Retina restored and brain abnormalities ameliorated by single-copy knock-in of human NR2E1 in null mice.

Nr2e1 encodes a stem cell fate determinant of the mouse forebrain and retina. Abnormal regulation of this gene results in retinal, brain, and behavioral abnormalities in mice. However, little is known about the functionality of human NR2E1. We investigated this functionality using a novel knock-in humanized-mouse strain carrying a single-copy bacterial artificial chromosome (BAC). We also documented, for the first time, the expression pattern of the human BAC, using an NR2E1-lacZ reporter strain. Unexpectedly, cerebrum and olfactory bulb hypoplasia, hallmarks of the Nr2e1-null phenotype, were not fully corrected in animals harboring one functional copy of human NR2E1. These results correlated with an absence of NR2E1-lacZ reporter expression in the dorsal pallium of embryos and proliferative cells of adult brains. Surprisingly, retinal histology and electroretinograms demonstrated complete correction of the retina-null phenotype. These results correlated with appropriate expression of the NR2E1-lacZ reporter in developing and adult retina. We conclude that the human BAC contained all the elements allowing correction of the mouse-null phenotype in the retina, while missing key regulatory regions important for proper spatiotemporal brain expression. This is the first time a separation of regulatory mechanisms governing NR2E1 has been demonstrated. Furthermore, candidate genomic regions controlling expression in proliferating cells during neurogenesis were identified.

Evidence for the homeostatic regulation of induced beta cell mass expansion.

AIMS/HYPOTHESIS: Diabetes results from an insufficient insulin-secreting beta cell mass. Restoration of beta cell mass through pharmaceutically induced endogenous beta cell mass expansion may revolutionise diabetes therapy. However, it remains to be determined whether the induced beta cell mass expansion is under homeostatic regulation. METHODS: Beta cell mass expansion rates were derived from three separate studies of continuous stimulation of islet neogenesis, including the partial duct obstruction of euglycaemic Syrian hamsters, administration of a pentadecapeptide with the same amino acid sequence as residues 104-118 of islet neogenesis-associated protein (INGAP(104-118)) to euglycaemic Syrian hamsters, as well as to euglycaemic CD-1 mice. The incidence of islet neogenesis, average beta cell size, and beta cell replication and apoptotic rates were determined. RESULTS: Partial duct obstruction led to a approximately 2.5-fold increase in endocrine tissue at day 56 (p<0.05). From day 0 to day 7 the average rate of change of islet area was 12.7% per day, and this rate decreased to 5.3% per day from day 7 to day 42, and to 2.8% per day from day 42 to day 56. Administration of INGAP(104-118) to adult hamsters led to a 31% increase in total beta cell mass at day 30 (p=0.031). From day 0 to day 10 the average rate of beta cell mass expansion was 148 mug/day, whereas from day 10 to day 30 it decreased to 45 mug/day. INGAP(104-118) administration to adult CD-1 mice resulted in an approximately twofold increase in beta cell mass after 31 days (p=0.021). However, at day 90, there was no significant difference vs age-matched control mice (p=0.30), even though the neogenic beta cell mass was approximately fourfold greater (p=0.026). Beta cell replication was decreased by 56% (p<0.048), whereas beta cell apoptosis was fourfold greater (p<0.003) in 90-day INGAP(104-118)-treated mice compared with age-matched control mice. CONCLUSIONS/INTERPRETATION: These data indicate that in the presence of ongoing islet neogenesis, homeostatic regulatory mechanisms intervene to regulate beta cell mass according to the prevailing metabolic requirements.

Autocrine insulin action activates Akt and increases survival of isolated human islets.

AIMS/HYPOTHESIS: The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in promoting the survival of pancreatic beta cells. Akt becomes activated in isolated human islets following overnight culture despite significant levels of cell death. The aim of the current study was to identify the cause of the observed increase in Akt phosphorylation in isolated islets. We hypothesised that a factor secreted by the islets in culture was acting in an autocrine manner to activate Akt. METHODS: In order to identify the stimulus of the PI3K/Akt pathway in culture, we examined the effects of different culture conditions on Akt phosphorylation and islet survival during the immediate post-isolation period. RESULTS: We demonstrated that islet-conditioned medium induced Akt phosphorylation in freshly isolated human islets, whereas frequent medium replacement decreased Akt phosphorylation. Following overnight culture, islet-conditioned medium contained significantly elevated levels of insulin, indicating that insulin may be responsible for the observed increase in Akt phosphorylation. Indeed, treatment with an anti-insulin antibody or with inhibitors of insulin receptor/IGF receptor 1 kinase activity suppressed Akt phosphorylation, leading to decreased islet survival. In addition, dispersion of islets into single cells also suppressed Akt phosphorylation and induced islet cell death, indicating that islet integrity is also required for maximal Akt phosphorylation. CONCLUSIONS/INTERPRETATION: Our findings demonstrate that insulin acts in an autocrine manner to activate Akt and mediate the survival of isolated human islets. These findings provide new information on how culturing islets prior to transplantation may be beneficial to their survival by allowing for autocrine activation of the pro-survival Akt pathway.

Endodontic, surgical and periodontal treatment of dens invaginatus. Case report.

The aim of this paper is to propose a single stage global treatment of endodontic, periapical and periodontal lesions in a lateral maxillary incisor with dens invaginatus. A 24 year-old woman presenting a lateral maxillary incisor with dens invaginatus in association with periapica1 and periodontal lesions underwent simultaneous surgical, endodontic and periodontal regenerative procedures. At 2, 6, 12, 18 months follow-up the radiographic healing appeared to be improved and the periapical lesion healed completely 1 year after surgical intervention. Surgery in association with endodontic and periodontal procedures represents the treatment of choice to maximize long term prognosis in cases of dens invaginatus with chronic periapical and periodontal lesions.

Surgical correction of gingival recessions associated with radicular carious lesions.

In this clinical report, six cases are presented in which radicular carious lesions and gingival recessions were treated concurrently. The combined treatment included the removal of caries, radicular planing, and various surgical techniques for root coverage. Traditional procedures, as well as newer procedures, such as guided tissue regeneration, showed successful results.