Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain.

A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of ß-amyloid-(Aß)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aß deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

The management of adult patients with severe chronic small intestinal dysmotility.

Adult patients with severe chronic small intestinal dysmotility are not uncommon and can be difficult to manage. This guideline gives an outline of how to make the diagnosis. It discusses factors which contribute to or cause a picture of severe chronic intestinal dysmotility (eg, obstruction, functional gastrointestinal disorders, drugs, psychosocial issues and malnutrition). It gives management guidelines for patients with an enteric myopathy or neuropathy including the use of enteral and parenteral nutrition.

Changes in neuromuscular structure and functions of human colon during ageing are region-dependent.

OBJECTIVE: To determine if human colonic neuromuscular functions decline with increasing age. DESIGN: Looking for non-specific changes in neuromuscular function, a standard burst of electrical field stimulation (EFS) was used to evoke neuronally mediated (cholinergic/nitrergic) contractions/relaxations in ex vivomuscle strips of human ascending and descending colon, aged 35-91 years (macroscopically normal tissue; 239 patients undergoing cancer resection). Then, to understand mechanisms of change, numbers and phenotype of myenteric neurons (30 306 neurons stained with different markers), densities of intramuscular nerve fibres (51 patients in total) and pathways involved in functional changes were systematically investigated (by immunohistochemistry and use of pharmacological tools) in elderly (≥70 years) and adult (35-60 years) groups. RESULTS: With increasing age, EFS was more likely to evoke muscle relaxation in ascending colon instead of contraction (linear regression: n=109, slope 0.49%±0.21%/year, 95% CI), generally uninfluenced by comorbidity or use of medications. Similar changes were absent in descending colon. In the elderly, overall numbers of myenteric and neuronal nitric oxide synthase-immunoreactive neurons and intramuscular nerve densities were unchanged in ascending and descending colon, compared with adults. In elderly ascending, not descending, colon numbers of cell bodies exhibiting choline acetyltransferase immunoreactivity increased compared with adults (5.0±0.6 vs 2.4±0.3 neurons/mm myenteric plexus, p=0.04). Cholinergically mediated contractions were smaller in elderly ascending colon compared with adults (2.1±0.4 and 4.1±1.1 g-tension/g-tissue during EFS; n=25/14; p=0.04); there were no changes in nitrergic function or in ability of the muscle to contract/relax. Similar changes were absent in descending colon. CONCLUSION: In ascending not descending colon, ageing impairs cholinergic function.

Inflammatory skin and bowel disease linked to ADAM17 deletion.

We performed genetic and immunohistochemical studies in a sister and brother with autosomal recessive neonatal inflammatory skin and bowel lesions. The girl died suddenly at 12 years of age from parvovirus B19-associated myocarditis; her brother had mild cardiomyopathy. We identified a loss-of-function mutation in ADAM17, which encodes a disintegrin and metalloproteinase 17 (also called tumor necrosis factor α [TNF-α]-converting enzyme, or TACE), as the probable cause of this syndrome. Peripheral-blood mononuclear cells (PBMCs) obtained from the brother at 17 years of age showed high levels of lipopolysaccharide-induced production of interleukin-1ß and interleukin-6 but impaired release of TNF-α. Despite repeated skin infections, this young man has led a relatively normal life. (Funded by Barts and the London Charity and the European Commission Seventh Framework Programme.).

Reduction in cycle time for a rapid polymerase chain reaction diagnostic test at the point of care.

Background: Rapid testing facilitates safe and effective diagnosis, but the true speed of the process is the time from collection of a sample to delivery of an accurate and reliable test result - 'end-to-end' time. Transport, unpacking and relaying of information can extend this time considerably beyond the minimum laboratory turnaround times as stipulated by PCR testing protocols. Aim/Objective: This study aimed to minimise time needed to ascertain SARS-CoV-2 status prior to treatment in a UK Dental Hospital using a novel, mobile, direct to polymerase chain reaction (PCR) workflow. Methods: Process flow analysis and PDSA (Plan, Do, Study, Act) cycles for rapid continuous improvement were employed in a service improvement programme. Primerdesign™ q16 rapid PCR instruments and PROmate® COVID-19 direct assays were used for molecular testing. Findings/Results: We showed a reduction in real-world end-to-end time for a diagnostic test from 240 min to 85 min (65% reduction) over a 4-week period. Discussion: New rapid technologies have become available that reduce analytical time to under 90 min, but the real-world clinical implementation of the test requires a fully integrated workflow from clinic to reporting.

Adult-onset megacolon with focal hypoganglionosis: A detailed phenotyping and prospective cohort study.

BACKGROUND: In this prospective cohort study, we evaluated features of "adult-onset megacolon with focal hypoganglionosis." METHODS: We assessed the radiologic, endoscopic, and histopathologic phenotyping and treatment outcomes of 29 patients between 2017 and 2020. Data from community controls, consisting of 19,948 adults undergoing health screenings, were analyzed to identify risk factors. Experts reviewed clinical features and pathological specimens according to the London Classification for gastrointestinal neuromuscular pathology. KEY RESULTS: The median age of the patients with adult-onset megacolon with focal hypoganglionosis at symptom onset was 59 years (range, 32.0-74.9 years), with mean symptom onset only 1 year before diagnosis. All patients had focal stenotic regions with proximal bowel dilatation (mean diameter, 78.8 mm; 95% confidence interval [CI], 72-86). The comparison with community controls showed no obvious risk factors. Ten patients underwent surgery, and all exhibited significant hypoganglionosis: 5.4 myenteric ganglion cells/cm (interquartile range [IQR], 3.7-16.4) in the stenotic regions compared to 278 cells/cm (IQR, 190-338) in the proximal and 95 cells/cm (IQR, 45-213) in the distal colon. Hypoganglionosis was associated with CD3+ T cells along the myenteric plexus. Colectomy was associated with significant symptom improvement compared to medical treatment [change in the Global Bowel Satisfaction score, -5.4 points (surgery) vs. -0.3 points (medical treatment); p < 0.001]. CONCLUSIONS AND INFERENCES: Adult-onset megacolon with focal hypoganglionosis has distinct features characterized by hypoganglionosis due to inflammation. Bowel resection appears to benefit these patients.

CTLA4Ig alters the course of autoimmune disease development in Lyn-/- mice.

Lyn-deficient (Lyn(-/-)) mice develop an age-dependent autoimmune disease similar to systemic lupus erythematosus, characterized by the production of IgG anti-nuclear Ab. To determine the extent to which this autoimmune phenotype is driven by T cell costimulation, we generated Lyn(-/-) mice expressing a soluble form of the T cell inhibitory molecule, CTLA4 (CTLA4Ig). Surprisingly, although CTLA4Ig prevented myeloid hyperplasia, splenomegaly and IgG anti-nuclear Ab production in Lyn(-/-) mice, it did not inhibit immune complex deposition and tissue destruction in the kidney. In fact, regardless of CTLA4Ig expression, Lyn(-/-) serum contained elevated titers of IgA anti-nuclear Ab, although generally IgA deposition in the kidney was only revealed in the absence of self-reactive IgG. This demonstrated that activation of autoreactive B cell clones in Lyn(-/-) mice can still occur despite impaired costimulation. Indeed, CTLA4Ig did not alter perturbed Lyn(-/-) B cell development and behavior, and plasma cell frequencies were predominantly unaffected. These results suggest that when self-reactive B cell clones are unimpeded in acquiring T cell help, they secrete pathogenic IgG autoantibodies that trigger the fulminant autoimmunity normally observed in Lyn(-/-) mice. The absence of these IgG immune complexes reveals an IgA-mediated axis of autoimmunity that is not sufficient to cause splenomegaly or extramedullary myelopoiesis, but which mediates destructive glomerulonephritis. These findings have implications for the understanding of the basis of Ab-mediated autoimmune diseases and for their treatment with CTLA4Ig.

Perceived feasibility, facilitators and barriers to incorporating point-of-care testing for SARS-CoV-2 into emergency medical services by ambulance service staff: a survey-based approach.

OBJECTIVES: This body of work aimed to elicit ambulance service staff's perceptions on the barriers and facilitators to adoption, and clinical utility of incorporating rapid SARS-CoV-2 testing during ambulance assessments. DESIGN: A mixed-methods survey-based project using a framework analysis method to organise qualitative data. SETTING: Emergency and non-emergency care ambulatory services in the UK were approached to take part. PARTICIPANTS: Current, practising members of the UK ambulance service (paramedics, technicians, assistants and other staff) were included in this body of work. RESULTS: Survey 1: 226 responses were collected between 3 December 2020 and 11 January 2021, 179 (79.2%) of which were completed in full. While the majority of respondents indicated that an ambulance-based testing strategy was feasible in concept (143/190, 75.3%), major barriers to adoption were noted. Many open-ended responses cited concerns regarding misuse of the service by the general public and other healthcare services, timing and conveyance issues, and increased workloads, alongside training and safety concerns. Survey 2: 26 responses were received between 8 February 2021 and 22 February 2021 to this follow-up survey. Survey 2 revealed conveyance decision-making, and risk stratification to be the most frequently prioritised use cases among ambulance service staff. Optimal test characteristics for clinical adoption according to respondents were; accuracy (above 90% sensitivity and specificity), rapidity (<30 min time to results) and ease of sample acquisition. CONCLUSIONS: The majority of commercially available lateral flow devices are unlikely to be supported by paramedics as their duty of care requires both rapid and accurate results that can inform clinical decision making in an emergency situation. Further investigation is needed to define acceptable test characteristics and criteria required for ambulance service staff to be confident and supportive of deployment of a SARS-CoV-2 test in an emergency care setting.

DYRK1A-dosage imbalance perturbs NRSF/REST levels, deregulating pluripotency and embryonic stem cell fate in Down syndrome.

Down syndrome (DS) is the most common cause of mental retardation. Many neural phenotypes are shared between DS individuals and DS mouse models; however, the common underlying molecular pathogenetic mechanisms remain unclear. Using a transchromosomic model of DS, we show that a 30%-60% reduced expression of Nrsf/Rest (a key regulator of pluripotency and neuronal differentiation) is an alteration that persists in trisomy 21 from undifferentiated embryonic stem (ES) cells to adult brain and is reproducible across several DS models. Using partially trisomic ES cells, we map this effect to a three-gene segment of HSA21, containing DYRK1A. We independently identify the same locus as the most significant eQTL controlling REST expression in the human genome. We show that specifically silencing the third copy of DYRK1A rescues Rest levels, and we demonstrate altered Rest expression in response to inhibition of DYRK1A expression or kinase activity, and in a transgenic Dyrk1A mouse. We reveal that undifferentiated trisomy 21 ES cells show DYRK1A-dose-sensitive reductions in levels of some pluripotency regulators, causing premature expression of transcription factors driving early endodermal and mesodermal differentiation, partially overlapping recently reported downstream effects of Rest +/-. They produce embryoid bodies with elevated levels of the primitive endoderm progenitor marker Gata4 and a strongly reduced neuroectodermal progenitor compartment. Our results suggest that DYRK1A-mediated deregulation of REST is a very early pathological consequence of trisomy 21 with potential to disturb the development of all embryonic lineages, warranting closer research into its contribution to DS pathology and new rationales for therapeutic approaches.

The role of humoral autoimmunity in gastrointestinal neuromuscular diseases.

Dysfunction of the gastrointestinal neuromuscular apparatus (including interstitial cells of Cajal) is presumed to underlie a heterogeneous group of disorders collectively termed gastrointestinal neuromuscular diseases (GINMDs). There is increasing experimental and clinical evidence that some GINMDs are immune-mediated, with cell-mediated dysfunction relatively well studied. Humoral (antibody)-mediated autoimmunity is associated with several well-established acquired neuromuscular diseases and is now implicated in an increasing number of less well-characterised disorders, particularly of the central nervous system. The role of autoimmunity in GINMDs has been less studied. Whilst most work has focused on the presence of antibodies directed to nuclear antigens, particularly in the context of secondary disorders such as paraneoplastic intestinal pseudo-obstruction, the possibility that 'functional' anti-neuronal antibodies directed to membrane-bound ion channels may cause disease (channelopathy) is now also being realised. The evidence for humoral autoimmunity as an etiologic factor in primary (idiopathic) and secondary GINMDs is systematically presented using the original paradigms previously applied to established autoimmune neuromuscular disorders. The presence of anti-enteric neuronal antibodies, although repeatedly demonstrated, still requires the identification of specific neuronal autoantigens and validated evidence of pathogenicity.

The London Classification of gastrointestinal neuromuscular pathology: report on behalf of the Gastro 2009 International Working Group.

OBJECTIVE: Guidelines on histopathological techniques and reporting for adult and paediatric gastrointestinal neuromuscular pathology have been produced recently by an international working group (IWG). These addressed the important but relatively neglected areas of histopathological practice of the general pathologist, including suction rectal biopsy and full-thickness intestinal tissue. Recommendations were presented for the indications, safe acquisition of tissue, histological techniques, reporting and referral of such histological material. DESIGN: Consensual processes undertaken by the IWG and following established guideline decision group methodologies. RESULTS AND CONCLUSION: This report presents a contemporary and structured classification of gastrointestinal neuromuscular pathology based on defined histopathological criteria derived from the existing guidelines. In recognition of its origins and first presentation in London at the World Congress of Gastroenterology 2009, this has been named 'The London Classification'. The implementation of this classification should allow some diagnostic standardisation, but should necessarily be viewed as a starting point for future modification as new data become available.

Megarectum: systematic histopathological evaluation of 35 patients and new common pathways in chronic rectal dilatation.

AIMS: Megarectum is well described in the surgical literature but few contemporary pathological studies have been undertaken. There is uncertainty whether 'idiopathic' megarectum is a primary neuromuscular disorder or whether chronic dilatation leads to previously reported and unreported pathological changes. We sought to answer this question. METHODS: Systematic histopathological evaluation (in accord with international guidance) of 35 consecutive patients undergoing rectal excision surgery for megarectum (primary: n=24) or megarectum following surgical correction of anorectal malformation (secondary: n=11) in a UK university hospital with adult/paediatric surgical and gastrointestinal neuropathology expertise. RESULTS: We confirmed some previously reported observations, notably hypertrophy of the muscularis propria (27 of 35, 77.1% of patients) and extensive fibrosis (30 of 35, 85.7% of patients). We also observed unique and previously unreported features including elastosis (19 of 33, 57.6%) and the presence of polyglucosan bodies (15 of 32, 46.9% of patients). In contrast to previous literature, few patients had any strong evidence of specific forms of visceral neuropathy (5 of 35, including 3 plexus duplications) or myopathy (6 of 35, including 3 muscle duplications). All major pathological findings were common to both primary and secondary forms of the disease, implying that these may be a response to chronic rectal distension rather than of primary aetiology. CONCLUSIONS: In the largest case series reported to date, we challenge the current perception of idiopathic megarectum as a primary neuromuscular disease and propose a cellular pathway model for the features present. The severe morphological changes account for some of the irreversibility of the condition and reinforce the need to prevent ongoing rectal distension when first identified.

Expression of p16 Within Myenteric Neurons of the Aged Colon: A Potential Marker of Declining Function.

Human colonic neuromuscular functions decline among the elderly. The aim was to explore the involvement of senescence. A preliminary PCR study looked for age-dependent differences in expression of CDKN1A (encoding the senescence-related p21 protein) and CDKN2A (encoding p16 and p14) in human ascending and descending colon (without mucosa) from 39 (approximately 50: 50 male: female) adult (aged 27-60 years) and elderly donors (70-89 years). Other genes from different aging pathways (e.g., inflammation, oxidative stress, autophagy) and cell-types (e.g., neurons, neuron axonal transport) were also examined. Unlike CDKN1A, CDKN2A (using primers for p16 and p14 but not when using p14-specific primers) was upregulated in both regions of colon. Compared with the number of genes appearing to upregulate in association with temporal age, more genes positively associated with increased CDKN2A expression (respectively, 16 and five of 44 genes studied for ascending and descending colon). Confirmation of increased expression of CDKN2A was sought by immunostaining for p16 in the myenteric plexus of colon from 52 patients, using a semi-automated software protocol. The results showed increased staining not within the glial cells (S100 stained), but in the cytoplasm of myenteric nerve cell bodies (MAP2 stained, with identified nucleus) of ascending, but not descending colon of the elderly, and not in the cell nucleus of either region or age group (5,710 neurons analyzed: n = 12-14 for each group). It was concluded that increased p16 staining within the cytoplasm of myenteric nerve cell bodies of elderly ascending (but not descending) colon, suggests a region-dependent, post-mitotic cellular senescence-like activity, perhaps involved with aging of enteric neurons within the colon.

Gastrointestinal neuromuscular pathology: guidelines for histological techniques and reporting on behalf of the Gastro 2009 International Working Group.

The term gastrointestinal neuromuscular disease describes a clinically heterogeneous group of disorders of children and adults in which symptoms are presumed or proven to arise as a result of neuromuscular, including interstitial cell of Cajal, dysfunction. Such disorders commonly have impaired motor activity, i.e. slowed or obstructed transit with radiological evidence of transient or persistent visceral dilatation. Whilst sensorimotor abnormalities have been demonstrated by a variety of methods in these conditions, standards for histopathological reporting remain relatively neglected. Significant differences in methodologies and expertise continue to confound the reliable delineation of normality and specificity of particular pathological changes for disease. Such issues require urgent clarification to standardize acquisition and handling of tissue specimens, interpretation of findings and make informed decisions on risk-benefit of full-thickness tissue biopsy of bowel or other diagnostic procedures. Such information will also allow increased certainty of diagnosis, facilitating factual discussion between patients and caregivers, as well as giving prognostic and therapeutic information. The following report, produced by an international working group, using established consensus methodology, presents proposed guidelines on histological techniques and reporting for adult and paediatric gastrointestinal neuromuscular pathology. The report addresses the main areas of histopathological practice as confronted by the pathologist, including suction rectal biopsy and full-thickness tissue obtained with diagnostic or therapeutic intent. For each, indications, safe acquisition of tissue, histological techniques, reporting and referral recommendations are presented.

Spontaneous Restoration of Nutrition Autonomy in a Case of Intestinal Failure Secondary to a Gastrointestinal Neuromuscular Disease.

Polyglucosan inclusion body myopathy (PIBM) is a recently described gastrointestinal neuromuscular disease. Given its rarity, very little is known of its natural history and prognosis. In this case report, we present the first case of PIBM and resultant intestinal failure in which, following 7 years of home parenteral nutrition, gastrointestinal symptoms subsided and nutrition autonomy was restored.

Early postnatal death and motor disorders in mice congenitally deficient in calnexin expression.

Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific.

An integrated genetic, radiation hybrid, physical and transcription map of a region of distal mouse chromosome 12, including an imprinted locus and the 'Legs at odd angles' (Loa) mutation.

A variety of loci with interesting patterns of regulation such as imprinted expression, and critical functions such as involvement in tumour necrosis factor pathways, map to a distal portion of mouse chromosome 12. This region also contains disease related loci including the 'Legs at odd angles' mutation (Loa) that we are pursuing in a positional cloning project. To further define the region and prepare for comparative sequencing projects, we have produced genetic, radiation hybrid, physical and transcript maps of the region, with probes providing anchors between the maps. We show a summary of 95 markers and 91 genomic clones that has enabled us to identify 18 transcripts including new genes and candidates for Loa which will help in future studies of gene context and regulation.