A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency.

Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3 × 1011 vg donor template and 1 × 1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1 × 1012 vg SP-B-donor template and 5 × 1011 vg nuclease significantly extended median survival (p = 0.0034) from 5 days in the untreated off doxycycline group to 16 days in the donor AAV and nuclease group, with one gene-edited mouse living 243 days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.

Optimizing delivery for efficient cardiac reprogramming.

Following heart injury, cardiomyocytes, are lost and are not regenerated. In their place, fibroblasts invade the dead tissue where they generate a scar, which reduces cardiac function. We and others have demonstrated that combinations of specific miRNAs (miR combo) or transcription factors (GMT), delivered by individual lenti-/retro-viruses in vivo, can convert fibroblasts into cardiomyocytes and improve cardiac function. However, the effects are relatively modest due to the low efficiency of delivery of miR combo or GMT. We hypothesized that efficiency would be improved by optimizing delivery. In the first instance, we developed a multicistronic system to express all four miRNAs of miR combo from a single construct. The order of each miRNA in the multicistronic construct gave rise to different levels of miRNA expression. A combination that resulted in equivalent expression levels of each of the four miRNAs of miR combo showed the highest reprogramming efficiency. Further efficiency can be achieved by directly targeting fibroblasts. Screening of several AAV serotypes indicated that AAV1 displayed tropism towards cardiac fibroblasts. Combining multicistronic expression with AAV1 delivery robustly reprogrammed cardiac fibroblasts into cardiomyocytes in vivo.

Long-term outcomes in patients with recurrent human papillomavirus-positive oropharyngeal cancer after upfront transoral robotic surgery.

BACKGROUND: Clinical course following failure of human papillomavirus (HPV)-positive oropharyngeal cancers (HPV + OPC) is poorly understood. This study aims to characterize disease course following failure after transoral robotic surgery (TORS). METHODS: We identified patients with HPV + OPC-treated upfront with TORS at our institution from 2007 to 2017. HPV status was confirmed with immunohistochemistry or HPV DNA polymerase chain reaction. Patient characteristics, treatment modalities, and post-recurrence outcomes were analyzed for the recurrent cohort. RESULTS: Of the 317 HPV + OPC patients, 28 (8.8%) experienced recurrence, all of HPV 16/18 subtypes. Median post-recurrence survival was 19.8 months (range 2.3-195.8 months) in the 12 locoregional and 16 months (range 2.4-79.5 months) in the 14 distant failures. Sixteen are alive with a median of 39.8 months (range 5.5-209.4 months) after retreatment. CONCLUSION: This is one of the largest series evaluating survival following TORS failure in HPV + OPC. Despite failure, long-term survival and durable remission are possible with single-modal or multiple-modal salvage treatment.

A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency.

Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.

Augmented curation of clinical notes from a massive EHR system reveals symptoms of impending COVID-19 diagnosis.

Understanding temporal dynamics of COVID-19 symptoms could provide fine-grained resolution to guide clinical decision-making. Here, we use deep neural networks over an institution-wide platform for the augmented curation of clinical notes from 77,167 patients subjected to COVID-19 PCR testing. By contrasting Electronic Health Record (EHR)-derived symptoms of COVID-19-positive (COVIDpos; n = 2,317) versus COVID-19-negative (COVIDneg; n = 74,850) patients for the week preceding the PCR testing date, we identify anosmia/dysgeusia (27.1-fold), fever/chills (2.6-fold), respiratory difficulty (2.2-fold), cough (2.2-fold), myalgia/arthralgia (2-fold), and diarrhea (1.4-fold) as significantly amplified in COVIDpos over COVIDneg patients. The combination of cough and fever/chills has 4.2-fold amplification in COVIDpos patients during the week prior to PCR testing, in addition to anosmia/dysgeusia, constitutes the earliest EHR-derived signature of COVID-19. This study introduces an Augmented Intelligence platform for the real-time synthesis of institutional biomedical knowledge. The platform holds tremendous potential for scaling up curation throughput, thus enabling EHR-powered early disease diagnosis.

Assessment of Expert-Level Automated Detection of Plasmodium falciparum in Digitized Thin Blood Smear Images.

Importance: Decades of effort have been devoted to establishing an automated microscopic diagnosis of malaria, but there are challenges in achieving expert-level performance in real-world clinical settings because publicly available annotated data for benchmark and validation are required. Objective: To assess an expert-level malaria detection algorithm using a publicly available benchmark image data set. Design, Setting, and Participants: In this diagnostic study, clinically validated malaria image data sets, the Taiwan Images for Malaria Eradication (TIME), were created by digitizing thin blood smears acquired from patients with malaria selected from the biobank of the Taiwan Centers for Disease Control from January 1, 2003, to December 31, 2018. These smear images were annotated by 4 clinical laboratory scientists who worked in medical centers in Taiwan and trained for malaria microscopic diagnosis at the national reference laboratory of the Taiwan Centers for Disease Control. With TIME, a convolutional neural network-based object detection algorithm was developed for identification of malaria-infected red blood cells. A diagnostic challenge using another independent data set within TIME was performed to compare the algorithm performance against that of human experts as clinical validation. Main Outcomes and Measures: Performance on detecting Plasmodium falciparum-infected blood cells was measured by average precision, and performance on detecting P falciparum infection at the image level was measured using sensitivity, specificity, and area under the receiver operating characteristic curve (AUC). Results: The TIME data sets contained 8145 images of 36 blood smears from patients with suspected malaria (30 P falciparum-positive and 6 P falciparum-negative smears) that had reliable annotations. For clinical validation, the average precision was 0.885 for detecting P falciparum-infected blood cells and 0.838 for ring form. For detecting P falciparum infection on blood smear images, the algorithm had expert-level performance (sensitivity, 0.995; specificity, 0.900; AUC, 0.997 [95% CI, 0.993-0.999]), especially in detecting ring form (sensitivity, 0.968; specificity, 0.960; AUC, 0.995 [95% CI, 0.990-0.998]) compared with experienced microscopists (mean sensitivity, 0.995 [95% CI, 0.993-0.998]; mean specificity, 0.955 [95% CI, 0.885-1.000]). Conclusions and Relevance: The findings suggest that a clinically validated expert-level malaria detection algorithm can be developed by using reliable data sets.

The Impact of Body Mass Index on Perioperative and Postoperative Outcomes for Endovascular Abdominal Aneurysm Repair.

BACKGROUND: There is varying evidence regarding the effects of body mass index (BMI) on outcomes of endovascular aneurysm repair (EVAR). This study investigates the effects of BMI on an index of perioperative and postoperative outcomes after EVAR. METHODS: Four hundred ninety-two patients who underwent elective EVAR at Mount Sinai Hospital were included in this study. Patients were classified as either normal weight (BMI = 18.5-25), overweight (BMI = 25-30), or obese (BMI>30). Chi-squared tests were used to determine significant differences between weight classes across an index of outcomes. The following outcomes were collected: intraoperative complications (e.g., conversion to open), perioperative complications (e.g., hematoma, bowel ischemia, and so forth), and postoperative outcomes (endoleak, sac enlargement, sac shrinkage, access site infection, prolonged postoperative length of stay, reintervention, stroke, claudication/lower extremity ischemia, deep vein thrombosis, limb occlusion, renal complications, abdominal aortic aneurysm (AAA) rupture, AAA-related mortality, and all-cause mortality). Kaplan-Meier survival analysis and a log-rank test were used to determine meaningful differences in all-cause mortality following EVAR between the respective weight classes. Subsequently, multivariate Cox proportional hazards were performed for selection of outcomes, with weight classes as predictors. Finally, a multivariate logistic regression was performed for postoperative hospital stay. Subgroup multivariate analysis was also performed examining only class I obese patients, rather than all obese patients. RESULTS: Overweight patients were significantly less likely to experience all-cause mortality up to 9 years after EVAR than normal-weight patients in both Kaplan-Meier and multivariable Cox proportional hazards models. Obese patients similarly had a lower risk of mortality in Kaplan-Meier analysis, but this did not persist in the multivariate analysis. Overweight patients were also significantly less likely to require a postoperative hospital stay longer than 1 day when compared with normal-weight patients. Finally, obese patients were less likely to have a sac shrinkage greater than 5 mm after EVAR, but were also less likely to have an endoleak. CONCLUSIONS: This study adds to the debate on the effects of BMI on outcomes of EVAR. Obesity was not a risk factor for negative perioperative or postoperative outcomes after EVAR with the exception of decreased sac shrinkage. Obese patients were less likely to have an endoleak, and overweight patients were protected against all-cause mortality and longer postoperative hospital stays.

Combination of Active Transfer Learning and Natural Language Processing to Improve Liver Volumetry Using Surrogate Metrics with Deep Learning.

PURPOSE: To determine if weakly supervised learning with surrogate metrics and active transfer learning can hasten clinical deployment of deep learning models. MATERIALS AND METHODS: By leveraging Liver Tumor Segmentation (LiTS) challenge 2017 public data (n = 131 studies), natural language processing of reports, and an active learning method, a model was trained to segment livers on 239 retrospectively collected portal venous phase abdominal CT studies obtained between January 1, 2014, and December 31, 2016. Absolute volume differences between predicted and originally reported liver volumes were used to guide active learning and assess accuracy. Overall survival based on liver volumes predicted by this model (n = 34 patients) versus radiology reports and Model for End-Stage Liver Disease with sodium (MELD-Na) scores was assessed. Differences in absolute liver volume were compared by using the paired Student t test, Bland-Altman analysis, and intraclass correlation; survival analysis was performed with the Kaplan-Meier method and a Mantel-Cox test. RESULTS: Data from patients with poor liver volume prediction (n = 10) with a model trained only with publicly available data were incorporated into an active learning method that trained a new model (LiTS data plus over- and underestimated active learning cases [LiTS-OU]) that performed significantly better on a held-out institutional test set (absolute volume difference of 231 vs 176 mL, P = .0005). In overall survival analysis, predicted liver volumes using the best active learning-trained model (LiTS-OU) were at least comparable with liver volumes extracted from radiology reports and MELD-Na scores in predicting survival. CONCLUSION: Active transfer learning using surrogate metrics facilitated deployment of deep learning models for clinically meaningful liver segmentation at a major liver transplant center.© RSNA, 2019Supplemental material is available for this article.

Outcomes of using endovascular aneurysm repair with active fixation in complex aneurysm morphology.

OBJECTIVE: The ideal treatment option for patients with complex aneurysm morphology remains highly debated. The aim of this study was to investigate the impact of endovascular aneurysm repair (EVAR) with active fixation on outcomes in patients with complex aneurysm morphology. METHODS: There were 340 consecutive patients who underwent EVAR using active fixation devices, 234 with active infrarenal fixation (AIF; Gore Excluder; W. L. Gore & Associates, Flagstaff, Ariz) and 106 with active suprarenal fixation (ASF; 85 Medtronic Endurant [Medtronic, Santa Rosa, Calif] and 21 Cook Zenith [Cook Medical, Bloomington, Ind]). Demographics, comorbidities, anatomic features, and outcomes were analyzed for patients receiving devices with active fixation. Outcomes of using active fixation in necks with <15-mm neck lengths, >60-degree infrarenal neck angle (ß), >30-mm infrarenal neck diameter, severe aortic neck calcification or thrombus, and nonstraight neck morphology were evaluated. RESULTS: Of the 340 patients, 106 (78 men; mean age, 74.5 ± 9.3 years at the time of surgery) received implants with ASF and 234 (191 men; mean age, 74.6 ± 8.9 years at the time of surgery) received implants with AIF. In comparing AIF and ASF devices, patients in the suprarenal fixation group had significantly shorter follow-up time (25 ± 17 months vs 44.3 ± 32 months; P < .0001). Patients in the ASF group had shorter aortic neck lengths (25.5 ± 15.1 mm vs 28.6 ± 14.9 mm; P = NS) and significantly larger infrarenal neck diameters (25.9 ± 6.3 mm vs 23.4 ± 3.2 mm; P < .0001) and aneurysm diameters (59.9 ± 11.6 mm v. 55.9 ± 10.0 mm; P = .002). Outcomes were similar between groups, with no significant differences in reintervention, proximal endoleak, sac growth, abdominal aortic aneurysm-related death, or rupture. Of the complex anatomic neck features investigated, neck diameter >30 mm and nonstraight neck morphology had the highest rates of reintervention in ASF devices. CONCLUSIONS: In cases of hostile infrarenal neck morphology, ASF appears to be used more frequently. Our data suggest that ASF may be useful for certain patients but may be unfavorable for others, such as those with wide necks or several difficult neck features. Nevertheless, further research is needed to evaluate more optimal treatment options, such as fenestrated EVAR, branched EVAR, and endovascular adjuncts such as EndoAnchors (Aptus Endosystems, Sunnyvale, Calif), in dealing with high-risk anatomic characteristics that may not be optimally managed with standard EVAR devices with active fixation.

Stem cell biology and regenerative medicine for neonatal lung diseases.

Lung diseases remain one of the main causes of morbidity and mortality in neonates. Cell therapy and regenerative medicine have the potential to revolutionize the management of life-threatening and debilitating lung diseases that currently lack effective treatments. Over the past decade, the repair capabilities of stem/progenitor cells have been harnessed to prevent/rescue lung damage in experimental neonatal lung diseases. Mesenchymal stromal cells and amnion epithelial cells exert pleiotropic effects and represent ideal therapeutic cells for bronchopulmonary dysplasia, a multifactorial disease. Endothelial progenitor cells are optimally suited to promote lung vascular growth and attenuate pulmonary hypertension in infants with congenital diaphragmatic hernia or a vascular bronchopulmonary dysplasia phenotype. Induced pluripotent stem cells (iPSCs) are one of the most exciting breakthroughs of the past decade. Patient-specific iPSCs can be derived from somatic cells and differentiated into any cell type. iPSCs can be capitalized upon to develop personalized regenerative cell products for surfactant protein deficiencies-lethal lung disorders without treatment-that affect a single gene in a single cell type and thus lend themselves to phenotype-specific cell replacement. While the clinical translation has begun, more needs to be learned about the biology of these repair cells to make this translation successful.

Human induced pluripotent stem cell-derived lung progenitor and alveolar epithelial cells attenuate hyperoxia-induced lung injury.

BACKGROUND AIMS: Bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by disrupted lung growth, is the most common complication in extreme premature infants. BPD leads to persistent pulmonary disease later in life. Alveolar epithelial type 2 cells (AEC2s), a subset of which represent distal lung progenitor cells (LPCs), promote normal lung growth and repair. AEC2 depletion may contribute to persistent lung injury in BPD. We hypothesized that induced pluripotent stem cell (iPSC)-derived AECs prevent lung damage in experimental oxygen-induced BPD. METHODS: Mouse AECs (mAECs), miPSCs/mouse embryonic stem sells, human umbilical cord mesenchymal stromal cells (hUCMSCs), human (h)iPSCs, hiPSC-derived LPCs and hiPSC-derived AECs were delivered intratracheally to hyperoxia-exposed newborn mice. Cells were pre-labeled with a red fluorescent dye for in vivo tracking. RESULTS: Airway delivery of primary mAECs and undifferentiated murine pluripotent cells prevented hyperoxia-induced impairment in lung function and alveolar growth in neonatal mice. Similar to hUCMSC therapy, undifferentiated hiPSCs also preserved lung function and alveolar growth in hyperoxia-exposed neonatal NOD/SCID mice. Long-term assessment of hiPSC administration revealed local teratoma formation and cellular infiltration in various organs. To develop a clinically relevant cell therapy, we used a highly efficient method to differentiate hiPSCs into a homogenous population of AEC2s. Airway delivery of hiPSC-derived AEC2s and hiPSC-derived LPCs, improved lung function and structure and resulted in long-term engraftment without evidence of tumor formation. CONCLUSIONS: hiPSC-derived AEC2 therapy appears effective and safe in this model and warrants further exploration as a therapeutic option for BPD and other lung diseases characterized by AEC injury.

ABCA8 Regulates Cholesterol Efflux and High-Density Lipoprotein Cholesterol Levels.

OBJECTIVE: High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate-binding cassette transporter A8 (ABCA8) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. APPROACH AND RESULTS: We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P=0.005). HDLc levels were significantly decreased by 29% (P=0.01) in Abca8b-/- mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P=0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate-binding cassette transporter A1 and further potentiates adenosine triphosphate-binding cassette transporter A1-mediated cholesterol efflux. CONCLUSIONS: ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.

Feasibility and reproducibility of liver surface nodularity quantification for the assessment of liver cirrhosis using CT and MRI.

PURPOSE: To assess intra-observer, inter-observer and inter-modality (CT vs. MRI) reproducibility of liver surface nodularity (LSN) scores measured with software used for detection of liver fibrosis. METHODS: This IRB-approved retrospective study included patients with both abdominal CT and MRI within 6 months of histopathologic sampling. Two independent observers used post-processing software to quantify LSN scores on axial non-contrast CT (NCT), axial contrast-enhanced CT (CECT), axial T2-weighted (T2W) HASTE, and axial and coronal post-gadoxetic acid T1-weighted (T1W) images obtained during the hepatobiliary phase (HBP). Ten slices were used to acquire the LSN scores. Intra-observer, inter-observer, and inter-modality (CT vs. MRI) reproducibility were assessed with intraclass correlation coefficient (ICC) and coefficients of variability (CV). Accuracy for detection of cirrhosis was evaluated for each technique. RESULTS: 26 patients (M/F 19/7, mean age 57 years), including 7 with cirrhosis (26.9%), were assessed. Technical failure occurred with NCT (1/23, 4.3%) and T2 HASTE (8/28, 28.6%). Intra-observer reproducibility was excellent for NCT, CECT, axial and coronal T1W HBP [ICC ≥ 0.92, CV ≤ 8%]. Inter-observer reproducibility was also excellent for NCT and CECT (ICC ≥ 0.95, CV ≤ 7.3%) and for coronal T1W HBP (ICC = 0.84, CV = 5.6%). There was fair to moderate agreement between CT and MRI (ICC 0.20-0.44). There were significant differences in mean LSN scores between non-cirrhotic and cirrhotic patients with NCT (2.6 vs. 4.2, p = 0.04) and T1W HBP (3.7 vs. 4.6; p = 0.01) images, with AUCs of 0.81 and 0.82, respectively. CONCLUSIONS: LSN measurement is highly reproducible with NCT and post-contrast T1W HBP on MRI, with different results obtained between CT and MRI.

Comparison Between 3-Scan Trace and Diagonal Body Diffusion-Weighted Imaging Acquisitions: A Phantom and Volunteer Study.

Diagonal diffusion-weighted imaging (dDWI) uses simultaneous maximized application of 3 orthogonal gradient systems as opposed to sequential acquisition in 3 directions in conventional 3-scan trace DWI (tDWI). Several theoretical advantages of dDWI vs. tDWI include reduced artifacts and increased sharpness. We compared apparent diffusion coefficient (ADC) quantification and image quality between monopolar dDWI and tDWI in a dedicated diffusion phantom (b = 0/500/900/2000 s/mm2) and in the abdomen (b = 50/400/800 s/mm2) and pelvis (b = 50/1000/1600 s/mm2) of 2 male volunteers at 1.5 T and 3.0 T. Phantom estimated signal-to-noise ratio (eSNR) was also measured. Two independent observers assessed the image quality on a 5-point scale. In the phantom, image quality was similar between tDWI and dDWI, with equivalent ADC quantification (mean coefficient of variation [CV] between sequences: 1.4% ± 1.2% at 1.5 T and 0.7% ± 0.7% at 3.0 T). Phantom eSNR was similar for both tDWI and dDWI, except for a significantly lower eSNR for b900 of dDWI at 3.0 T (P = .006). In the volunteers, the CV values between tDWI and dDWI were higher than those in the phantom (CV range: abdominal organs, 1.3%-13.3%; pelvic organs, 0.6%-5.7%). A trend toward significant better image quality for dDWI compared with tDWI was observed for b800 (abdomen) at 3.0 T and for b1000 and b1600 (pelvis) at 1.5 T (P = .063 to .066). Our data suggest that dDWI may provide better image quality than tDWI without affecting ADC quantification, needing confirmation in a future clinical study.

MicroRNAs and Cardiac Regeneration.

The human heart has a limited capacity to regenerate lost or damaged cardiomyocytes after cardiac insult. Instead, myocardial injury is characterized by extensive cardiac remodeling by fibroblasts, resulting in the eventual deterioration of cardiac structure and function. Cardiac function would be improved if these fibroblasts could be converted into cardiomyocytes. MicroRNAs (miRNAs), small noncoding RNAs that promote mRNA degradation and inhibit mRNA translation, have been shown to be important in cardiac development. Using this information, various researchers have used miRNAs to promote the formation of cardiomyocytes through several approaches. Several miRNAs acting in combination promote the direct conversion of cardiac fibroblasts into cardiomyocytes. Moreover, several miRNAs have been identified that aid the formation of inducible pluripotent stem cells and miRNAs also induce these cells to adopt a cardiac fate. MiRNAs have also been implicated in resident cardiac progenitor cell differentiation. In this review, we discuss the current literature as it pertains to these processes, as well as discussing the therapeutic implications of these findings.

Direct intracerebral delivery of a miR-33 antisense oligonucleotide into mouse brain increases brain ABCA1 expression. [Corrected].

The ATP-binding cassette transporter A1 (ABCA1) is a membrane bound protein that serves to efflux cholesterol and phospholipids onto lipid poor apolipoproteins during HDL biogenesis. Increasing the expression and activity of ABCA1 have beneficial effects in experimental models of various neurologic and cardiovascular diseases including Alzheimer's disease. Despite the beneficial effects of liver X receptor (LXR) agonists--compounds that increase ABCA1 expression--in preclinical studies, their therapeutic utility is limited by systemic adverse effects on lipid metabolism. Interestingly, microRNA-33 (miR-33) inhibition increases ABCA1 expression and activity in rodents and non-human primates without severe metabolic adverse effects. Herein, we demonstrate that treatment of cultured mouse neurons, astrocytes and microglia with an antisense oligonucleotide (ASO) targeting miR-33 increased ABCA1 expression, which was accompanied by increased cholesterol efflux and apoE secretion in astrocytic cultures. We also show that intracerebral delivery of an ASO targeting miR-33 leads to increased ABCA1 expression in cerebral cortex or subcortical structures such as hippocampus. These findings highlight an effective strategy for increasing brain ABCA1 expression/activity for relevant mechanistic studies. [Corrected]

Clinical, Biochemical, and Molecular Characterization of Novel Mutations in ABCA1 in Families with Tangier Disease.

Tangier disease is a rare, autosomal recessive disorder caused by mutations in the ABCA1 gene and is characterized by near absence of plasma high-density lipoprotein cholesterol, accumulation of cholesterol in multiple tissues, peripheral neuropathy, and accelerated atherosclerosis. Here we report three new kindreds with Tangier disease harboring both known and novel mutations in ABCA1. One patient was identified to be homozygous for a nonsense mutation, p.Gln1038*. In a remarkably large Tangier disease pedigree with four affected siblings, we identified compound heterozygosity for previously reported missense variants, p.Arg937Val and p.Thr940Met, and show that both of these mutations result in significantly impaired cholesterol efflux in transfected cells. In a third pedigree, the proband was identified to be compound heterozygous for two novel mutations, a frameshift (p.Ile1200Hisfs*4) and an intronic variant (c.4176-11T>G), that lead to the creation of a cryptic splice site acceptor and premature truncation, p.Ser1392Argfs*6. We demonstrate that this mutation arose de novo, the first demonstration of a pathogenic de novo mutation in ABCA1 associated with Tangier disease. We also report results of glucose tolerance testing in a Tangier disease kindred for the first time, showing a gene-dose relationship between ABCA1 activity and glucose tolerance and suggesting that Tangier disease patients may have substantially impaired islet function. Our findings provide insight into the diverse phenotypic manifestations of this rare disorder, expand the list of pathogenic mutations in ABCA1, and increase our understanding of how specific mutations in this gene lead to abnormal cellular and physiological phenotypes.

ABCA1 in adipocytes regulates adipose tissue lipid content, glucose tolerance, and insulin sensitivity.

Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1(-ad/-ad)). When fed a high-fat, high-cholesterol diet, ABCA1(-ad/-ad) mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1(-ad/-ad) mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis.

Validation of automatic CPAP leak algorithm.

Obstructive Sleep Apnoea (OSA) is a prevalent disease. Reliable estimation of respiratory parameters by devices used to treat OSA is important for therapy initiation and maintenance. This is achieved by estimating patient flow in the presence of inadvertent leak from the total flow measured by the device. A method of validating the patient flow estimation of an Automatic Continuous Positive Airway Pressure (APAP) device is described. Novel techniques of a multi-composite simulant head and recorded patient flows are used. The APAP device tested was shown to reliably estimate patient flow across a range of therapy pressures, leak conditions and breath types.

Regulation of ABCA1 protein expression and function in hepatic and pancreatic islet cells by miR-145.

OBJECTIVE: The ATP-binding cassette transporter A1 (ABCA1) protein maintains cellular cholesterol homeostasis in several different tissues. In the liver, ABCA1 is crucial for high-density lipoprotein biogenesis, and in the pancreas ABCA1 can regulate insulin secretion. In this study, our aim was to identify novel microRNAs that regulate ABCA1 expression in these tissues. APPROACH AND RESULTS: We combined multiple microRNA prediction programs to identify 8 microRNAs that potentially regulate ABCA1. A luciferase reporter assay demonstrated that 5 of these microRNAs (miR-148, miR-27, miR-144, miR-145, and miR-33a/33b) significantly repressed ABCA1 3'-untranslated region activity with miR-145 resulting in one of the larger decreases. In hepatic HepG2 cells, miR-145 can regulate both ABCA1 protein expression levels and cholesterol efflux function. In murine islets, an increase in miR-145 expression decreased ABCA1 protein expression, increased total islet cholesterol levels, and decreased glucose-stimulated insulin secretion. Inhibiting miR-145 produced the opposite effect of increasing ABCA1 protein levels and improving glucose-stimulated insulin secretion. Finally, increased glucose levels in media significantly decreased miR-145 levels in cultured pancreatic beta cells. These findings suggest that miR-145 is involved in glucose homeostasis and is regulated by glucose concentration. CONCLUSIONS: Our studies demonstrate that miR-145 regulates ABCA1 expression and function, and inhibiting this microRNA represents a novel strategy for increasing ABCA1 expression, promoting high-density lipoprotein biogenesis in the liver, and improving glucose-stimulated insulin secretion in islets.