Tracking in situ checkpoint inhibitor-bound target T cells in patients with checkpoint-induced colitis.

The success of checkpoint inhibitors (CPIs) for cancer has been tempered by immune-related adverse effects including colitis. CPI-induced colitis is hallmarked by expansion of resident mucosal IFNγ cytotoxic CD8+ T cells, but how these arise is unclear. Here, we track CPI-bound T cells in intestinal tissue using multimodal single-cell and subcellular spatial transcriptomics (ST). Target occupancy was increased in inflamed tissue, with drug-bound T cells located in distinct microdomains distinguished by specific intercellular signaling and transcriptional gradients. CPI-bound cells were largely CD4+ T cells, including enrichment in CPI-bound peripheral helper, follicular helper, and regulatory T cells. IFNγ CD8+ T cells emerged from both tissue-resident memory (TRM) and peripheral populations, displayed more restricted target occupancy profiles, and co-localized with damaged epithelial microdomains lacking effective regulatory cues. Our multimodal analysis identifies causal pathways and constitutes a resource to inform novel preventive strategies.

Diagnostic accuracy of blood tests of inflammation in paediatric appendicitis: a systematic review and meta-analysis.

OBJECTIVE: Possible childhood appendicitis is a common emergency presentation. The exact value of blood tests is debated. This study sought to determine the diagnostic accuracy of four blood tests (white cell count (WCC), neutrophil(count or percentage), C reactive protein (CRP) and/or procalcitonin) for childhood appendicitis. DESIGN: A systematic review and diagnostic meta-analysis. Data sources included MEDLINE, EMBASE, Central, Web of Science searched from inception-March 2022 with reference searching and authors contacted for missing/unclear data. Eligibility criteria was studies reporting the diagnostic accuracy of the four blood tests compared to the reference standard (histology or follow-up). Risk of bias was assessed (QUADAS-2), pooled sensitivity and specificity were generated for each test and commonly presented cut-offs. To provide insight into clinical impact, we present strategies using a hypothetical cohort. RESULTS: 67 studies were included (34 839 children, 13 342 with appendicitis), all in the hospital setting. The most sensitive tests were WCC (≥10 000 cells/µL, 53 studies sensitivity 0.85 (95% CI 0.80 to 0.89)) and absolute neutrophil count (ANC) (≥7500 cells/µL, five studies sensitivity 0.90 (95% CI 0.85 to 0.94)). Combination of WCC or CRP increased sensitivity further(≥10 000 cells/µL or ≥10 mg/L, individual patient data (IPD) of 6 studies, 0.97 (95% CI 0.93 to 0.99)).Applying results to a hypothetical cohort(1000 children with appendicitis symptoms, of whom 400 have appendicitis) 60 and 40 children would be wrongly discharged based solely on WCC and ANC, respectively, 12 with combination of WCC or CRP.The most specific tests were CRP alone (≥50 mg/L, 38 studies, specificity 0.87 (95% CI 0.80 to 0.91)) or combined with WCC (≥10 000 cells/µL and ≥50 mg/L, IPD of six studies, 0.93 (95% CI 0.91 to 0.95)). CONCLUSIONS: The best performing single blood tests for ruling-out paediatric appendicitis are WCC or ANC; with accuracy improved combining WCC and CRP. These tests could be used at the point of care in combination with clinical prediction rules. We provide insight into the best cut-offs for clinical application. PROSPERO REGISTRATION NUMBER: CRD42017080036.

Isolation of human fetal intestinal cells for single-cell RNA sequencing.

The intestine has a large number of cell types. Thus, digestion of pure and viable populations is necessary for downstream techniques including single-cell RNA sequencing. We outline a protocol to isolate both epithelial and non-epithelial cells from human fetal samples at high viability, which was used to produce a full thickness atlas of intestinal cells across human development. This protocol can also be adapted to adult endoscopy and surgical specimens. For details on the use of this protocol, please refer to Fawkner-Corbett et al. (2021).

Bilateral Wilms' tumour: An international comparison of treatments and outcomes.

INTRODUCTION: Wilms' tumour is the most common childhood renal malignancy, with 5-10% of cases presenting bilaterally 1. However, there is currently no consensus between centres on optimal management of bilateral Wilms' tumours. This is an international multi-centre case series comparing management and outcomes of bilateral Wilms' tumours between low-income centres (LIC) and high-income centres (HIC). METHODS: Patients with bilateral Wilms' tumour were identified from four tertiary referral centres internationally. Data were collected on baseline characteristics, disease status, treatment used and clinical outcomes. Results were compared between individual centres as well as between groups of low-income centres (LIC) and high-income centres (HIC). RESULTS: Data were collected for forty patients. Most patients received preoperative chemotherapy (n = 38, 95%). The most common surgical procedures were bilateral nephron-sparing surgery (n = 10, 25%) and nephrectomy with partial nephrectomy (n = 20, 50%). Ten-year survival after treatment was as follows: LIC's n = 13 (65%); HIC's n = 20 (100%) (p = 0.01). DISCUSSION: Ten-year survival was significantly higher in HIC's. Our results show this may be caused by patient factors such as later presentation with more advanced disease in low-income centres. This comparative case series is the first to report on a large number of cases from multiple international centres, and to compare key outcomes.

Spatiotemporal analysis of human intestinal development at single-cell resolution.

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

Single-cell atlas of colonic CD8+ T cells in ulcerative colitis.

Colonic antigen-experienced lymphocytes such as tissue-resident memory CD8+ T cells can respond rapidly to repeated antigen exposure. However, their cellular phenotypes and the mechanisms by which they drive immune regulation and inflammation remain unclear. Here we compiled an unbiased atlas of human colonic CD8+ T cells in health and ulcerative colitis (UC) using single-cell transcriptomics with T-cell receptor repertoire analysis and mass cytometry. We reveal extensive heterogeneity in CD8+ T-cell composition, including expanded effector and post-effector terminally differentiated CD8+ T cells. While UC-associated CD8+ effector T cells can trigger tissue destruction and produce tumor necrosis factor (TNF)-α, post-effector cells acquire innate signatures to adopt regulatory functions that may mitigate excessive inflammation. Thus, we identify colonic CD8+ T-cell phenotypes in health and UC, define their clonal relationships and characterize terminally differentiated dysfunctional UC CD8+ T cells expressing IL-26, which attenuate acute colitis in a humanized IL-26 transgenic mouse model.

Colonic epithelial cell diversity in health and inflammatory bowel disease.

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.

Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Microbiome, pattern recognition receptor function in health and inflammation.

The innate immune system plays an important role in shaping the microbiota into configurations that are tolerated and beneficial to the host, thereby playing a crucial role in human health. Innate immunity is based on the fundamental principle that Pattern Recognition Receptors (PRRs) recognise pathogen associated molecular patterns as non-self-entities and trigger intracellular signalling pathways that lead to the induction of numerous cytokines and chemokines that help maintain host resistance to infections. Dysregulation of this interaction has been identified as the core defect that leads to chronic intestinal inflammation allowing certain microbiota to be harmful to host health. This dysbiosis of the microbiome is found associated with numerous chronic diseases. A logical explanation would be that genetic defects in the recognition and response pathways that the host uses to identify these microbial pathogens could lead to altered microbial colonisation or mis-recognition of normal bacteria leading to diseases. The interaction between pattern recognition receptors, microbial traits and human health with respect to the gut are now rapidly resolved and will be the subject of this review.

Enteric nervous system stem cells associated with thickened extrinsic fibers in short segment aganglionic Hirschsprung's disease gut are absent in the total colonic and intestinal variants of disease.

BACKGROUND/PURPOSE: Despite current treatments patients with Hirschsprung's disease (HSCR) suffer significant long-term morbidity. Therefore, there is increasing interest in adjunctive therapies, such as using enteric nervous system stem cells (ENSSC), isolated from typical aganglionic bowel. The source of these cells is unclear however it is hypothesized that they are present in the thickened nerve trunks in aganglionic short and long segment HSCR gut. These cells should therefore be absent in total colonic and pan intestinal HSCR where these thickened fibers are absent. METHODS: Cells were isolated from samples of short segment HSCR gut (n=18) and total colonic and total intestinal HSCR gut (n=2). Acetylcholinesterase histochemistry confirmed the presence/absence of thickened nerve trunks. P75 immunofluorescence highlighted ENSSC at isolation and after 10days in culture in both groups. RESULTS: ENSSC were not isolated or cultured from total colonic and total intestinal HSCR gut where thickened nerve trunks were absent. In contrast 10.0% (+/-1.9 SEM) of cells from short segment HSCR gut were ENSSC at isolation rising to 22.7% (+/-2.9 SEM) after 10days in culture. CONCLUSIONS: These results associate ENSCC with thickened nerve trunks and also suggest that the aganglionic bowel segment in total colonic and intestinal HSCR cannot be used as a source of ENSCC for adjunctive therapy.

Emergency abdominal wall defects in neonates: saved by distress.

This report presents two cases of neonatal patients with abdominal wall defects requiring emergency intervention, a closing gastroschisis and a pedunculated exomphalos with eviscerated liver. Both presented as pre-partum fetal distress and were delivered in a tertiary centre, where they received antenatal care. Coordination in the multidisciplinary team and prompt surgical intervention prevented loss of the eviscerated abdominal contents and prevented mortality in both cases.

Malrotation: Age-Related Differences in Reoperation Rate.

OBJECTIVE: Intestinal malrotation classically presents in the neonatal period with bilious vomiting. However, population studies suggest that up to two-thirds of these patients are diagnosed later in childhood or in adulthood. Increased morbidity in the adult population has been reported. Local experience suggested that surgery was technically more difficult in older children and led to the hypothesis that it would be associated with increased morbidity. METHODS: A retrospective case note analysis was performed on all children presenting with intestinal malrotation to a tertiary referral center between January 2002 and November 2014. Case notes and operation records were reviewed and those who underwent laparotomy for confirmed malrotation were included. Children were grouped as infants (< 1 year) and older (> 1 year). The primary outcome was total emergency reoperation rate. Secondary outcomes were requirement for a bypass at reoperation and mortality. RESULTS: A total of 131 children with malrotation were identified (104 infants, 27 older children; 78 males; age range, 0-16 years). Overall, 13 patients had emergency reoperation following initial Ladd procedure (6 infants and 7 older children). Risk for reoperation was significantly higher in older children (p = 0.005) and additionally a bypass procedure was more often required in older children than infants (4 children, 2 infants, p = 0.016). Adhesiolysis was required on four occasions and redo Ladd procedure in two; these were evenly distributed between both groups. One child was found to have distal bowel obstruction at reoperation. There were three deaths (2.3%), all in the infant group. One was directly associated with malrotation with extensive bowel necrosis. The other two died of unrelated sepsis several months later. CONCLUSIONS: Malrotation surgery in older children is associated with a significantly higher emergency reoperation rate. The primary duodenal bypass procedure should always be considered with longstanding chronic intermittent obstruction associated with malrotation if the simple Ladd procedure is deemed inadequate.

Rhinovirus-C detection in children presenting with acute respiratory infection to hospital in Brazil.

Human rhinovirus (RV) is a common cause of acute respiratory infection (ARI) in children. We aimed to characterize the clinical and demographic features associated with different RV species detected in children attending hospital with ARI, from low-income families in North-east Brazil. Nasopharyngeal aspirates were collected from 630 children <5 years with ARI. Clinical diagnosis and disease severity were also recorded. Samples were analyzed by multiplex PCR for 18 viral and atypical bacterial pathogens; RV positive samples underwent partial sequencing to determine species and type. RV was the fourth commonest pathogen accounting for 18.7% of pathogens detected. RV was commonly detected in children with bronchiolitis, pneumonia, and asthma/episodic viral wheeze (EVW). Species and type were assigned in 112 cases (73% RV-A; 27% RV-C; 0% RV-B). Generally, there were no differences in clinical or demographic characteristics between those infected with RV-A and RV-C. However, in children with asthma/EVW, RV-C was detected relatively more frequently than RV-A (23% vs. 5%; P = 0.04). Our findings highlight RV as a potentially important pathogen in this setting. Generally, clinical and demographic features were similar in children in whom RV-A and C species were detected. However, RV-C was more frequently found in children with asthma/EVW than RV-A.

Wilms' tumor--lessons and outcomes--a 25-year single center UK experience.

Wilms' tumor (WT) is a common childhood renal cancer. A 25-year single center UK experience is reported. During 1985-2010, 97 children underwent immediate nephrectomy or delayed resection of tumor after chemotherapy. Survival, morbidity, and late effects following treatment are described. Tumor distribution was: Stage I, 25.7% (n = 25); Stage II, 24.7% (n = 24); Stage III, 26.8% (n = 26); Stage IV, 17.5% (n = 17); and Stage V, 5.2% (n = 5). Immediate nephrectomy was performed in 39% (n = 38) patients with elective delayed resection in 61% (n = 59) cases. Ten patients had cavotomy to excise tumor involving vena cava territory. Two cases required cardiopulmonary bypass. Tumor rupture was recorded in eight (8.5%) total operated cases-after immediate (n = 5/37), 13.5% vs delayed nephrectomy-(n = 3/57), 5.2%; X(2) P = .154. From 2001 onwards, one case of tumor rupture was recorded at this center after the universal adoption of UKW3 and SIOP guidelines advocating preoperative chemotherapy and delayed nephrectomy for all WT. Three treatment-related deaths occurred-hepatic veno-occlusive disease (n = 2) with actinomycin D and a single WT fatality due to vascular injury. Overall survival was 84.5% (82/97 cases). Two patients developed "late malignancies" -thyroid cancer and a basal cell carcinoma. This study demonstrates excellent survival for WT comparable with national outcomes and international cooperative studies. Adverse events with chemotherapy and surgery, including "late onset," second malignancies deserve special consideration.

Interval appendectomy in children clinical outcomes, financial costs and patient benefits.

BACKGROUND: Elective interval appendectomy (IA) is traditionally advocated for the management of appendiceal mass (AM) in children. Surgeons have debated the evidence and 'risks' vs. 'benefits' to support IA. There are currently no randomised controlled trials and guiding best practice and financial costings for IA are lacking. We herein report clinical outcomes, patient benefits and tariff charges linked with the provision of IA at a regional UK paediatric surgical centre. METHODS: Hospital case records of patients with AM were identified using pathology records and hospital admission codes during a 15-year period (1997-2011). Tariff costs (£ Sterling) were calculated for all admissions during the era 2007-2011. RESULTS: 69 children were admitted with AM (61% female, median age 10.5 years, range 2.1-16 years). Median initial hospital stay with resolution of symptoms was 8 days (range 3-14 days). 61 children (88%) had elective IA (median interval 76 days, range 29-230 days). Eight (12 %) patients required emergency readmission for early appendectomy (median interval 21 days, range 6-51 days). Hospital stay for emergency readmission appendectomy in these children was significantly longer than IA (median 6 vs. 3 days, p < 0.01). Laparoscopic appendectomy vs. 'open' appendectomy was associated with shorter length of stay in the IA cohort (median 3 vs. 2 days p < 0.01). No intra-operative morbidity was recorded in the study with only a single case developing a post-operative wound infection. Median cost for IA was £1,936. Costings were higher in the emergency appendectomy group-£2,171 vs. 1,936; p = 0.09, NS. CONCLUSION: Only 12% of children at this centre develop recurrent appendicitis after primary admission with AM. Interval and emergency appendectomy were associated with low morbidity. Parents should be informed that IA may be 'non essential' surgery. Paediatric surgeons not routinely advocating IA can potentially save the NHS £1,936 per patient. Future randomised studies are warranted to confirm or refute these findings.

The impact of the H1N1 influenza pandemic on clinical presentations and viral epidemiology of acute respiratory infection in preschool children in Brazil.

We assessed the impact of the H1N1 influenza pandemic on acute respiratory infection in young children from low-income families in Brazil. Influenza (specifically H1N1) detection in acute respiratory infection quintupled during the pandemic and, during its peak, it was associated with 30% of all acute respiratory infection visits to the emergency department. H1N1 was also associated with increased risk of hospitalization and coinfection.