Paediatric negative appendicectomy rates at a regional Western Australian Centre: a five-year cohort study.

BACKGROUND: In acute appendicitis, decision-making around operative intervention for paediatric patients differs from adults due to a higher weight placed on clinical assessment and reduced rates of cross-sectional imaging. In regional settings, non-paediatric emergency doctors, general surgeons, and radiologists usually assess and manage this patient group. Differences have been observed in paediatric negative appendicectomy rates between general and paediatric centres. METHODS: A retrospective cohort study was performed, identifying paediatric patients undergoing emergency appendicectomy at the Southwest Health Campus (Bunbury, Western Australia) from 2017 to 2021. The primary outcome measure was histopathology confirming the absence of transmural inflammation of the appendix. In addition, clinical, biochemical and radiological data were collected to identify predictors of negative appendicectomy (NA). Secondary outcome measures were hospital length-of-stay and post-operative complication rates. RESULTS: Four hundred and twenty-one patients were identified, of which 44.9% had a negative appendicectomy. Statistically significant associations between female gender, white cell count less than 10 × 109 , neutrophil ratio less than 75%, low CRP and NA were observed. NA was not associated with a lower risk of re-admission or complications compared with appendicectomy for appendicitis. CONCLUSIONS: Our centre's NA rate is higher than that observed in the literature at both non-paediatric and paediatric surgical centres. NA has similar morbidity risk to appendicectomy for uncomplicated appendicitis and offers a timely reminder that diagnostic laparoscopy in children is not benign.

Vesicle-associated membrane protein 8 (VAMP8) is a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) selectively required for sequential granule-to-granule fusion.

Compound exocytosis is found in many cell types and is the major form of regulated secretion in acinar and mast cells. Its key characteristic is the homotypic fusion of secretory granules. These then secrete their combined output through a single fusion pore to the outside. The control of compound exocytosis remains poorly understood. Although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as syntaxin 2, SNAP23 (synaptosome-associated protein of 23 kDa), and SNAP25 have been suggested to play a role, none has been proven. Vesicle-associated membrane protein 8 (VAMP8) is a SNARE first associated with endocytic processes but more recently has been suggested as an R-SNARE in regulated exocytosis. Secretion in acinar cells is reduced when VAMP8 function is inhibited and is less in VAMP8 knock-out mice. Based on electron microscopy experiments, it was suggested that VAMP8 may be involved in compound exocytosis. Here we have tested the hypothesis that VAMP8 controls homotypic granule-to-granule fusion during sequential compound exocytosis. We use a new assay to distinguish primary fusion events (fusion with the cell membrane) from secondary fusion events (granule-granule fusion). Our data show the pancreatic acinar cells from VAMP8 knock-out animals have a specific reduction in secondary granule fusion but that primary granule fusion is unaffected. Furthermore, immunoprecipitation experiments show syntaxin 2 association with VAMP2, whereas syntaxin 3 associates with VAMP8. Taken together our data indicate that granule-to-granule fusion is regulated by VAMP8 containing SNARE complexes distinct from those that regulate primary granule fusion.

Protons released during pancreatic acinar cell secretion acidify the lumen and contribute to pancreatitis in mice.

BACKGROUND & AIMS: Secretory granules are acidic; cell secretion will therefore lead to extracellular acidification. We propose that during secretion, protons co-released with proteins from secretory granules of pancreatic acinar cells acidify the restricted extracellular space of the pancreatic lumen to regulate normal physiological and pathophysiological functions in this organ METHODS: Extracellular changes in pH were quantified in real time using 2-photon microscopy analysis of pancreatic tissue fragments from mouse models of acute pancreatitis (mice given physiological concentrations [10 -20 pM] of cholecystokinin or high concentrations of [100 nM] cerulein). The effects of extracellular changes in pH on cell behavior and structures were measured. RESULTS: With physiological stimulation, secretory granule fusion (exocytosis) caused acidification of the pancreatic lumen. Acidifications specifically affected intracellular calcium responses and accelerated the rate of recovery from agonist-evoked calcium signals. Protons therefore appear to function as negative-feedback, extracellular messengers during coupling of cell stimuli with secretion. At high concentrations of cerulein, large increases in secretory activity were associated with extreme, prolonged acidification of the luminal space. These pathological changes in pH led to disruption of intercellular junctional coupling, measured by movement of occludin and E-cadherin. CONCLUSIONS: By measuring changes in extracellular pH in pancreas of mice, we observed that luminal acidification resulted from exocytosis of zymogen granules from acinar cells. This process is part of normal organ function but could contribute to the tissue damage in cases of acute pancreatitis.

Exocytosis, dependent on Ca2+ release from Ca2+ stores, is regulated by Ca2+ microdomains.

The relationship between the cellular Ca2+ signal and secretory vesicle fusion (exocytosis) is a key determinant of the regulation of the kinetics and magnitude of the secretory response. Here, we have investigated secretion in cells where the exocytic response is controlled by Ca2+ release from intracellular Ca2+ stores. Using live-cell two-photon microscopy that simultaneously records Ca2+signals and exocytic responses, we provide evidence that secretion is controlled by changes in Ca2+ concentration [Ca2+] in relatively large-volume microdomains. Our evidence includes: (1) long latencies (>2 seconds) between the rise in [Ca2+] and exocytosis, (2) observation of exocytosis all along the lumen and not clustered around Ca2+ release hot-spots, (3) high affinity (Kd=1.75 microM) Ca2+dependence of exocytosis, (4) significant reduction in exocytosis in the presence of cytosolic EGTA, (5) spatial exclusion of secretory granules from the cell membrane by the endoplasmic reticulum, and (6) inability of local Ca2+ responses to trigger exocytosis. These results strongly indicate that the control of exocytosis, triggered by Ca2+ release from stores, is through the regulation of cytosolic[Ca2+] within a microdomain.

Local dynamic changes in confined extracellular environments within organs.

1. Herein we review past work that has studied the composition of luminal fluid in organs, with a focus on measures of calcium and pH in the exocrine glands. This luminal environment is 'external' to the mammalian body and is not subject to the usual mechanisms of homeostatic control. Instead, it is controlled by the behaviour of the cells that line the lumen. 2. We discuss the likely possibility that rapid and local changes in calcium and pH occur within microdomains in the lumen. Further, we present preliminary evidence, using live cell imaging of intact pancreatic fragments, that supports the idea that pH changes do occur. Our evidence indicates that exocytosis of secretory granules in pancreatic acinar cells leads to a loss of protons from the granule and a subsequent local acidification of the lumen. 3. These changes in luminal composition are placed in the context of diseases of the pancreas, such as cystic fibrosis and pancreatitis, both of which are known to result in perturbations of luminal fluid composition.