A prospective observational study of the effect of critical illness on ultrastructural and microscopic morphology of duodenal mucosa.

OBJECTIVE: Disturbed intestinal barrier function due to 'leaky' tight junctions may cause secondary sepsis via paracellular translocation across the gut wall. Our objective was to describe the effects of critical illness on duodenal morphology and ultrastructure. DESIGN, SETTING AND PARTICIPANTS: Prospective observational study of 12 mechanically ventilated critically ill patients in an intensive care unit and 15 control participants in an outpatient endoscopy suite. INTERVENTION: We took six endoscopic biopsy samples of the duodenum from each participant for analysis by electron and light microscopy. MAIN OUTCOME MEASURES: Our primary outcome was tight junction morphology, examined with electron microscopy. Secondary outcomes were microvillus length and density, vascular endothelium morphology and mitochondrial density and morphology, examined with electron microscopy, and morphology examined with light microscopy. RESULTS: We observed no abnormalities of tight junction ultrastructure in either group. There was a tendency towards shorter microvilli in the critically ill group: mean length in critically ill patients, 1.17 µm (interquartile range [IQR], 1.05-1.60 µm) v mean length in control patients, 1.58 µm (IQR, 1.30-1.72 µm); P = 0.07. There was a tendency towards less dense microvilli in the critically ill group: mean density in critically ill patients, 7.29 microvilli/µm (IQR, 6.83-8.05 microvilli/µm) v mean density in control patients, 8.23 microvilli/µm (IQR, 7.34-9.11 microvilli/µm); P = 0.07. Vascular endothelium appeared normal in all critically ill patients and abnormal in one control participant. Abnormal mitochondrial morphology was noted in one critically ill patient and one control participant, and no differences were seen in mitochondrial density. Using light microscopy, we saw more apoptotic cells in the critically ill patients (P = 0.018), but villus height, crypt depth and lymphocyte density were normal. CONCLUSIONS: We did not detect any morphological abnormalities of duodenal tight junctions in critically ill patients. Our results should be interpreted with caution because of the small sample population, but our observations challenge the concept that paracellular translocation facilitates secondary sepsis.

Identification of KLHL41 Mutations Implicates BTB-Kelch-Mediated Ubiquitination as an Alternate Pathway to Myofibrillar Disruption in Nemaline Myopathy.

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.

Heterogeneity of fibrosis patterns in non-alcoholic fatty liver disease supports the presence of multiple fibrogenic pathways.

BACKGROUND: Adult non-alcoholic fatty liver disease (NAFLD) involves lobular necroinflammatory activity and fibrosis is typically centrilobular, whereas paediatric NAFLD has predominantly portal fibrosis. The reasons for these differences are unclear. We aimed to determine (a) how centrilobular and portal fibrosis in children relate to histological parameters; and (b) whether atypical fibrosis patterns exist in adults that are unexplained by current fibrogenesis models. METHODS: Histological features of paediatric (n = 38) and adult (n = 56) NAFLD were assessed using conventional scoring systems. Keratin-7 immunostaining was used to assess hepatic progenitor cell numbers and the ductular reaction. Centrilobular and portal components of fibrosis were independently scored and fibrosis patterns were classified according to accepted types. Post-treatment (rosiglitazone/gastric banding) biopsies were also examined in adults. RESULTS: Twenty-six children (68.4%) had portal-predominant fibrosis, although the typical "adult" pattern was seen in 11 (28.9%). Portal fibrosis was associated with a ductular reaction (P = 0.021) and hepatic progenitor cell expansion (P < 0.001), whereas centrilobular fibrosis was associated with lobular inflammation (P = 0.026) and ballooning (P = 0.001). Before intervention, six adults (10.7%) had atypical fibrosis including 3 (5.4%) with a previously unrecognized pattern of very fine, non-zonal sinusoidal fibrosis. Despite improvements in steatosis and inflammation, more patients developed this unusual pattern after intervention with most having had surgery (9 of 10 adults; P < 0.001). CONCLUSION: Differing associations with portal and centrilobular fibrosis in children and atypical fibrosis patterns in adults suggest that multiple fibrogenic pathways exist in NAFLD. This has implications for therapy and understanding pathogenesis.

Whole exome sequencing in foetal akinesia expands the genotype-phenotype spectrum of GBE1 glycogen storage disease mutations.

The clinically and genetically heterogenous foetal akinesias have low rates of genetic diagnosis. Exome sequencing of two siblings with phenotypic lethal multiple pterygium syndrome identified compound heterozygozity for a known splice site mutation (c.691+2T>C) and a novel missense mutation (c.956A>G; p.His319Arg) in glycogen branching enzyme 1 (GBE1). GBE1 mutations cause glycogen storage disease IV (GSD IV), including a severe foetal akinesia sub-phenotype. Re-investigating the muscle pathology identified storage material, consistent with GSD IV, which was confirmed biochemically. This study highlights the power of exome sequencing in genetically heterogeneous diseases and adds multiple pterygium syndrome to the phenotypic spectrum of GBE1 mutation.