Patients with moderate-severe Crohn's disease (CD) who are treated with antitumor necrosis factor alpha (TNF-α) agents may be subjected to primary nonresponse or partial response. We aimed to ...identify tissue markers that may predict response to these agents.
Pediatric patients (6-18 years) with either ileal or ileo-colonic CD who were treated with anti-TNF-α were stratified into three different groups based on their overall response to therapy at the end of induction including clinical and laboratory parameters (group 1-full responders FR, group 2-partial responders PR, group 3-nonresponders NR). Seven tissue markers (fibronectin, interleukin IL-23R, IL-23, TNF-α, collagen-III, IL-13R, and hypoxia-inducible factors HIF-1α) were evaluated. Immunofluorescence (IF) analyses were performed on biopsies from the terminal ileum, which were retrieved up to 6 months before treatment initiation.
Twenty-six CD patients (16 61.5% males; age 13.9 ± 2.9 years), including 8 (30.8%) with ileal disease and 18 (69.2%) with ileo-colonic disease, were enrolled. Terminal ileum biopsies from nine patients from group 1, nine from group 2, and eight from group 3 were evaluated. Three antibodies were found to be significantly different between NR and FR groups; Collagen III and fibronectin stains were significantly more prominent in NR patients, while TNF-α stain was significantly more pronounced in FR, p < 0.05 for each. PR could not have been predicted with neither of markers.
Decreased tissue IF intensity of fibronectin and collagen III and increased intensity of TNF-α may predict response to anti-TNF-α treatment.
Transdifferentiation (TD) is the direct reprogramming of adult cells into cells of alternate fate and function. We have previously shown that liver cells can be transdifferentiated into beta‐like, ...insulin‐producing cells through ectopic expression of pancreatic transcription factors (pTFs). However, the efficiency of the process was consistently limited to <15% of the human liver cells treated in culture. The data in the current study suggest that liver‐to‐pancreas TD is restricted to a specific population of liver cells that is predisposed to undergo reprogramming. We isolated TD‐predisposed subpopulation of liver cells from >15 human donors using a lineage tracing system based on the Wnt response element, part of the pericentral‐specific promoter of glutamine synthetase. The cells, that were propagated separately, consistently exhibited efficient fate switch and insulin production and secretion in >60% of the cells upon pTF expression. The rest of the cells, which originated from 85% of the culture, resisted TD. Both populations expressed the ectopic pTFs with similar efficiencies, followed by similar repression of hepatic genes. Our data suggest that the TD‐predisposed cells originate from a distinct population of liver cells that are enriched for Wnt signaling, which is obligatory for efficient TD. In TD‐resistant populations, Wnt induction is insufficient to induce TD. An additional step of chromatin opening enables TD of these cells. Conclusion: Liver‐to‐pancreas TD occurs in defined predisposed cells. These cells' predisposition is maintained by Wnt signaling that endows the cells with the plasticity needed to alter their transcriptional program and developmental fate when triggered by ectopic pTFs. These results may have clinical implications by drastically increasing the efficacy of TD in future clinical uses. (Hepatology 2018).
Biliary atresia is a neonatal liver disease with extrahepatic bile duct obstruction and progressive liver fibrosis. The etiology and pathogenesis of the disease are unknown. We previously identified ...a plant toxin, biliatresone, responsible for biliary atresia in naturally-occurring animal models, that causes cholangiocyte destruction in in-vitro models. Decreases in reduced glutathione (GSH) mimic the effects of biliatresone, and agents that replenish cellular GSH ameliorate the effects of the toxin. The goals of this study were to define signaling pathways downstream of biliatresone that lead to cholangiocyte destruction and to determine their relationship to GSH. Using cholangiocyte culture and 3D cholangiocyte spheroid cultures, we found that biliatresone and decreases in GSH upregulated RhoU/Wrch1, a Wnt signaling family member, which then mediated an increase in Hey2 in the NOTCH signaling pathway, causing downregulation of the transcription factor Sox17. When these genes were up- or down-regulated, the biliatresone effect on spheroids was phenocopied, resulting in lumen obstruction. Biopsies of patients with biliary atresia demonstrated increased RhoU/Wrch1 and Hey2 expression in cholangiocytes. We present a novel pathway of cholangiocyte injury in a model of biliary atresia, which is relevant to human BA and may suggest potential future therapeutics.
Bile duct integrity is essential for the maintenance of the structure and function of the biliary tree. We previously showed that cholangiocyte injury in a toxic model of biliary atresia leads to ...increased monolayer permeability. Increased epithelial permeability was also shown in other cholangiopathies. We hypothesized that after initial cholangiocyte injury, leakage of bile acids into the duct submucosa propagates cholangiocyte damage and fibrosis. We thus aimed to determine the impact of bile acid exposure on cholangiocytes and the potential therapeutic effect of a non-toxic bile acid.
Extrahepatic bile duct explants were isolated from adult and neonatal BALB/c mice. Explants were cultured with or without glycochenodeoxycholic acid and ursodeoxycholic acid. They were then fixed and stained.
Explants treated with glycochenodeoxycholic acid demonstrated cholangiocyte injury with monolayer disruption and partial lumen obstruction compared to control ducts. Masson's trichrome stains revealed increased collagen fibers. Myofibroblast marker α-SMA stains were significantly elevated in the periductal region. The addition of ursodeoxycholic acid resulted in decreased cholangiocyte injury and reduced fibrosis.
Bile acid leakage into the submucosa after initial cholangiocyte injury may serve as a possible mechanism of disease propagation and progressive fibrosis in cholangiopathies.
The molecular basis for primary hereditary hypertriglyceridemia has been identified in fewer than 5% of cases. Investigation of monogenic dyslipidemias has the potential to expose key metabolic ...pathways. We describe a hitherto unreported disease in ten individuals manifesting as moderate to severe transient childhood hypertriglyceridemia and fatty liver followed by hepatic fibrosis and the identification of the mutated gene responsible for this condition. We performed SNP array-based homozygosity mapping and found a single large continuous segment of homozygosity on chromosomal region 12q13.12. The candidate region contained 35 genes that are listed in Online Mendelian Inheritance in Man (OMIM) and 27 other genes. We performed candidate gene sequencing and screened both clinically affected individuals (children and adults with hypertriglyceridemia) and also a healthy cohort for mutations in GPD1, which encodes glycerol-3-phosphate dehydrogenase 1. Mutation analysis revealed a homozygous splicing mutation, c.361−1G>C, which resulted in an aberrantly spliced mRNA in the ten affected individuals. This mutation is predicted to result in a truncated protein lacking essential conserved residues, including a functional site responsible for initial substrate recognition. Functional consequences of the mutation were evaluated by measuring intracellular concentrations of cholesterol and triglyceride as well as triglyceride secretion in HepG2 (hepatocellular carcinoma) human cells lines overexpressing normal and mutant GPD1 cDNA. Overexpression of mutant GPD1 in HepG2 cells, in comparison to overexpression of wild-type GPD1, resulted in increased secretion of triglycerides (p = 0.01). This finding supports the pathogenicity of the identified mutation.
Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function ...of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.
Abstract Background The combination of microcephaly, pyramidal signs, abnormal corpus callosum, and intellectual disability presents a diagnostic challenge. We describe an autosomal recessive ...disorder characterized by microcephaly, pyramidal signs, thin corpus callosum, and intellectual disability. Methods We previously mapped the locus for this disorder to 8q23.2-q24.12; the candidate region included 22 genes. We performed Sanger sequencing of 10 candidate genes; to ensure other genes in the candidate region do not harbor mutations, we sequenced the exome of one affected individual. Results We identified two homozygous missense changes, p.Thr186Arg and p.Pro416His in TAF2, which encodes a multisubunit cofactor for TFIID-dependent RNA polymerase II–mediated transcription, in all affected individuals. Conclusions We propose that the disorder is caused by the more conserved mutation p.Thr186Arg, with the second sequence change identified, p.Pro416His, possibly further negatively affecting the function of the protein. However, it is unclear which of the two changes, or maybe both, represents the causative mutation. A single missense mutation in TAF2 in a family with microcephaly and intellectual disability was described in a large-scale study reporting on the identification of 50 novel genes. We suggest that a mutation in TAF2 can cause this syndrome.
Abstract
Background
Insulin producing cells generated by liver cell transdifferentiation, could serve as an attractive source for regenerative medicine. The present study assesses the relationship ...between DNA methylation pTFs induced liver to pancreas transdifferentiation.
Results
The transdifferentiation process is associated with DNA demethylation, mainly at gene regulatory sites, and with increased expression of these genes. Active inhibition of DNA methylation promotes the pancreatic transcription factor-induced transdifferentiation process, supporting a causal role for DNA demethylation in this process.
Conclusions
Transdifferentiation is associated with global DNA hypomethylation, and with increased expression of specific demethylated genes. A combination of epigenetic modulators may be used to increase chromatin accessibility of the pancreatic transcription factors, thus promoting the efficiency of the developmental process.
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