3D spheroids versus 2D-cultured human adipose stem cells to generate smooth muscle cells in an internal anal sphincter-targeting cryoinjured mouse model.

BACKGROUND: The efficacy of cell implantation via 3D-spheroids to treat basal tone in fecal incontinence remains unclear. To address this, in this study, we aimed to identify cell differentiation and assess the development of a contractile phenotype corresponding to smooth muscle cells (SMCs) following implantation of 3D-spheroid and 2D-cultured human adipose stem cells (hASCs) in an in vivo internal anal sphincter (IAS)-targeted mouse model. METHODS: We developed an IAS-targeted in vivo model via rapid freezing (at - 196 °C) of the dorsal layers of the region of interest (ROI) of the IAS ring posterior quarter, between the submucosal and muscular layers, following submucosal dissection (n = 60 rats). After implantation of tetramethylindocarbocyanine perchlorate (Dil)-stained 3D and 2D-cells into randomly allocated cryoinjured rats, the entire sphincter ring or only the cryoinjured ROI was harvested. Expression of SMC markers, RhoA/ROCKII and its downstream molecules, and fibrosis markers was analyzed. Dil, α-smooth muscle actin (α-SMA), and RhoA signals were used for cell tracking. RESULTS: In vitro, 3D-spheroids exhibited higher levels of SMC markers and RhoA/ROCKII-downstream molecules than 2D-hASCs. The IAS-targeted cryoinjured model exhibited substantial loss of SMC layers of the squamous epithelium lining of the anal canal, as well as reduced expression of SMC markers and RhoA-related downstream molecules. In vivo, 3D-spheroid implantation induced SMC markers and contractile molecules weakly at 1 week. At 2 weeks, the mRNA expression of aSma, Sm22a, Smoothelin, RhoA, Mypt1, Mlc20, Cpi17, and Pp1cd increased, whereas that of fibrosis markers reduced significantly in the 3D-spheroid implanted group compared to those in the sham, non-implanted, and 2D-hASC implanted groups. Protein levels of RhoA, p-MYPT1, and p-MLC20 were higher in the 3D-spheroid-implanted group than in the other groups. At 2 weeks, in the implanted groups, the cryoinjured tissues (which exhibited Dil, α-SMA, and RhoA signals) were restored, while they remained defective in the sham and non-implanted groups. CONCLUSIONS: These findings demonstrate that, compared to 2D-cultured hASCs, 3D-spheroids more effectively induce a contractile phenotype that is initially weak but subsequently improves, inducing expression of RhoA/ROCKII-downstream molecules and SMC differentiation associated with IAS basal tone.

Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter.

The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca2+ activities in the IAS. The present study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs), indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type, whereas the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (l-NNA) abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by l-NNA but not MRS2500, suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)+ cells but not ICC-IM expressed P2Y1 receptors (P2Y1R) and small-conductance Ca2+-activated K+ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca2+ transients in Type II ICC-IM via PDGFRα+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS, with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC, and PDGFRα+ cells via the SIP syncytium.NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca2+ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα+ cells).

Interstitial cells of Cajal: clinical relevance in pediatric gastrointestinal motility disorders.

Interstitial cells of Cajal (ICCs) are pacemaker cells of gastrointestinal motility that generate and transmit electrical slow waves to smooth muscle cells in the gut wall, thus inducing phasic contractions and coordinated peristalsis. Traditionally, tyrosine-protein kinase Kit (c-kit), also known as CD117 or mast/stem cell growth factor receptor, has been used as the primary marker of ICCs in pathology specimens. More recently, the Ca2+-activated chloride channel, anoctamin-1, has been introduced as a more specific marker of ICCs. Over the years, various gastrointestinal motility disorders have been described in infants and young children in which symptoms of functional bowel obstruction arise from ICC-related neuromuscular dysfunction of the colon and rectum. The current article provides a comprehensive overview of the embryonic origin, distribution, and functions of ICCs, while also illustrating the absence or deficiency of ICCs in pediatric patients with Hirschsprung disease intestinal neuronal dysplasia, isolated hypoganglionosis, internal anal sphincter achalasia, and congenital smooth muscle cell disorders such as megacystis microcolon intestinal hypoperistalsis syndrome.

HPV virus and biomarkers of resistance to chemoradiation in circulating tumor cells from patients with squamous cell carcinoma of the anus.

Localized anal cancer is mostly represented by squamous cell carcinoma of the anus (SCCA) and is cured in ≥80 % of cases by chemoradiation (CRT). Development of techniques for detection/evaluating circulating tumor cells (CTCs) for diagnosis/ prognosis/response to therapy can change the manner we treat/follow SCCA patients. OBJECTIVE: to detect CTCs from patients with SCCA and evaluate the presence of HPV virus, p16 expression and markers related to resistance to CRT (RAD23B/ ERCC1/ TYMS) in CTCs at baseline and after CRT. METHODS: CTCs were isolated/quantified by ISET®, protein expressions were analyzed by immunocytochemistry and HPV DNA was detected by chromogenic in situ hybridization. RESULTS: We enrolled 15 patients: median age was 61 (43-73) years, the majority was women (10/15). CTCs were detected in all patients at baseline (median= 0.4 (0.4-3.33) CTCs/mL) and in 8/9 patients, after CRT (median= 2.33 (0-7.0) CTCs/mL). DNA from HPV was found in CTCs in 14/15 patients (93.33 %) at baseline and in 7/9 (77.7 %) after treatment. At a median follow-up of 22.20 (1.45-38.55) months, three patients expressed ERCC1 in CTCs after treatment, with one of them having disease recurrence. CONCLUSION: We showed that detection of HPV in CTCs from patients with non-metastatic SCCA is feasible and appears to be a sensitive diagnostic method. These results may be clinically useful for better monitoring these patients. However, future larger cohorts may demonstrate whether there is any correlation between the presence of HPV and the expression of screening markers for CRT in SCCA.

Characterisation of anal intraepithelial neoplasia and anal cancer in HIV-positive men by immunohistochemical markers p16, Ki-67, HPV-E4 and DNA methylation markers.

Human papillomavirus (HPV)-induced anal intraepithelial neoplasia (AIN, graded 1-3) is highly prevalent in HIV-positive (HIV+) men who have sex with men (MSM), but only a minority of lesions progresses to cancer. Our study aimed to characterise comprehensively anal tissue samples from a cross-sectional series (n = 104) of HIV+ MSM and longitudinal series (n = 40) of AIN2/3 progressing to cancer using different biomarkers. The cross-sectional series consisted of 8 normal, 26 AIN1, 45 AIN2, 15 AIN3 and 10 anal squamous cell carcinoma. Tissue sections were immunohistochemically (IHC) stained for p16 (viral transformation marker), Ki-67 (cellular proliferation marker) and HPV-E4 (viral production marker). We evaluated the expression of IHC markers and compared it with DNA methylation, a marker for malignant transformation. E4 positivity decreased, whereas p16 and Ki-67 scores and methylation marker positivity increased (P values < .001) with increasing severity of anal lesions. Within AIN2, a heterogeneous biomarker pattern was observed concerning E4, p16 and methylation status, reflecting the biological heterogeneity of these lesions. In the longitudinal series, all AIN2/3 and carcinomas showed high p16 and Ki-67 expression, strong methylation positivity and occasional E4 positivity. We earlier showed that high methylation levels are associated with progression to cancer. The observed E4 expression in some AIN2/3 during the course of progression to cancer and absence of E4 in a considerable number of AIN1 lesions make the potential clinical significance of E4 expression difficult to interpret. Our data show that IHC biomarkers can help to characterise AIN; however, their prognostic value for cancer risk stratification, next to objective methylation analysis, appears to be limited.

The transcriptome of anal papillae of Aedes aegypti reveals their importance in xenobiotic detoxification and adds significant knowledge on ion, water and ammonia transport mechanisms.

The anal papillae of mosquito larvae are osmoregulatory organs in direct contact with the external aquatic environment that actively sequester ions and take up water in dilute freshwater. In the disease vector Aedes aegypti mechanisms of ion, water and ammonia transport have only been partially resolved. Furthermore, A. aegypti larvae are known to reside in high ammonia sewage and high salt brackish waters, and understanding of anal papillae function in these conditions is in its infancy. The objective of this study was to identify the complement of ion and water transport genes expressed by the anal papillae of freshwater larvae by sequencing their transcriptome, and comparing their expression in anal papillae of larvae abruptly transferred to brackish water for 24 h. Results identified a number of ion and water transport proteins, ammonia detoxifying enzymes, a full suite of xenobiotic detoxifying enzymes and transporters, and G-protein coupled receptors of specific hormones. We identified a marked increase in transcript and protein abundance of aquaporin AaAQP2 in the anal papillae with abrupt transfer to brackish water. We present an updated and more comprehensive model for ion and water transport with additional putative transporters for Na+ and Cl- uptake in the anal papillae. These are organs which are actively engaged in Na+, Cl- and water uptake and regulation when the aquatic larvae encounter fluctuating salinities over the course of their development. Furthermore the transcriptome of the anal papillae includes a full set of xenobiotic detoxification genes suggesting that these are important detoxification organs which is particularly important when larvae reside in polluted water.

Oscillating calcium signals in smooth muscle cells underlie the persistent basal tone of internal anal sphincter.

A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence; its inhibition via the rectoanal inhibitory reflex (RAIR) is required for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2-photon imaging, and cell-specific gene-modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca2+ signals (i.e., synchronized Ca2+ oscillations [SCaOs] and asynchronized Ca2+ oscillations [ACaOs]) that last for hours. RyRs, TMEM16A, L-type Ca2+ channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca2+ signals, leading to IAS's full relaxation. Our results show that the oscillating nature of Ca2+ signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation.

Neoadjuvant PD-1 Blockade Combined With Chemotherapy Followed by Concurrent Immunoradiotherapy in Locally Advanced Anal Canal Squamous Cell Carcinoma Patients: Antitumor Efficacy, Safety and Biomarker Analysis.

Background: Anal canal squamous cell carcinoma (ACSCC) is an exceedingly rare malignant neoplasm with challenges in sphincter preservation, treatment toxicities and long-term survival. Little is known concerning the activity of PD-1 antibodies in locally advanced ACSCC. This study reports on the efficacy and toxicities of a neoadjuvant PD-1 blockade combined with chemotherapy followed by concurrent immunoradiotherapy in ACSCC patients, and describes biomarkers expression and mutation signatures. Methods: In this cohort study, patients were treated as planned, including four cycles of neoadjuvant PD-1 antibody toripalimab combined with docetaxol and cisplatin, followed by radiotherapy and two cycles of concurrent toripalimab. Multiplex immunofluorescence staining (mIHC) with PD-L1, CD8, CD163, Pan-Keratin and DAPI was performed with the pretreatment tumor tissue. Whole exome sequencing was performed for the primary tumor and peripheral blood mononuclear cells. The primary endpoint was the complete clinical response (cCR) rate at 3 months after overall treatment. Acute and late toxicities graded were assessed prospectively. Results: Five female patients with a median age of 50 years old (range, 43-65 years old), finished treatment as planned. One patient had grade 3 immune related dermatitis. Two patients had grade 3 myelosuppression during neoadjuvant treatment. No severe radiation-related toxicities were noted. Four patients with PD-L1 expression >1% achieved a cCR after neoadjuvant treatment. and the other patient with negative PD-L1 expression also achieved a cCR at 3 months after radiotherapy. All the patients were alive and free from disease and had a normal quality of life, with 19.6-24 months follow up. Inconsistent expression of PD-L1 and CD163 was detected in 3 and 5 patients, respectively. TTN, POLE, MGAM2 were the top mutation frequencies, and 80 significant driver genes were identified. Pathway analysis showed enrichment of apoptosis, Rap1, Ras, and pathways in cancer signaling pathways. Eight significantly deleted regions were identified. Conclusions: This small cohort of locally advanced ACSCC patients had quite satisfactory cCR and sphincter preservation rate, after neoadjuvant PD-1 antibody toripalimab combined with chemotherapy followed by concurrent immunoradiotherapy, with mild acute and long-term toxicities.

Physiological 18F-FDG uptake in the normal adult anal canal: evaluation by PET/CT.

OBJECTIVE: Despite their benefit for detecting primary tumors, data for normal 18F-fluoro-2-deoxy-D-glucose (FDG) uptake in the anal canal are insufficient. Here we used positron emission tomography-computed tomography (PET/CT) to determine the uptake of FDG in the normal adult anal canal (AC) and to evaluate its clinical significance compared with that of anal cancer. METHODS: We conducted a retrospective study of-PET/CT images in the anal region, of 201 consecutive patients without symptoms or pathology taken from January 2015 to August 2019, after excluding two patients (one each with Crohn's disease and hemorrhoid). These patients were included in the normal group, and data of eight patients with anal cancer were collected from January 2011 to August 2019 for comparison. FDG uptake was quantitatively evaluated (compared with the maximum standardized uptake value [SUVmax] to the SUVmax values of liver and distal rectum) and qualitatively (compared with background) in early and delayed phases. Normal grade 3 uptake was qualitatively defined as FDG uptake higher than the surrounding muscles. RESULTS: In the normal group, mean anal canal SUVmax of early phase was: 2.26 (range 1.00-6.30), and delayed phase: 2.52 (range 1.00-8.80). Their ratios to liver SUVmax were early: 0.74 (range 0.24-2.25), and delayed: 0.81 (range 0.23-2.32); ratios to rectal SUVmax were early: 0.87 (range 0.30-1.89), and delayed: 0.90 (range 0.30-1.27). Qualitatively, 25 patients (15.4%) had normal grade 3 uptake during the early and delayed phases. In contrast, qualitative data showed that all patients with anal cancer exhibited high FDG uptake in the anal canal. The mean early- and delayed-phase values of SUVmax of the anal canal and anal cancer group were 11.09 (range 5.40-17.73) and 14.23 (range 6.70-22.85), respectively. There was a significant difference between the mean-early and -delayed anal SUVmax values of the normal grade 3 and anal cancer groups. Furthermore, the ratios to liver SUVmax were significantly different between the two groups. CONCLUSIONS: PET/CT scans occasionally showed high FDG uptake in the anal canal of healthy adults. Comparing the SUVmax values of liver FDG uptake may help differentiate between normal tissue and anal cancer.

A rigorous exploration of anal HPV genotypes using a next-generation sequencing (NGS) approach in HIV-infected men who have sex with men at risk for developing anal cancer.

BACKGROUND: There are no HPV-based measures for managing anal cancer (AC) in HIV-infected (HIV+) men who have sex with men (MSM) because of the high positivity of high-risk (HR)-HPVs. As next-generation sequencing (NGS) is able to describe the composition of HPVs as percent (%) reads rather than positive vs negative results, we used NGS approach to detect HPVs in anal samples of HIV+ MSM to test its ability to differentiate those who are diagnosed with atypical squamous cells of unknown significance or greater (ASCUS+) from those who are free of such lesions and to understand the burden of HPV infections in relation to HPV vaccines. METHODS: Study included 81 HIV+ MSM characterized for demographics, patient-reported outcome measures, HIV related laboratory measures and anal cytology. We summarized NGS HPV data using % read cut points (>0%->30%) and tested the relationship between % reads of HR-HPVs and risk of ASCUS+ using logistic regression. RESULTS: Forty-six HPVs were detected at the >0% read cut point. The prevalence of any HR-HPVs varied from 100% to 40% with >0% to >30% reads while ≥99% were infected with HR-HPVs included or not included in the 9 valent HPV vaccine at the >0% read cut point. MSM with >30% HR-HPV reads were 4.5 times more likely to be diagnosed with ASCUS+ compared to ≤30% reads (P = .033). CONCLUSION: NGS-based approach is more accurate than PCR-based HPV testing for identifying HIV+ MSM at risk for developing AC. We raise the concern regarding the efficacy of current HPV vaccines for preventing AC in this high-risk population.

Development of Aedes aegypti (Diptera: Culicidae) mosquito larvae in high ammonia sewage in septic tanks causes alterations in ammonia excretion, ammonia transporter expression, and osmoregulation.

Larvae of the disease vector mosquito, Aedes aegypti (L.) readily develop in ammonia rich sewage in the British Virgin Islands. To understand how the larvae survive in ammonia levels that are lethal to most animals, an examination of ammonia excretory physiology in larvae collected from septic-water and freshwater was carried out. A. aegypti larvae were found to be remarkably plastic in dealing with high external ammonia through the modulation of NH4+ excretion at the anal papillae, measured using the scanning ion-selective electrode technique (SIET), and NH4+ secretion in the primary urine by the Malpighian tubules when developing in septicwater. Ammonia transporters, Amt and Rh proteins, are expressed in ionoregulatory and excretory organs, with increases in Rh protein, Na+-K+-ATPase, and V-type-H+-ATPase expression observed in the Malpighian tubules, hindgut, and anal papillae in septic-water larvae. A comparative approach using laboratory A. aegypti larvae reared in high ammonia septic-water revealed similar responses to collected A. aegypti with regard to altered ammonia secretion and hemolymph ion composition. Results suggest that the observed alterations in excretory physiology of larvae developing in septic-water is a consequence of the high ammonia levels and that A. aegypti larvae may rely on ammonia transporting proteins coupled to active transport to survive in septic-water.

Knowing Me, Knowing You: Anal Gland Secretion of European Badgers (Meles meles) Codes for Individuality, Sex and Social Group Membership.

European badgers, Meles meles, are group-living in the UK, and demarcate their ranges with shared latrines. As carnivores, badgers possess paired anal glands, but olfactory information on the content of badger anal gland secretion (AGS) is largely uninvestigated. Here, we examined the volatile organic compounds (VOCs) of AGS samples from 57 free-living badgers using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry. AGS was rich in alkanes (C7-C15, 14.3% of identified compounds), aldehydes (C5-C14, 9.7%), phenols (C6-C15, 9.5%), alcohols (C5-C10, 7.3%), aromatic hydrocarbons (C6-C13, 6.8%), ketones (C6-C13, 6.3%) and carboxylic acids (C3-C12, 5.6%) and contained a variety of esters, sulfurous and nitrogenous compounds, and ethers. The number of VOCs per profile ranged from 20 to 111 (mean = 65.4; ± 22.7 SD), but no compound was unique for any of the biological categories. After normalization of the raw data using Probabilistic Quotient Normalization, we produced a resemblance matrix by calculating the Euclidian distances between all sample pairs. PERMANOVA revealed that AGS composition differs between social groups, and concentration and complexity in terms of number of measurable VOCs varies between seasons and years. AGS VOC profiles encode individual identity, sex and vary with female reproductive state, indicating an important function in intraspecific communication. Because AGS is excreted together with fecal deposits, we conclude that chemical complexity of AGS enables particularly latrine-using species, such as badgers, to advertise more complex individual-specific information than in feces alone.

Perianal and perineal keratoacanthoma: Two cases demonstrating histologic similarity to subungual keratoacanthoma.

Perianal keratoacanthomas are rare, with 10 cases reported to date. Perineal keratoacanthoma has not previously been described. In this report, we describe two cases of keratoacanthoma, one perianal and one perineal. Both lesions show prominent dyskeratotic keratinocytes, with striking and curious histologic resemblance to subungual keratoacanthoma.

Downregulation of thromboxane A2 and angiotensin II type 1 receptors associated with aging-related decrease in internal anal sphincter tone.

Aging-associated decrease in internal anal sphincter (IAS) tone (AADI) is a major contributor in the rectoanal incontinence (RI). To determine the pathogenesis of AADI, we investigated the effect of aging on GPCR activation and related downstream signaling. We particularly investigated two GPCRs that characterize IAS smooth muscle cells (SMCs): thromboxane A2 and angiotensin II type 1. Two groups of Fischer 344 rats (6-month-old [young group] and 26-month-old [old group]) were employed to determine the GPCR function by isometric contraction, the expressions of GPCRs, and their downstream regulatory signaling proteins (regulator of G-protein signaling 2, RGS2; GPCR Kinase 5, GRK5; and ß-arrestin, Arrb2) using RT-PCR, qPCR, and western blot analyses. We used reversible biotinylation to monitor the GPCR trafficking using SMCs. Aging selectively attenuated thromboxane A2 and Ang II-induced IAS contraction. RT-PCR, qPCR, and WB data revealed a significant decrease in the expressions of the GPCRs and increase in the expression of RGS2, GRK5, and Arrb2. The increased GPCR internalization and decreased recycling under aging were validated by reversible biotinylation. We conclude that downregulation of GPCR, accompanied by upregulation of regulatory proteins, plays an important role in receptor desensitization and may be important underlying mechanisms of RI in certain aging patients.

Human Papillomavirus (HPV)-associated Lymphoepithelioma-like Carcinoma of the Vagina and Anal Canal: A Rare Variant of Squamous Cell Carcinoma.

Lymphoepithelioma-like carcinoma (LELC) is an uncommon variant of squamous cell carcinoma, which is histologically identical to lymphoepithelial carcinoma of the nasopharynx. LELCs have been reported at a variety of sites, including the stomach, salivary gland, thymus, cervix, endometrium, breast, skin, bladder, and lung. We report 2 LELCs of the vagina and 1 of the anal canal, the first report of LELC at the latter site. All 3 neoplasms were diffusely positive with p16 (block-type immunoreactivity) and the anal canal lesion contained high-risk human papillomavirus type 16; the 2 vaginal neoplasms underwent human papillomavirus testing but were unsuitable for analysis. All cases were Epstein-Barr virus negative. In reporting these cases, we highlight the potential for misdiagnosis and suggest an association with human papillomavirus infection similar to LELCs in the uterine cervix.

Effect of high-fat diet-induced obesity on the small-conductance Ca2+-activated K+ channel function affecting the contractility of rat detrusor smooth muscle.

PURPOSE: Obesity usually induces overactive bladder (OAB) associated with detrusor overactivity, which is related to increased contractility of the detrusor smooth muscle (DSM). Small-conductance Ca2+-activated K+ (SK) channels play a constitutive role in the regulation of DSM contractility. However, the role of SK channels in the DSM changes in obesity-related OAB is still unknown. Here, we tested the hypothesis that obesity-related OAB is associated with reduced expression and activity of SK channels in DSM and that SK channels activation is a potential treatment for OAB. METHODS: Female Sprague-Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD) and weighed after 12 weeks. Urodynamic studies, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and isometric tension recording were performed. RESULTS: Increased average body weights and urodynamically demonstrated OAB were observed in HFD rats. qRT-PCR experiments revealed a decrease in the mRNA expression level of SK channel in DSM tissue of the HFD rats. Isometric tension recordings indicated an attenuated relaxation effect of NS309 on the spontaneous phasic and electrical field stimulation-induced contractions that occurred via SK channel activation in HFD DSM strips. CONCLUSIONS: Reduced expression and activity of SK channels in the DSM contribute to obesity-related OAB, indicating that SK channels are a potential therapeutic target for OAB.

Isolation of internal and external sphincter progenitor cells from the human anal sphincter with or without radiotherapy.

AIM: We aimed to isolate and propagate internal and external anal sphincter progenitor cells from the human anal sphincter, with or without radiotherapy, for tailored cell therapy of faecal incontinence. METHODS: Sphincter progenitor cells were isolated from normal internal and external anal sphincters collected from 10 patients with rectal cancer who had undergone abdominoperineal resection with (n = 6) or without (n = 4) preoperative chemoradiotherapy. The isolated cells and differentiated muscle fibres were identified using immunofluorescence assay, western blotting and reverse transcription polymerase chain reaction (RT-PCR). The proliferation of progenitor cells with and without radiotherapy was compared by quantitative 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The immunofluorescence assay before differentiation confirmed that the internal anal sphincter progenitor cells expressed CD34 and neural-glial antigen 2 (NG2), whereas the external anal sphincter progenitor cells expressed CD34 and PAX7. After differentiation, the internal anal sphincter progenitor cells expressed desmin, calponin and α-smooth muscle actin, whereas the external anal sphincter progenitor cells expressed desmin, myogenic factor 4 and myosin heavy chain. The differential expression profiles of both cell types were confirmed by western blotting and RT-PCR. MTT assays showed that the viability of internal and external anal sphincter progenitor cells was significantly lower in the radiotherapy group than that in the nonradiotherapy group. CONCLUSIONS: This study describes the differential harvest internal and external sphincter muscle progenitor cells from human anal sphincters. We confirm that radiotherapy decreases the viability of internal and external anal sphincter progenitor cells.

Innervation of the entire internal anal sphincter in a mouse model of Hirschsprung's disease: a first report.

BACKGROUND: Impaired function of the internal anal sphincter (IAS) may be implicated in postoperative obstructed defecation (POD) that may complicate Hirschsprung's disease (HD) patients. While innervation of part of the IAS in HD has been reported, accurate details based on anatomic landmarks that can explain the clinical morbidity seen in POD are lacking, and there appear to be no studies that specifically document the innervation of the "entire" IAS in HD. We used endothelin receptor-B knockout mice to represent HD (HD-mice) and C57B6 wild mice as controls (C-mice) to investigate the innervation of the entire IAS to assess the pathophysiology of POD experimentally. METHODS: The end-point of the longitudinal muscle layer was used to define the border between the IAS and the circular muscle layer (CML). Specimens of anorectum from HD- and C-mice were immunostained with PGP 9.5 and S100 as general nerve markers, nNOS and VIP as parasympathetic nerve markers, TH as a sympathetic nerve marker, and calretinin as a reliable diagnostic marker for HD. Immunostained cells/fibers were quantified using ImageJ. RESULTS: On fluorescence microscopy, PGP 9.5, nNOS, and calretinin were significantly lower in the IAS of HD-mice than in C-mice (p < 0.05, respectively), while there were no significant differences between HD-mice and C-mice for S100, VIP, or TH. CONCLUSION: We are the first to confirm that the expression of histochemical markers of innervation is abnormal throughout the "entire" IAS in HD-mice. Application of this finding may be beneficial for preventing POD and requires further research.

Changes in murine anorectum signaling across the life course.

BACKGROUND: Increasing age is associated with an increase in the incidence of chronic constipation and fecal impaction. The contribution of the natural aging process to these conditions is not fully understood. This study examined the effects of increasing age on the function of the murine anorectum. METHODS: The effects of increasing age on cholinergic, nitrergic, and purinergic signaling pathways in the murine anorectum were examined using classical organ bath assays to examine tissue function and electrochemical sensing to determine age-related changes in nitric oxide and acetylcholine release. KEY RESULTS: Nitrergic relaxation increased between 3 and 6 months, peaked at 12 months and declined in the 18 and 24 months groups. These changes were in part explained by an age-related decrease in nitric oxide (NO) release. Cholinergic signaling was maintained with age by an increase in acetylcholine (ACh) release and a compensatory decrease in cholinesterase activity. Age-related changes in purinergic relaxation were qualitatively similar to nitrergic relaxation although the relaxations were much smaller. Increasing age did not alter the response of the anorectum smooth muscle to exogenously applied ACh, ATP, sodium nitroprusside or KCl. Similarly, there was no change in basal tension developed by the anorectum. CONCLUSIONS AND INFERENCES: The decrease in nitrergic signaling with increasing age may contribute to the age-related fecal impaction and constipation previously described in this model by partially obstructing defecation.

Characterization of cadmium and calcium fluxes along the gut, malpighian tubules, and anal papillae of the dipteran Chironomus riparius.

Chironomids are often one of the dominant organisms in significantly polluted freshwater. Many invertebrate studies have characterized whole-organism mechanisms of toxicity, for example, assessing cadmium (Cd) uptake via calcium (Ca) channels. However, with the use of the scanning ion-selective electrode technique and an innovative Cd-selective microelectrode, we analyze this relationship at the organ level using a realistic concentration of Cd and Ca in the hemolymph (blood). Generally, Cd fluxes follow the same directional pattern as Ca, although Ca fluxes are approximately 5 times higher than those of Cd. These results correlate well with previous studies indicating that chironomids have a higher affinity for Ca over Cd, which affords them tolerance to Cd toxicity. When saline Ca concentration was increased to 10 times physiological levels, Cd fluxes from the gut lumen into the cells of the midgut regions were reduced by 50 to 80%. Transport of Cd from hemolymph to tissue for the posterior midgut, Malpighian tubule, and proximal ceca was also reduced by approximately 50%. The present results indicate that Cd fluxes into or across the gut and Malpighian tubules are reduced by high Ca, suggesting that Cd may be transported in some cells by similar mechanisms. However, Cd was actively excreted at the anal papillae after a 48-h waterborne exposure to Cd, but this process was independent of Ca and instead may involve a P-glycoprotein-related pump to detoxify Cd. Environ Toxicol Chem 2018;37:2542-2549. © 2018 SETAC.