Anti-Gephyrin Antibodies: A Novel Specificity in Patients With Systemic Sclerosis and Lower Bowel Dysfunction.

OBJECTIVE: Autoantibodies are clinically useful in phenotyping patients with systemic sclerosis (SSc). Gastrointestinal (GI) function is regulated by the enteric nervous system (ENS) and commonly impaired in SSc, suggesting that the SSc autoimmune response may target ENS antigens. We sought to identify novel anti-ENS autoantibodies with an aim to clinically phenotype SSc GI dysfunction. METHODS: Serum from a patient with SSc with GI dysfunction but without defined SSc-associated autoantibodies was used for autoantibody discovery. Immunoprecipitations performed with murine myenteric plexus lysates were on-bead digested, and autoantigens were identified by mass spectrometry. Prevalence was determined, and clinical features associated with novel autoantibodies were evaluated in a SSc cohort using regression analyses. The expression of gephyrin in human GI tract tissue was examined by immunohistochemistry. RESULTS: We identified gephyrin as a novel SSc autoantigen. Anti-gephyrin antibodies were present in 9% of patients with SSc (16/188) and absent in healthy controls (0/46). Anti-gephyrin antibody-positive patients had higher constipation scores (1.00 vs 0.50, P = 0.02) and were more likely to have severe constipation and severe distention/bloating (46% vs 15%, P = 0.005; 54% vs 25%, P = 0.023, respectively). Anti-gephyrin antibody levels were significantly higher among patients with severe constipation (0.04 vs 0.00; P = 0.001) and severe distention and bloating (0.03 vs 0.004; P = 0.010). Severe constipation was associated with anti-gephyrin antibodies even in the adjusted model. Importantly, gephyrin was expressed in the ENS, which regulates gut motility. CONCLUSION: Gephyrin is a novel ENS autoantigen that is expressed in human myenteric ganglia. Anti-gephyrin autoantibodies are associated with the presence and severity of constipation in patients with SSc.

Age-associated changes in lineage composition of the enteric nervous system regulate gut health and disease.

The enteric nervous system (ENS), a collection of neural cells contained in the wall of the gut, is of fundamental importance to gastrointestinal and systemic health. According to the prevailing paradigm, the ENS arises from progenitor cells migrating from the neural crest and remains largely unchanged thereafter. Here, we show that the lineage composition of maturing ENS changes with time, with a decline in the canonical lineage of neural-crest derived neurons and their replacement by a newly identified lineage of mesoderm-derived neurons. Single cell transcriptomics and immunochemical approaches establish a distinct expression profile of mesoderm-derived neurons. The dynamic balance between the proportions of neurons from these two different lineages in the post-natal gut is dependent on the availability of their respective trophic signals, GDNF-RET and HGF-MET. With increasing age, the mesoderm-derived neurons become the dominant form of neurons in the ENS, a change associated with significant functional effects on intestinal motility which can be reversed by GDNF supplementation. Transcriptomic analyses of human gut tissues show reduced GDNF-RET signaling in patients with intestinal dysmotility which is associated with reduction in neural crest-derived neuronal markers and concomitant increase in transcriptional patterns specific to mesoderm-derived neurons. Normal intestinal function in the adult gastrointestinal tract therefore appears to require an optimal balance between these two distinct lineages within the ENS.

A comparative study of the effect of two different delivery techniques (conventional versus microplegia) of del Nido cardioplegia on myocardium in paeditric congenital heart disease.

INTRODUCTION: del Nido cardioplegia was developed for immature myocardium to prevent myocardial damage by Ca+2 in traditional blood cardioplegia. But due to increased hemodilution and decreased colloid oncotic pressure it may cause myocardial edema and increased cardiac morbidity. Microplegia may have better cardioprotection in comparison to del Nido as there is less hemodilution. MATERIAL AND METHODS: 60 patients from the age group of 1 to 14 years were divided into two groups i.e. del Nido based microplegia group and conventional del Nido group for studying two different cardioplegia technique. Data were collected and compared for intraoperative Hb, CPK-MB and Trop-I levels changes and requirement for defibrillation in intraoperative period. Demographic data, CPB time and ACC time were also collected. RESULTS: Marked elevation in CPK-MB and Trop-I levels were seen in both groups. Statistically significant difference was seen in CPK-MB levels after 6 h of surgery where del Nido group has higher value in comparison to microplegia group. No statistical difference was seen in Trop-I levels in both groups. Strength of correlation (r) was also stronger for CPK-MB rise in association with CPB time and ACC time, in del Nido group but not for Trop-I. Significantly higher hemodilution was also seen in del Nido group after delivering cardioplegia. None of the patients required defibrillation in any group. CONCLUSION: Lesser hemodilution was seen in microplegia group. Significant cardioprotection is associated with use of microplegia solution in pediatric age group.

Systemic sclerosis gastrointestinal dysmotility: risk factors, pathophysiology, diagnosis and management.

Nearly all patients with systemic sclerosis (SSc) are negatively affected by dysfunction in the gastrointestinal tract, and the severity of gastrointestinal disease in SSc correlates with high mortality. The clinical complications of this dysfunction are heterogeneous and include gastro-oesophageal reflux disease, gastroparesis, small intestinal bacterial overgrowth, intestinal pseudo-obstruction, malabsorption and the requirement for total parenteral nutrition. The abnormal gastrointestinal physiology that promotes the clinical manifestations of SSc gastrointestinal disease throughout the gastrointestinal tract are diverse and present a range of therapeutic targets. Furthermore, the armamentarium of medications and non-pharmacological interventions that can benefit affected patients has substantially expanded in the past 10 years, and research is increasingly focused in this area. Here, we review the details of the gastrointestinal complications in SSc, tie physiological abnormalities to clinical manifestations, detail the roles of standard and novel therapies and lay a foundation for future investigative work.

Relationship Between Gastrointestinal Transit, Medsger Gastrointestinal Severity, and University of California-Los Angeles Scleroderma Clinical Trial Consortium Gastrointestinal Tract 2.0 Symptoms in Patients With Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc)-associated gastrointestinal (GI) complications are attributed to a variety of factors, including diet, microbiota dysbiosis, or GI transit abnormalities. Our objective was to examine the contribution of abnormal GI transit to SSc Medsger GI severity scores and/or University of California Los Angeles Scleroderma Clinical Trial Consortium Gastrointestinal Tract (UCLA GIT) 2.0 symptoms. METHODS: Patients with SSc and GI symptoms (n = 71) and healthy controls (n = 18) underwent whole gut transit (WGT) scintigraphy to assess transit from the esophagus to the colon. The presence of delayed transit and percent emptying in each GI region were measured. We compared the WGT measurements between categories of the Medsger GI severity score (0-4) and across UCLA GIT 2.0 domains and total score (0-3). RESULTS: A total of 88% of patients had >1 abnormal region of the gut on WGT scintigraphy. All patients requiring total parenteral nutrition had delayed small bowel transit, compared to only approximately 11% of patients in other Medsger GI severity groups (P ≤ 0.01). Severe colonic transit delays were more likely in patients with Medsger GI scores of 3 (pseudo-obstruction and/or malabsorption) compared to other Medsger GI groups (P = 0.02). Seventy-percent of these patients had ≤30% colonic emptying at 72 hours. Modest associations were noted between gastroesophageal reflux disease symptoms and delayed esophageal (r = -0.31, P = 0.05) and gastric emptying (r = -0.32, P = 0.05). CONCLUSION: These data are important in providing evidence that SSc bowel disease affects transit of GI content and that delay in transit accounts in part for both bowel symptoms and Medsger GI severity. Prospective studies examining the benefit of early therapeutic intervention targeting GI transit abnormalities in patients at high risk for severe GI complications are needed.

COUNTEN, an AI-Driven Tool for Rapid and Objective Structural Analyses of the Enteric Nervous System.

The enteric nervous system (ENS) consists of an interconnected meshwork of neurons and glia residing within the wall of the gastrointestinal (GI) tract. While healthy GI function is associated with healthy ENS structure, defined by the normal distribution of neurons within ganglia of the ENS, a comprehensive understanding of normal neuronal distribution and ganglionic organization in the ENS is lacking. Current methodologies for manual enumeration of neurons parse only limited tissue regions and are prone to error, subjective bias, and peer-to-peer discordance. There is accordingly a need for robust, and objective tools that can capture and quantify enteric neurons within multiple ganglia over large areas of tissue. Here, we report on the development of an AI-driven tool, COUNTEN (COUNTing Enteric Neurons), which is capable of accurately identifying and enumerating immunolabeled enteric neurons, and objectively clustering them into ganglia. We tested and found that COUNTEN matches trained humans in its accuracy while taking a fraction of the time to complete the analyses. Finally, we use COUNTEN's accuracy and speed to identify and cluster thousands of ileal myenteric neurons into hundreds of ganglia to compute metrics that help define the normal structure of the ileal myenteric plexus. To facilitate reproducible, robust, and objective measures of ENS structure across mouse models, experiments, and institutions, COUNTEN is freely and openly available to all researchers.

Neurodegenerative disorders and gut-brain interactions.

Neurodegenerative disorders (NDs) affect essential functions not only in the CNS, but also cause persistent gut dysfunctions, suggesting that they have an impact on both CNS and gut-innervating neurons. Although the CNS biology of NDs continues to be well studied, how gut-innervating neurons, including those that connect the gut to the brain, are affected by or involved in the etiology of these debilitating and progressive disorders has been understudied. Studies in recent years have shown how CNS and gut biology, aided by the gut-brain connecting neurons, modulate each other's functions. These studies underscore the importance of exploring the gut-innervating and gut-brain connecting neurons of the CNS and gut function in health, as well as the etiology and progression of dysfunction in NDs. In this Review, we discuss our current understanding of how the various gut-innervating neurons and gut physiology are involved in the etiology of NDs, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, to cause progressive CNS and persistent gut dysfunction.

Parkinson's disease and the gut: Models of an emerging relationship.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by a progressive loss of fine motor function that impacts 1-2 out of 1,000 people. PD occurs predominately late in life and lacks a definitive biomarker for early detection. Recent cross-disciplinary progress has implicated the gut as a potential origin of PD pathogenesis. The gut-origin hypothesis has motivated research on gut PD pathology and transmission to the brain, especially during the prodromal stage (10-20 years before motor symptom onset). Early findings have revealed several possible triggers for Lewy pathology - the pathological hallmark of PD - in the gut, suggesting that microbiome and epithelial interactions may play a greater than appreciated role. But the mechanisms driving Lewy pathology and gut-brain transmission in PD remain unknown. Development of artificial α-Synuclein aggregates (α-Syn preformed fibrils) and animal disease models have recapitulated features of PD progression, enabling for the first time, controlled investigation of the gut-origin hypothesis. However, the role of specific cells in PD transmission, such as neurons, remains limited and requires in vitro models for controlled evaluation and perturbation. Human cell populations, three-dimensional organoids, and microfluidics as discovery platforms inch us closer to improving existing treatment for patients by providing platforms for discovery and screening. This review includes a discussion of PD pathology, conventional treatments, in vivo and in vitro models, and future directions. STATEMENT OF SIGNIFICANCE: Parkinson's Disease remains a common neurodegenerative disease with palliative versus causal treatments. Recently, the gut-origin hypothesis, where Parkinson's disease is thought to originate and spread from the gut to the brain, has gained traction as a field of investigation. However, despite the wealth of studies and innovative approaches to accelerate the field, there remains a need for in vitro tools to enable fundamental biological understanding of disease progression, and compound screening and efficacy. In this review, we present a historical perspective of Parkinson's Disease pathogenesis, detection, and conventional therapy, animal and human models investigating the gut-origin hypothesis, in vitro models to enable controlled discovery, and future outlooks for this blossoming field.

Neuro-innate immune interactions in gut mucosal immunity.

The gastrointestinal (GI) tract performs a set of vital physiological functions related to food and water consumption. To help regulate these complex physiological processes, the GI tract is innervated by extensive neural networks. The GI tract also serves as the largest immune organ aimed to protect hosts from harmful microbes and toxins ingested with food. It emerges that the enteric nervous and immune systems are highly integrated to optimize digestion while reinforcing immune protection. In this review, we will discuss key cellular players involved in the neuro-immune interactions within the GI mucosa with the focus on the recently uncovered neural pathways that regulate mucosal immunity in a context relevant to GI health and disease.

Cholinergic-induced anion secretion in murine jejunal enteroids involves synergy between muscarinic and nicotinic pathways.

Acetylcholine induces robust electrogenic anion secretion in mammalian intestine and it has long been hypothesized that it mediates the epithelial response through the M3 and, to a lesser extent, the M1 muscarinic receptors in the mouse. However, nicotinic receptors have recently been identified in intestinal enterocytes by quantitative real-time (qRT)-PCR/RNAseq, although any direct influence on intestinal transport has not been identified. We tested the hypothesis that cholinergic-induced anion secretion in the intestine is a result of both muscarinic and nicotinic pathways that are intrinsic to the intestinal epithelia. We developed a method to generate mouse jejunal enteroid monolayers which were used to measure active electrogenic anion secretion by the Ussing chamber/voltage-clamp technique. Here, we show that the cholinergic agonist carbachol (CCh) and the muscarinic agonist bethanechol (BCh) stimulate short-lived, concentration-dependent anion secretion in the epithelial cell-only enteroid monolayers. The muscarinic antagonist atropine completely inhibited CCh- and BCh-induced secretion, while the nicotinic antagonist hexamethonium reduced the CCh response by ~45%. While nicotine alone did not alter anion secretion, it increased the BCh-induced increase in short-circuit current in a concentration-dependent manner; this synergy was prevented by pretreatment with hexamethonium. In addition to being sensitive to hexamethonium, monolayers express both classes of cholinergic receptor by qRT-PCR, including 13 of 16 nicotinic receptor subunits. Our findings indicate that an interaction between muscarinic and nicotinic agonists synergistically stimulates anion secretion in mouse jejunal epithelial cells and identify a role for epithelial nicotinic receptors in anion secretion.

Intestinal Bacteria Maintain Adult Enteric Nervous System and Nitrergic Neurons via Toll-like Receptor 2-induced Neurogenesis in Mice.

BACKGROUND & AIMS: The enteric nervous system (ENS) exists in close proximity to luminal bacteria. Intestinal microbes regulate ENS development, but little is known about their effects on adult enteric neurons. We investigated whether intestinal bacteria or their products affect the adult ENS via toll-like receptors (TLRs) in mice. METHODS: We performed studies with conventional C57/BL6, germ-free C57/BL6, Nestin-creERT2:tdTomato, Nestin-GFP, and ChAT-cre:tdTomato. Mice were given drinking water with ampicillin or without (controls). Germ-free mice were given drinking water with TLR2 agonist or without (controls). Some mice were given a blocking antibody against TLR2 or a TLR4 inhibitor. We performed whole gut transit, bead latency, and geometric center studies. Feces were collected and analyzed by 16S ribosomal RNA gene sequencing. Longitudinal muscle myenteric plexus (LMMP) tissues were collected, analyzed by immunohistochemistry, and levels of nitric oxide were measured. Cells were isolated from colonic LMMP of Nestin-creERT2:tdTomato mice and incubated with agonists of TLR2 (receptor for gram-positive bacteria), TLR4 (receptor for gram-negative bacteria), or distilled water (control) and analyzed by flow cytometry. RESULTS: Stool from mice given ampicillin had altered composition of gut microbiota with reduced abundance of gram-positive bacteria and increased abundance of gram-negative bacteria, compared with mice given only water. Mice given ampicillin had reduced colon motility compared with mice given only water, and their colonic LMMP had reduced numbers of nitrergic neurons, reduced neuronal nitric oxide synthase production, and reduced colonic neurogenesis. Numbers of colonic myenteric neurons increased after mice were switched from ampicillin to plain water, with increased markers of neurogenesis. Nestin-positive enteric neural precursor cells expressed TLR2 and TLR4. In cells isolated from the colonic LMMP, incubation with the TLR2 agonist increased the percentage of neurons originating from enteric neural precursor cells to approximately 10%, compared with approximately 0.01% in cells incubated with the TLR4 agonist or distilled water. Mice given an antibody against TLR2 had prolonged whole gut transit times; their colonic LMMP had reduced total neurons and a smaller proportion of nitrergic neurons per ganglion, and reduced markers of neurogenesis compared with mice given saline. Colonic LMMP of mice given the TLR4 inhibitor did not have reduced markers of neurogenesis. Colonic LMMP of germ-free mice given TLR2 agonist had increased neuronal numbers compared with control germ-free mice. CONCLUSIONS: In the adult mouse colon, TLR2 promotes colonic neurogenesis, regulated by intestinal bacteria. Our findings indicate that colonic microbiota help maintain the adult ENS via a specific signaling pathway. Pharmacologic and probiotic approaches directed towards specific TLR2 signaling processes might be developed for treatment of colonic motility disorders related to use of antibiotics or other factors.

'Nervous' Immunity: Walking the Tightrope.

There is a major gap in our understanding of how the intestinal immune and nervous systems are integrated to regulate protective adaptations to enteric infections while maintaining tissue homeostasis. Three recent complementary reports published in Cell (2020) provide new mechanistic insights into how this enteric neuro-immune crosstalk may occur.

Lymphatic vessel remodeling and invasion in pancreatic cancer progression.

BACKGROUND: The lymphatic system is involved in metastasis in pancreatic cancer progression. In cancer staging, lymphatic spread has been used to assess the invasiveness of tumor cells. However, from the endothelium's perspective, the analysis downplays the peri-lesional activities of lymphatic vessels. This unintended bias is largely due to the lack of 3-dimensional (3-D) tissue information to depict the lesion microstructure and vasculature in a global and integrated fashion. METHODS: We targeted the pancreas as the model organ to investigate lymphatic vessel remodeling in cancer lesion progression. Transparent pancreases were prepared by tissue clearing to facilitate deep-tissue, tile-scanning microscopy for 3-D lymphatic network imaging. FINDINGS: In human pancreatic ductal adenocarcinoma, we identify the close association between the pancreatic intraepithelial neoplasia (PanIN) lesions and the lymphatic network. In mouse models of PanIN (elastase-CreER;LSL-KrasG12D and elastase-CreER;LSL-KrasG12D;p53+/-), the 3-D image data reveal the peri-lesional lymphangiogenesis, endothelial invagination, formation of the bridge/valve-like luminal tubules, vasodilation, and luminal invasion. In the orthotopic mouse model of pancreatic cancer, we identify the localized, graft-induced lymphangiogenesis and the peri- and intra-tumoral lymphatic vessel invasion. INTERPRETATION: The integrated view of duct lesions and vascular remodeling suggests an active role, rather than a passive target, of lymphatic vessels in the metastasis of pancreatic cancer. Our 3-D image data provide insights into the pancreatic cancer microenvironment and establish the technical and morphological foundation for systematic detection and 3-D analysis of lymphatic vessel invasion. FUND: Taiwan Academia Sinica (AS-107-TP-L15 and AS-105-TP-B15), Ministry of Science and Technology (MOST 106-2321-B-001-048, 106-0210-01-15-02, 106-2321-B-002-034, and 106-2314-B-007-004-MY2), and Taiwan National Health Research Institutes (NHRI EX107-10524EI).

Transneuronal Propagation of Pathologic α-Synuclein from the Gut to the Brain Models Parkinson's Disease.

Analysis of human pathology led Braak to postulate that α-synuclein (α-syn) pathology could spread from the gut to brain via the vagus nerve. Here, we test this postulate by assessing α-synucleinopathy in the brain in a novel gut-to-brain α-syn transmission mouse model, where pathological α-syn preformed fibrils were injected into the duodenal and pyloric muscularis layer. Spread of pathologic α-syn in brain, as assessed by phosphorylation of serine 129 of α-syn, was observed first in the dorsal motor nucleus, then in caudal portions of the hindbrain, including the locus coeruleus, and much later in basolateral amygdala, dorsal raphe nucleus, and the substantia nigra pars compacta. Moreover, loss of dopaminergic neurons and motor and non-motor symptoms were observed in a similar temporal manner. Truncal vagotomy and α-syn deficiency prevented the gut-to-brain spread of α-synucleinopathy and associated neurodegeneration and behavioral deficits. This study supports the Braak hypothesis in the etiology of idiopathic Parkinson's disease (PD).

Advances in Enteric Neurobiology: The "Brain" in the Gut in Health and Disease.

The enteric nervous system (ENS) is a large, complex division of the peripheral nervous system that regulates many digestive, immune, hormonal, and metabolic functions. Recent advances have elucidated the dynamic nature of the mature ENS, as well as the complex, bidirectional interactions among enteric neurons, glia, and the many other cell types that are important for mediating gut behaviors. Here, we provide an overview of ENS development and maintenance, and focus on the latest insights gained from the use of novel model systems and live-imaging techniques. We discuss major advances in the understanding of enteric glia, and the functional interactions among enteric neurons, glia, and enteroendocrine cells, a large class of sensory epithelial cells. We conclude by highlighting recent work on muscularis macrophages, a group of immune cells that closely interact with the ENS in the gut wall, and the importance of neurological-immune system communication in digestive health and disease.

Age-dependent shift in macrophage polarisation causes inflammation-mediated degeneration of enteric nervous system.

OBJECTIVE: The enteric nervous system (ENS) undergoes neuronal loss and degenerative changes with age. The cause of this neurodegeneration is poorly understood. Muscularis macrophages residing in close proximity to enteric ganglia maintain neuromuscular function via direct crosstalk with enteric neurons and have been implicated in the pathogenesis of GI motility disorders like gastroparesis and postoperative ileus. The aim of this study was to assess whether ageing causes alterations in macrophage phenotype that contributes to age-related degeneration of the ENS. DESIGN: Longitudinal muscle and myenteric plexus from small intestine of young, mid-aged and old mice were dissected and prepared for whole mount immunostaining, flow cytometry, Luminex immunoassays, western blot analysis, enteric neural stem cell (ENSC) isolation or conditioned media. Bone marrow derived macrophages were prepared and polarised to classic (M1) or alternative (M2) activation states. Markers for macrophage phenotype were measured using quantitative RT-PCR. RESULTS: Ageing causes a shift in macrophage polarisation from anti-inflammatory 'M2' to proinflammatory 'M1' that is associated with a rise in cytokines and immune cells in the ENS. This phenotypic shift is associated with a neural response to inflammatory signals, increase in apoptosis and loss of enteric neurons and ENSCs, and delayed intestinal transit. An age-dependent decrease in expression of the transcription factor FoxO3, a known longevity gene, contributes to the loss of anti-inflammatory behaviour in macrophages of old mice, and FoxO3-deficient mice demonstrate signs of premature ageing of the ENS. CONCLUSIONS: A shift by macrophages towards a proinflammatory phenotype with ageing causes inflammation-mediated degeneration of the ENS.