Clinical Relevance of Pathological Diagnosis of Hirschsprung's Disease with Acetylcholine-Esterase Histochemistry or Calretinin Immunohistochemistry.

INTRODUCTION: Hirschsprung disease (HD) manifests as a developmental anomaly affecting the enteric nervous system, where there is an absence of ganglion cells in the lower part of the intestine. This deficiency leads to functional blockages within the intestines. HD is usually confirmed or ruled out through rectal biopsy. The identification of any ganglion cells through hematoxylin and eosin (H&E) staining rules out HD. If ganglion cells are absent, further staining with acetylcholine-esterase (AChE) histochemistry or calretinin immunohistochemistry (IHC) forms part of the standard procedure for determining a diagnosis of HD. In 2017, our Institute of Pathology at University Hospital of Heidelberg changed our HD diagnostic procedure from AChE histochemistry to calretinin IHC. In this paper, we report the impact of the diagnostic procedure change on surgical HD therapy procedures and on the clinical outcome of HD patients. METHODS: We conducted a retrospective review of the diagnostic procedures, clinical data, and postoperative progress of 29 patients who underwent surgical treatment for HD in the Department of Pediatric Surgery, University of Heidelberg, between 2012 and 2021. The patient sample was divided into two groups, each covering a treatment period of 5 years. In 2012-2016, HD diagnosis was performed exclusively using AChE histochemistry (AChE group, n = 17). In 2017-2021, HD diagnosis was performed exclusively using calretinin IHC (CR group, n = 12). RESULTS: There were no significant differences between the groups in sex distribution, weeks of gestation, birth weight, length of the aganglionic segment, or associated congenital anomalies. Almost half of the children in the AChE group, twice as many as in the CR group, required an enterostomy before transanal endorectal pull-through procedure (TERPT). In the AChE group, 4 patients (23.5%) required repeat bowel sampling to confirm the diagnosis. Compared to the AChE group, more children in the CR group suffered from constipation post TERPT. DISCUSSION: Elevated AChE expression is linked to hypertrophied extrinsic cholinergic nerve fibers in the aganglionic segment in the majority of patients with HD. The manifestation of increased AChE expression develops over time. Therefore, in neonatal patients with HD, especially those in the first 3 weeks of life, an increase in AChE reaction is not detected. Calretinin IHC reliably identifies the presence or absence of ganglion cells and offers multiple benefits over AChE histochemistry. These include the ability to perform the test on paraffin-embedded tissue sections, a straightforward staining pattern, a clear binary interpretation (negative or positive), cost-effectiveness, and utility regardless of patient age. CONCLUSIONS: The ability of calretinin IHC to diagnose HD early and time-independently prevented repeated intestinal biopsies in our patient population and allowed us to perform a one-stage TERPT in the first months of life, reducing the number of enterostomies and restoring colonic continuity early. Patients undergoing transanal pull-through under the age of 3 months require a close follow-up to detect cases with bowel movement problems.

The serotonin receptor 3E variant is a risk factor for female IBS-D.

Irritable bowel syndrome (IBS) is a gut-brain disorder of multifactorial origin. Evidence of disturbed serotonergic function in IBS accumulated for the 5-HT3 receptor family. 5-HT3Rs are encoded by HTR3 genes and control GI function, and peristalsis and secretion, in particular. Moreover, 5-HT3R antagonists are beneficial in the treatment of diarrhea predominant IBS (IBS-D). We previously reported on functionally relevant SNPs in HTR3A c.-42C > T (rs1062613), HTR3C p.N163K (rs6766410), and HTR3E c.*76G > A (rs56109847 = rs62625044) being associated with IBS-D, and the HTR3B variant p.Y129S (rs1176744) was also described within the context of IBS. We performed a multi-center study to validate previous results and provide further evidence for the relevance of HTR3 genes in IBS pathogenesis. Therefore, genotype data of 2682 IBS patients and 9650 controls from 14 cohorts (Chile, Germany (2), Greece, Ireland, Spain, Sweden (2), the UK (3), and the USA (3)) were taken into account. Subsequent meta-analysis confirmed HTR3E c.*76G > A (rs56109847 = rs62625044) to be associated with female IBS-D (OR = 1.58; 95% CI (1.18, 2.12)). Complementary expression studies of four GI regions (jejunum, ileum, colon, sigmoid colon) of 66 IBS patients and 42 controls revealed only HTR3E to be robustly expressed. On top, HTR3E transcript levels were significantly reduced in the sigma of IBS patients (p = 0.0187); more specifically, in those diagnosed with IBS-D (p = 0.0145). In conclusion, meta-analysis confirmed rs56109847 = rs62625044 as a risk factor for female IBS-D. Expression analysis revealed reduced HTR3E levels in the sigmoid colon of IBS-D patients, which underlines the relevance of HTR3E in the pathogenesis of IBS-D.

Serotonin type 3 receptor subunit gene polymorphisms associated with psychosomatic symptoms in irritable bowel syndrome: A multicenter retrospective study.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) of the serotonin type 3 receptor subunit (HTR3) genes have been associated with psychosomatic symptoms, but it is not clear whether these associations exist in irritable bowel syndrome (IBS). AIM: To assess the association of HTR3 polymorphisms with depressive, anxiety, and somatization symptoms in individuals with IBS. METHODS: In this retrospective study, 623 participants with IBS were recruited from five specialty centers in Germany, Sweden, the United States, the United Kingdom, and Ireland. Depressive, anxiety, and somatization symptoms and sociodemographic characteristics were collected. Four functional SNPs - HTR3A c.-42C>T, HTR3B c.386A>C, HTR3C c.489C>A, and HTR3E c.*76G>A - were genotyped and analyzed using the dominant and recessive models. We also performed separate analyses for sex and IBS subtypes. SNP scores were calculated as the number of minor alleles of the SNPs above. The impact of HTR3C c.489C>A was tested by radioligand-binding and calcium influx assays. RESULTS: Depressive and anxiety symptoms significantly worsened with increasing numbers of minor HTR3C c.489C>A alleles in the dominant model (F depressive = 7.475, P depressive = 0.006; F anxiety = 6.535, P anxiety = 0.011). A higher SNP score (range 0-6) was linked to a worsened depressive symptoms score (F = 7.710, P-linear trend = 0.006) in IBS. The potential relevance of the HTR3C SNP was corroborated, showing changes in the expression level of 5-HT3AC variant receptors. CONCLUSION: We have provided the first evidence that HTR3C c.489C>A is involved in depressive and anxiety symptoms in individuals with IBS. The SNP score indicated that an increasing number of minor alleles is linked to the worsening of depressive symptoms in IBS.

Treatment dependent impact of plasma-derived exosomes from head and neck cancer patients on the epithelial-to-mesenchymal transition.

Background: Epithelial to mesenchymal transition (EMT) is a key process in carcinogenesis of head and neck squamous cell carcinoma (HNSCC), contributing to tumor invasiveness, distant metastasis, and recurrence. Exosomes are known mediators and regulators of EMT. Here, we analyze the impact of exosomes that were primed by conventional therapy on EMT modulation. Methods: Plasmas of n = 22 HNSCC patients were collected before and after standard of care surgery and adjuvant or primary (chemo)radiotherapy. Exosomes were isolated by size exclusion chromatography. Upon co-incubation of exosomes with HNSCC cells, the cellular EMT profile was analyzed by flow cytometry and RT-qPCR. Wound healing assays were performed to evaluate migratory potential of exosome-treated cells. Results: Reduction of total exosome protein after therapy and in vitro exosome induced EMT profiles were dependent on the type of treatment. Exosomal TFG-ß and miRNA cargo were partly responsible for observed exosome induced EMT changes. Exosomes from recurrent patients induced higher tumor cell migration after therapy than exosomes from disease-free patients. Conclusions: HNSCC patients' exosomes from timepoints before and after therapy were able to confer therapy induced EMT modulation in vitro and have the potential to monitor the EMT process. Exosome induced changes in migratory potential emerged as discriminants of therapy outcome.

The alternative serotonin transporter promoter P2 impacts gene function in females with irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a gut-brain disorder in which symptoms are shaped by serotonin acting centrally and peripherally. The serotonin transporter gene SLC6A4 has been implicated in IBS pathophysiology, but the underlying genetic mechanisms remain unclear. We sequenced the alternative P2 promoter driving intestinal SLC6A4 expression and identified single nucleotide polymorphisms (SNPs) that were associated with IBS in a discovery sample. Identified SNPs built different haplotypes, and the tagging SNP rs2020938 seems to associate with constipation-predominant IBS (IBS-C) in females. rs2020938 validation was performed in 1978 additional IBS patients and 6,038 controls from eight countries. Meta-analysis on data from 2,175 IBS patients and 6,128 controls confirmed the association with female IBS-C. Expression analyses revealed that the P2 promoter drives SLC6A4 expression primarily in the small intestine. Gene reporter assays showed a functional impact of SNPs in the P2 region. In silico analysis of the polymorphic promoter indicated differential expression regulation. Further follow-up revealed that the major allele of the tagging SNP rs2020938 correlates with differential SLC6A4 expression in the jejunum and with stool consistency, indicating functional relevance. Our data consolidate rs2020938 as a functional SNP associated with IBS-C risk in females, underlining the relevance of SLC6A4 in IBS pathogenesis.

Parkinson mice show functional and molecular changes in the gut long before motoric disease onset.

BACKGROUND: There is increasing evidence that Parkinson's disease (PD) might start in the gut, thus involving and compromising also the enteric nervous system (ENS). At the clinical onset of the disease the majority of dopaminergic neurons in the midbrain is already destroyed, so that the lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use a transgenic A30P-α-synuclein-overexpressing PD mouse model to identify appropriate candidate markers in the gut before hallmark symptoms begin to manifest. METHODS: Based on a gait analysis and striatal dopamine levels, we defined 2-month-old A30P mice as pre-symptomatic (psA30P), since they are not showing any motoric impairments of the skeletal neuromuscular system and no reduced dopamine levels, but an intestinal α-synuclein pathology. Mice at this particular age were further used to analyze functional and molecular alterations in both, the gastrointestinal tract and the ENS, to identify early pathological changes. We examined the gastrointestinal motility, the molecular composition of the ENS, as well as the expression of regulating miRNAs. Moreover, we applied A30P-α-synuclein challenges in vitro to simulate PD in the ENS. RESULTS: A retarded gut motility and early molecular dysregulations were found in the myenteric plexus of psA30P mice. We found that i.e. neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are significantly altered in the psA30P, thus representing potential biomarkers for early PD. Many of the dysregulated miRNAs found in the psA30P mice are reported to be changed in PD patients as well, either in blood, cerebrospinal fluid or brain tissue. Interestingly, the in vitro approaches delivered similar changes in the ENS cultures as seen in the transgenic animals, thus confirming the data from the mouse model. CONCLUSIONS: These findings provide an interesting and novel approach for the identification of appropriate biomarkers in men.

A complementary study approach unravels novel players in the pathoetiology of Hirschsprung disease.

Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR.

Molecular Characterization of Embryonic Stem Cell-Derived Cardiac Neural Crest-Like Cells Revealed a Spatiotemporal Expression of an Mlc-3 Isoform.

BACKGROUND AND OBJECTIVES: Pluripotent embryonic stem (ES) cells represent a perfect model system for the investigation of early developmental processes. Besides their differentiation into derivatives of the three primary germ layers, they can also be differentiated into derivatives of the 'fourth' germ layer, the neural crest (NC). Due to its multipotency, extensive migration and outstanding capacity to generate a remarkable number of different cell types, the NC plays a key role in early developmental processes. Cardiac neural crest (CNC) cells are a subpopulation of the NC, which are of crucial importance for precise cardiovascular and pharyngeal glands' development. CNC-associated malformations are rare, but always severe and life-threatening. Appropriate cell models could help to unravel underlying pathomechanisms and to develop new therapeutic options for relevant heart malformations. METHODS: Murine ES cells were differentiated according to a mesodermal-lineage promoting protocol. Expression profiles of ES cell-derived progeny at various differentiation stages were investigated on transcript and protein level. RESULTS: Comparative expression profiling of murine ES cell multilineage progeny versus undifferentiated ES cells confirmed differentiation into known cell derivatives of the three primary germ layers and provided evidence that ES cells have the capacity to differentiate into NC/CNC-like cells. Applying the NC/CNC cell-specific marker, 4E9R, an unambiguous identification of ES cell-derived NC/CNC-like cells was achieved. CONCLUSIONS: Our findings will facilitate the establishment of an ES cell-derived CNC cell model for the investigation of molecular pathways during cardiac development in health and disease.

Expression Analysis of ATP-Binding Cassette Transporters ABCB11 and ABCB4 in Primary Sclerosing Cholangitis and Variety of Pediatric and Adult Cholestatic and Noncholestatic Liver Diseases.

ATP-binding cassette (ABC) transporters are the members of the efflux pumps that are responsible for the removal of cytotoxic substances by active transport. ABCB11, the bile salt efflux pump of hepatocytes, coordinates cellular excretion of numerous conjugated bile salts into the bile canaliculi, whereas ABCB4 acts as an ATP-dependent floppase translocating phosphatidylcholine from the inner to the outer leaflet of the bile canalicular membrane. Loss of functional ABCB11 and ABCB4 proteins causes early-onset refractory cholestasis or cholangiopathy. In this study, we investigated the expression and localization pattern of ABCB11 and ABCB4 using immunohistochemistry and RNA profiling in liver samples from patients with different types and stages of chronic cholestatic liver disease, with emphasis on primary sclerosing cholangitis (PSC), compared to a variety of cholestatic and noncholestatic hepatopathies. Therefore, ABCB11 and ABCB4 expressions were investigated on formalin-fixed and paraffin-embedded (FFPE) material in a patient cohort of total 43 patients with or without cholestatic liver diseases, on protein level using immunohistochemistry and on RNA level using nanoString technology. Intriguingly, our results demonstrated increased expression of ABCB11 and ABCB4 on protein as well as RNA level in PSC, and the expression pattern correlated with disease progression. We concluded from our study that patients with PSC demonstrate altered expression levels and pattern of ABCB11 and ABCB4 which correlated with disease progression; thereby, ABCB11 and ABCB4 analysis may be a useful tool for assessment of disease stages in PSC.

Postnatal human enteric neurospheres show a remarkable molecular complexity.

BACKGROUND: The enteric nervous system (ENS), a complex network of neurons and glial cells, coordinates major gastrointestinal functions. Impaired development or secondary aberrations cause severe enteric neuropathies. Neural crest-derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms and to develop novel therapy options. However, so far little is known about the detailed cellular composition and the proportional distribution of enteric neurospheres. Comprehensive knowledge will not only be essential for basic research but also for prospective cell replacement therapies to restore or to improve enteric neuronal dysfunction. METHODS: Human enteric neurospheres were generated from three individuals with varying age. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Corresponding archived paraffin-embedded individuals' specimens were analyzed accordingly. KEY RESULTS: Our data revealed a remarkable molecular complexity of enteric neurospheres and archived specimens. Amongst the expression of multipotent stem cell, progenitor cell, neuronal, glial, muscle and epithelial cell markers, moderate levels for the pluripotency marker POU5F1 were observed. Furthermore, besides the interindividual variability, we identified highly distinct intraindividual expression profiles. CONCLUSIONS & INFERENCES: Our results emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors, as the formation of tumors. Our study pipeline may serve as a blueprint implemented into the characterization procedure of enteric neurospheres for various future applications.

Murine transgenic embryonic stem cell lines for the investigation of sinoatrial node-related molecular pathways.

The elucidation of molecular mechanisms that restrict the potential of pluripotent stem cells and promote cardiac lineage differentiation is of crucial relevance, since embryonic stem cells (ESCs) hold great potential for cell based heart therapies. The homeodomain transcription factor Shox2 is essential for the development and proper function of the native cardiac pacemaker, the sinoatrial node. This prompted us to develop a cardiac differentiation model using ESC lines isolated from blastocysts of Shox2-deficient mice. The established cell model provides a fundamental basis for the investigation of molecular pathways under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

miR-16 and miR-103 impact 5-HT4 receptor signalling and correlate with symptom profile in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a gut-brain disorder involving alterations in intestinal sensitivity and motility. Serotonin 5-HT4 receptors are promising candidates in IBS pathophysiology since they regulate gut motor function and stool consistency, and targeted 5-HT4R selective drug intervention has been proven beneficial in subgroups of patients. We identified a single nucleotide polymorphism (SNP) (rs201253747) c.*61 T > C within the 5-HT4 receptor gene HTR4 to be predominantly present in diarrhoea-IBS patients (IBS-D). It affects a binding site for the miR-16 family and miR-103/miR-107 within the isoforms HTR4b/i and putatively impairs HTR4 expression. Subsequent miRNA-profiling revealed downregulation of miR-16 and miR-103 in the jejunum of IBS-D patients correlating with symptoms. In vitro assays confirmed expression regulation via three 3'UTR binding sites. The novel isoform HTR4b_2 lacking two of the three miRNA binding sites escapes miR-16/103/107 regulation in SNP carriers. We provide the first evidence that HTR4 expression is fine-tuned by miRNAs, and that this regulation is impaired either by the SNP c.*61 T > C or by diminished levels of miR-16 and miR-103 suggesting that HTR4 might be involved in the development of IBS-D.

Comparative expression analysis of Shox2-deficient embryonic stem cell-derived sinoatrial node-like cells.

The homeodomain transcription factor Shox2 controls the development and function of the native cardiac pacemaker, the sinoatrial node (SAN). Moreover, SHOX2 mutations have been associated with cardiac arrhythmias in humans. For detailed examination of Shox2-dependent developmental mechanisms in SAN cells, we established a murine embryonic stem cell (ESC)-based model using Shox2 as a molecular tool. Shox2+/+ and Shox2-/- ESC clones were isolated and differentiated according to five different protocols in order to evaluate the most efficient enrichment of SAN-like cells. Expression analysis of cell subtype-specific marker genes revealed most efficient enrichment after CD166-based cell sorting. Comparative cardiac expression profiles of Shox2+/+ and Shox2-/- ESCs were examined by nCounter technology. Among other genes, we identified Nppb as a novel putative Shox2 target during differentiation in ESCs. Differential expression of Nppb could be confirmed in heart tissue of Shox2-/- embryos. Taken together, we established an ESC-based cardiac differentiation model and successfully purified Shox2+/+ and Shox2-/- SAN-like cells. This now provides an excellent basis for the investigation of molecular mechanisms under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

No association between the common calcium-sensing receptor polymorphism rs1801725 and irritable bowel syndrome.

BACKGROUND: The calcium-sensing receptor (CaSR) is a calcium (Ca(2+)) sensitive G protein-coupled receptor implicated in various biological processes. In particular, it regulates Ca(2+)/Mg(2+)- homeostasis and senses interstitial Ca(2+) levels and thereby controls downstream signalling cascades. Due to its expression in the gut epithelium, the enteric nervous system and smooth muscles and its key function in regulation and coordination of muscular contraction and secretion, it represents an excellent candidate gene to be investigated in the pathophysiology of irritable bowel syndrome (IBS). Disturbed CaSR structure and function may impact gastrointestinal regulation of muscular contraction, neuronal excitation and secretion and consequently contribute to symptoms seen in IBS, such as disordered defecation as well as disturbed gut motility and visceral sensitivity. METHODS: We have therefore genotyped the functional CASR SNP rs1801725 in three case control samples from the UK, Belgium and the USA. RESULTS: Genotype frequencies showed no association in the three genotyped case-control samples, neither with IBS nor with IBS subtypes. CONCLUSIONS: Although we could not associate the SNP to any of the established bowel symptom based IBS subtypes we cannot rule out association to altered Ca(2+) levels and disturbed secretion and gut motility which were unfortunately not assessed in the patients genotyped. This underlines the necessity of a more detailed phenotyping of IBS patients and control individuals in future studies.

Transcription factor lbx1 expression in mouse embryonic stem cell-derived phenotypes.

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. During in vitro differentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation of Lbx1 at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

Shox2 mediates Tbx5 activity by regulating Bmp4 in the pacemaker region of the developing heart.

Heart formation requires a highly balanced network of transcriptional activation of genes. The homeodomain transcription factor, Shox2, is essential for the formation of the sinoatrial valves and for the development of the pacemaking system. The elucidation of molecular mechanisms underlying the development of pacemaker tissue has gained clinical interest as defects in its patterning can be related to atrial arrhythmias. We have analyzed putative targets of Shox2 and identified the Bmp4 gene as a direct target. Shox2 interacts directly with the Bmp4 promoter in chromatin immunoprecipitation assays and activates transcription in luciferase-reporter assays. In addition, ectopic expression of Shox2 in Xenopus embryos stimulates transcription of the Bmp4 gene, and silencing of Shox2 in cardiomyocytes leads to a reduction in the expression of Bmp4. In Tbx5(del/+) mice, a model for Holt-Oram syndrome, and Shox2(-/-) mice, we show that the T-box transcription factor Tbx5 is a regulator of Shox2 expression in the inflow tract and that Bmp4 is regulated by Shox2 in this compartment of the embryonic heart. In addition, we could show that Tbx5 acts cooperatively with Nkx2.5 to regulate the expression of Shox2 and Bmp4. This work establishes a link between Tbx5, Shox2 and Bmp4 in the pacemaker region of the developing heart and thus contributes to the unraveling of the intricate interplay between the heart-specific transcriptional machinery and developmental signaling pathways.