in detrusor smooth muscle cells and myofibroblasts of normal human bladder Expression of smoothelin in the normal and the overactive human bladder Diagnostic utility of antibody to smoothelin in the distinction of muscularis mucosa from muscularis propria of the urinary bladder (abstract) Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins Alpha-smooth muscle actin as a marker for soft tissue tumours: a comparison with desmin The myofibroblast: phenotypic characterization as a prerequisite to understanding its functions in translational medicine Is anti-h-caldesmon useful for distinguishing smooth muscle and myofibroblastic tumors The myofibroblast and its tumours: a review J Clin Pathol; published online: 17 October 2008 [e-pub ahead of print] Differentiation of the smooth muscle cell phenotypes during embryonic development of coronary vessels in the rat Smoothelin in adult and developing human arteries and myocardium Differentiation of smooth muscle cells in human blood vessels as defined by smoothelin a novel marker for the contractile phenotype Arterioscler Thromb Vasc Biol 1997;17:665–671 Download references This work was supported in part by a research grant from the Robert B and Jean G Adams Foundation to Dr Leona Council Presented in part at the 97th Annual Meeting of the United States and Canadian Academy of Pathology Download citation DOI: https://doi.org/10.1038/modpathol.2009.9 I legitimately believed in the offering and what they were trying to do," Oczkowski says "It’s unfortunate that some actions by a few actors at the senior level of the company reflected poorly on the entire company." In the wake of a data privacy debacle involving the misappropriation of as many as 87 million Facebook users' personal data, Cambridge Analytica has since ceased operations and become the subject of international investigations Though Oczkowski left the company in April 2017 he now joins hundreds of former employees trying to start over in a world that has begun looking at the field of data science not as novel and innovative 'We’re trying to figure out what shapes your worldview.' working out of Data Propria's San Antonio headquarters with some of his colleagues from that 2016 run Oczkowski hopes to continue the data analytics work he started back then giving consumers more ownership over their data and requiring businesses to get users' explicit consent to use and collect their data These changes will necessarily inform the way Data Propria and other data analytics firms operate Oczkowski acknowledges there will be plenty of "overlap" with Cambridge Analytica Data Propria will focus on behavioral data science which is essentially the practice of using data to target people with ads and marketing based on people's "motivational behavioral triggers." "We’re trying to figure out what shapes your worldview," he says Data Propria will conduct its own research and polling for clients develop its own targeting models based on what it learns from those polls and other datasets and work with a creative team to help them develop ads that are most likely to appeal to people based on those models Oczkowski believes the work he did helping sell a candidate in those states easily translates to helping commercial clients sell products "There are few people who understand middle America like we do," Oczkowski says of himself and Parscale He also plans on building out products similar to the ones Cambridge Analytica's team built for the campaign One in particular used data to determine what cities then-candidate Trump should visit based on local support in that place Oczkowski argues the same tool could help businesses determine where to expand Despite Cambridge Analytica's pariah status Oczkowski remains an advocate for the company's work As regulators increasingly force these companies to rethink their business models firms like Data Propria will need to focus on ways to more accurately target people based on the groups they're affiliated with not the personally identifying information that can be gleaned from data brokers companies like Data Propria still have plenty of data streams to pull from but as Facebook and others overhaul their privacy practices it's unclear how long those streams will last "There's a healthy discussion going on about privacy and I think that’s a discussion absolutely worth having Then there’s another conversation about convenience and being able to get messaging that matters to you and appeals to you," Oczkowski says "I think there’s a happy middle ground in between there that the public has to reconcile because I don't think the answer’s going to come from the government or some regulation any time soon." Parscale, who now sits on the CloudCommerce board, did not respond to WIRED's request for comment. Oczkowski maintains he was unaware of this history before joining the company. "The stuff with CloudCommerce happened long before I was involved," he says. "The stuff I've read in the press isn't really representative of what I know these guys to be." Oczkowski hopes to prove his critics, and critics of behavioral data science writ large, wrong. "The reaction to this shouldn’t be for us to peel back and say, 'Hey this isn't a good thing. We shouldn't be doing this,'" he says. "It should be figuring out what are the fair ethical standards that align marketers with consumers?" Exactly what those standards will entail remains an open question, one that Oczkowski and the rest of his industry are still trying to answer—even as they expand their data-driven pursuits. 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