AAV cis-regulatory sequences are correlated with ocular toxicity.

Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1ß were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.

Hepatitis B virus evades innate immunity of hepatocytes but activates cytokine production by macrophages.

Hepatitis B virus (HBV) infects hepatocytes specifically and causes immune-mediated liver damage. How HBV interacts with the innate immunity at the early phase of infection, either with hepatocytes or other cells in the liver, remains controversial. To address this question, we utilized various human cell-culture models and humanized Alb-uPA/SCID mice. All these models were unable to mount an interferon (IFN) response despite robust HBV replication. To elucidate the mechanisms involved in the lack of IFN response, we examined whether HBV actively inhibits innate immune functions of hepatocytes. By treating HBV-infected cells with known inducers of the IFN signaling pathway, we observed no alteration of either sensing or downstream IFN response by HBV. We showed that the DNA innate sensing pathways are poorly active in hepatocytes, consistent with muted innate immune recognition of HBV. Upon exposure to high-level HBV, human macrophages could be activated with increased inflammatory cytokine expressions. CONCLUSION: HBV behaves like a "stealth" virus and is not sensed by, nor actively interferes with, the intrinsic innate immunity of infected hepatocytes. Macrophages are capable of sensing HBV, but require exposure to high HBV titers, potentially explaining the long "window period" during acute infection and HBV's propensity to chronic infection. (Hepatology 2017;66:1779-1793).

The Association of Patient-centered Medical Home Designation With Quality of Care of HRSA-funded Health Centers: A Longitudinal Analysis of 2012-2015.

OBJECTIVES: (1) To evaluate the relationship between Patient-centered Medical Home (PCMH) recognition and quality of clinical care among health centers, and (2) to determine whether the duration of recognition is positively associated with cumulative quality improvement over time. METHODS: Data came from the 2012 to 2015 Uniform Data System, health centers' PCMH recognition status, and the Area Resource File. Health center was the unit of observation. The outcome variables included 11 measures of clinical quality. We pooled all years of data and modeled longitudinal data with generalized estimating equations to examine the degree of improvement in health care quality in health centers with and without PCMH recognition over the years 2012-2015. RESULTS: Health centers with PCMH recognition generally performed better on clinical quality measures than health centers that did not have PCMH recognition for all years studied. After accounting for health center and county-level potential confounders, health centers with longer periods of PCMH recognition were more likely to have improved their clinical quality on 9 of 11 measures, than health centers with fewer years of PCMH recognition. CONCLUSIONS: Health centers' length of time with PCMH recognition was positively associated with additive quality improvement. Adoption of the PCMH model of care may serve as a strategy to enhance quality of primary care services.

Should Renal Mass Biopsy Be Performed prior to or Concomitantly with Thermal Ablation?

PURPOSE: To determine diagnostic yield of renal biopsies performed in patients referred for image-guided tumor ablation (IGTA) and the frequency with which biopsy results would have obviated the need for subsequent ablation. MATERIALS AND METHODS: Retrospective review of an internal ablation database of a single institution revealed 401 consecutive percutaneous renal mass IGTAs performed from April 2000 to April 2015. Of 401 ablations, 32 were excluded, yielding 369 ablation events in 342 patients, which represented the study cohort. Patients were subdivided into groups according to whether or not biopsy was performed. Lesions were categorized according to size, malignancy/benignity, and pathology. RESULTS: IGTA was performed with biopsy for 317/369 (85.9%) and without biopsy for 52/369 (14.1%) lesions. Overall diagnostic yield for percutaneous biopsy was 94.3% (299/317). Based on biopsy results, 82.6% (262/317) were classified as malignant or suspicious, 9.5% (30/317) were classified as likely benign, and 2.2% (7/317) were classified as definitively benign. Only definitively benign lesions were designated as obviating the need for IGTA. IGTA was supported by biopsy results in the remaining 97.8% (310/317), including renal cell carcinomas, oncocytic neoplasms, metastases, and nondiagnostic biopsy results. CONCLUSIONS: Biopsy of renal masses with suspicious imaging features rarely (2.2%) obviated the need for IGTA. For patients who have undergone counseling and have elected to forgo active surveillance and surgical options, biopsy can safely be performed concomitantly with ablation.

Urine clusterin/apolipoprotein J is linked to tubular damage and renal outcomes in patients with type 2 diabetes mellitus.

OBJECTIVE: The objective of this study was to evaluate the association of urine clusterin/apolipoprotein J (Apo J) with the development and/or progression of diabetic kidney disease (DKD) in type 2 diabetes. MATERIALS AND METHODS: A total of 159 type 2 diabetic patients and 20 nondiabetic subjects with estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m2 were enrolled. The baseline values of urine clusterin and tubular damage markers were measured. The primary outcome was the annual decline rate in eGFR, and secondary outcomes were the development of chronic kidney disease (CKD) stage 3 or greater and the persistence/progression of albuminuria. The median follow-up duration of enrolled patients was 3.0 (1.0-5.9) years. RESULTS: Baseline clusterin levels in urine were significantly increased in type 2 diabetic subjects compared with those of nondiabetic subjects. The levels of urine clusterin had a significant correlation with urine tubular damage markers. A positive correlation between the annual rate of decline in eGFR and urine clusterin after adjusting for clinical confounding factors was detected. Multivariate analysis further indicated that urine clusterin correlated with the development of CKD stage 3 or greater and persistence/progression of albuminuria. In type 2 diabetic subjects with albuminuria, urine clusterin remained associated with the annual decline rate in eGFR and the progression of CKD stage. CONCLUSIONS: Urine clusterin reflects tubular damage in the early stage of DKD. The increase in urine clusterin along with albuminuria could be an independent predictive marker for the progression of DKD in type 2 diabetes.

Glutamine-driven oxidative phosphorylation is a major ATP source in transformed mammalian cells in both normoxia and hypoxia.

Mammalian cells can generate ATP via glycolysis or mitochondrial respiration. Oncogene activation and hypoxia promote glycolysis and lactate secretion. The significance of these metabolic changes to ATP production remains however ill defined. Here, we integrate LC-MS-based isotope tracer studies with oxygen uptake measurements in a quantitative redox-balanced metabolic flux model of mammalian cellular metabolism. We then apply this approach to assess the impact of Ras and Akt activation and hypoxia on energy metabolism. Both oncogene activation and hypoxia induce roughly a twofold increase in glycolytic flux. Ras activation and hypoxia also strongly decrease glucose oxidation. Oxidative phosphorylation, powered substantially by glutamine-driven TCA turning, however, persists and accounts for the majority of ATP production. Consistent with this, in all cases, pharmacological inhibition of oxidative phosphorylation markedly reduces energy charge, and glutamine but not glucose removal markedly lowers oxygen uptake. Thus, glutamine-driven oxidative phosphorylation is a major means of ATP production even in hypoxic cancer cells.

Probably benign breast MRI lesions: frequency, lesion type, and rate of malignancy.

PURPOSE: To determine the frequency, malignancy rate, and imaging characteristics of BI-RADS (Breast Imaging Reporting and Data System) 3 MRI detected lesions. MATERIALS AND METHODS: A total of 4370 consecutive contrast-enhanced breast MRI exams from March 1, 2004, to March 1, 2009, were retrospectively reviewed. The study was HIPAA (Health Insurance Portability Accountability Act) compliant and Institutional Review Board approved. The frequency of BI-RADS 3 MRI interpretations and lesion type, as well as malignancy rate at follow-up were calculated. RESULTS: There were 348/4370 (8%) BI-RADS 3 exams performed on 345 patients. There were 125 foci (35.9%), 52 non-masslike enhancements (14.9%), and 171 masses (49.1%). There were 207 lesions with biopsy or imaging follow-up of at least 24 months (mean, 32 months; range, 6-77 months) of the BI-RADS 3 lesion. Five of these 207 (2.4%) lesions were found to be malignant. CONCLUSION: Rate of malignancy among BI-RADS 3 MRI exams with follow-up was 2.4%. Types of lesions assigned as probably benign were 35.9% foci, 14.9% non-masslike enhancements, and 49.1% masses.

Formative Perceptions of a Digital Pill System to Measure Adherence to Heart Failure Pharmacotherapy: Mixed Methods Study.

BACKGROUND: Heart failure (HF) affects 6.2 million Americans and is a leading cause of hospitalization. The mainstay of the management of HF is adherence to pharmacotherapy. Despite the effectiveness of HF pharmacotherapy, effectiveness is closely linked to adherence. Measuring adherence to HF pharmacotherapy is difficult; most clinical measures use indirect strategies such as calculating pharmacy refill data or using self-report. While helpful in guiding treatment adjustments, indirect measures of adherence may miss the detection of suboptimal adherence and co-occurring structural barriers associated with nonadherence. Digital pill systems (DPSs), which use an ingestible radiofrequency emitter to directly measure medication ingestions in real-time, represent a strategy for measuring and responding to nonadherence in the context of HF pharmacotherapy. Previous work has demonstrated the feasibility of using DPSs to measure adherence in other chronic diseases, but this strategy has yet to be leveraged for individuals with HF. OBJECTIVE: We aim to explore through qualitative interviews the facilitators and barriers to using DPS technology to monitor pharmacotherapy adherence among patients with HF. METHODS: We conducted individual, semistructured qualitative interviews and quantitative assessments between April and August 2022. A total of 20 patients with HF who were admitted to the general medical or cardiology service at an urban quaternary care hospital participated in this study. Participants completed a qualitative interview exploring the overall acceptability of and willingness to use DPS technology for adherence monitoring and perceived barriers to DPS use. Quantitative assessments evaluated HF history, existing medication adherence strategies, and attitudes toward technology. We analyzed qualitative data using applied thematic analysis and NVivo software (QSR International). RESULTS: Most participants (12/20, 60%) in qualitative interviews reported a willingness to use the DPS to measure HF medication adherence. Overall, the DPS was viewed as useful for increasing accountability and reinforcing adherence behaviors. Perceived barriers included technological issues, a lack of need, additional costs, and privacy concerns. Most were open to sharing adherence data with providers to bolster clinical care and decision-making. Reminder messages following detected nonadherence were perceived as a key feature, and customization was desired. Suggested improvements are primarily related to the design and usability of the Reader (a wearable device). CONCLUSIONS: Overall, individuals with HF perceived the DPS to be an acceptable and useful tool for measuring medication adherence. Accurate, real-time ingestion data can guide adherence counseling to optimize adherence management and inform tailored behavioral interventions to support adherence among patients with HF.

Prolonged and Recurrent Intrahepatic Cholestasis of Pregnancy.

Intrahepatic cholestasis of pregnancy is one of the most common disorders of pregnancy, which typically resolves in the postpartum period. Intrahepatic cholestasis is characterized by elevated bile acid levels that present as pruritus. The maternal clinical significance of recurrent and prolonged cholestasis is unknown. We discuss the longest reported case of postpartum cholestasis of 125 weeks.

Utility of targeted sonography in management of probably benign breast lesions identified on magnetic resonance imaging.

OBJECTIVES: The purpose of this study was to determine the utility of targeted sonography in the management of probably benign breast lesions detected on magnetic resonance imaging (MRI). METHODS: A total of 4370 consecutive contrast-enhanced breast MRI examinations from March 1, 2004, to March 1, 2009, were retrospectively reviewed. The study was Health Insurance Portability and Accountability Act compliant and Institutional Review Board approved. When targeted sonography was recommended for a Breast Imaging Reporting and Data System (BI-RADS) category 3 examination, results of the sonography and any subsequent breast pathologic examinations were recorded. The frequency of identifying the MRI-detected lesions and the rate at which the BI-RADS category was changed by sonography were calculated for mass and non-mass-like lesions. RESULTS: Of the 4370 examinations, 349 (8%) had BI-RADS 3 findings in 346 patients. One hundred eighteen lesions underwent targeted sonography for evaluation of 85 masses and 33 areas of non-mass-like enhancement. Of these 118 lesions, 54 (46%) were seen on sonography. No cancers were detected on sonography in the areas of non-mass-like enhancement. Two of the 85 masses (2.4%) evaluated with targeted sonography had a malignant diagnosis before initiation of follow-up. CONCLUSIONS: Selective use of targeted sonography, particularly in masses, may help identify some malignancies before initiating short-interval follow-up for MRI-detected BI-RADS 3 lesions.

Engagement in Trauma-Specific CBT for Youth Post-9/11.

Treatment participation was examined among youth enrolled in an evaluation of cognitive behavioral therapy (CBT) for trauma following the 9/11 World Trade Center disaster. Staff at nine agencies serving a predominantly low-income ethnically diverse population were trained to deliver CBT and structured engagement strategies. Four hundred and forty-five youth ages 5-19 were eligible for CBT, and 417 (94%) received at least one treatment session. Pretreatment and treatment show rates and overall dose were examined. Treatment participation rates were higher than those typically reported in community studies of children's mental health services. Regression analyses indicated variability across sites in treatment show rates with the highest rates at where services were delivered in schools. However, sites, demographic factors and trauma symptoms accounted for a small amount of variance in treatment participation overall. The study suggests structured engagement strategies, linked to evidence-based treatments may improve treatment participation for youth.

Refinement of an Emergency Department-Based, Advance Care Planning Intervention for Nurses.

Background: Most older adults visit the emergency department (ED) near the end of life without advance care planning (ACP) and thus are at risk of receiving care that does not align with their wishes and values. ED GOAL is a behavioral intervention administered by ED clinicians, which is designed to engage seriously ill older adults in serious illness conversations in the ED. Seriously ill older adults found it acceptable in the ED. However, its potential to be used by nurses remains unclear. Objective: The aim of this study is to identify refinements to adapt an ED-based ACP intervention by eliciting the perspectives of nurses. Design: This is a qualitative study using semistructured interviews. Data were analyzed using axial coding methods. Setting/Subjects: We recruited a purposeful sample of ED nurses in one urban academic ED and one urban community ED in the northeastern region of the United States. Results: Twenty-five nurses were interviewed (mean age 46 years, 84% female, and mean clinical experience of 16 years). Emerging themes were identified within six domains: (1) nurses' prior experience with serious illness conversations, (2) overall impression of ED GOAL, (3) refinements to ED GOAL, (4) implementation of ED GOAL by ED nurses, (5) specially trained nursing model, and (6) use of telehealth with ED GOAL. Conclusions: ED nurses were generally supportive of using ED GOAL and provided insight into how to best adapt and implement it in their clinical practice. Empirical evidence for adapting ED GOAL to the nursing practice remains to be seen.

Nrf2 overexpression rescues the RPE in mouse models of retinitis pigmentosa.

Nrf2, a transcription factor that regulates the response to oxidative stress, has been shown to rescue cone photoreceptors and slow vision loss in mouse models of retinal degeneration (rd). The retinal pigment epithelium (RPE) is damaged in these models, but whether it also could be rescued by Nrf2 has not been previously examined. We used an adeno-associated virus (AAV) with an RPE-specific (Best1) promoter to overexpress Nrf2 in the RPE of rd mice. Control rd mice showed disruption of the regular array of the RPE, as well as loss of RPE cells. Cones were lost in circumscribed regions within the cone photoreceptor layer. Overexpression of Nrf2 specifically in the RPE was sufficient to rescue the RPE, as well as the disruptions in the cone photoreceptor layer. Electron microscopy showed compromised apical microvilli in control rd mice but showed preserved microvilli in Best1-Nrf2-treated mice. The rd mice treated with Best1-Nrf2 had slightly better visual acuity. Transcriptome profiling showed that Nrf2 upregulates multiple oxidative defense pathways, reversing declines seen in the glutathione pathway in control rd mice. In summary, Nrf2 overexpression in the RPE preserves RPE morphology and survival in rd mice, and it is a potential therapeutic for diseases involving RPE degeneration, including age-related macular degeneration (AMD).

Opioid prescribing trends in a Veterans Affairs emergency department and dental clinic before and after implementation of opioid-prescribing guidelines.

INTRODUCTION: Opioid overdose is highly prevalent among veterans. The Opioid Safety Initiative (OSI) and Centers for Disease Control and Prevention (CDC) issued prescribing guidelines for managing chronic pain. The purpose of this study was to investigate the impact of the 2013 OSI and 2016 CDC guidelines on opioid-prescribing trends in the emergency department and dental clinic within the Veterans Affairs Salt Lake City Health Care System. METHODS: In this retrospective, cohort study, opioid prescriptions were queried from January 1, 2013, through March 31, 2017, and separated into 3 groups: pre-OSI, post-OSI, and post-CDC. The primary outcome was to determine a decrease in opioid prescribing. Secondary outcomes included changes in concurrent benzodiazepine and naloxone prescriptions and prescriber status. Analysis of variance was used to determine a difference between study periods. RESULTS: There were 7339 opioid prescriptions identified. A statistically significant difference was found between the 3 groups in average number of opioids prescribed, morphine milligram equivalents per prescription, days' supplied, and medication quantity per prescription (P < .01). There was no significant difference between the 3 groups regarding morphine milligram equivalents per day (P = .24). Benzodiazepine prescribing remained the same. Concurrent naloxone prescriptions increased. DISCUSSION: The results demonstrate that days' supply, quantity, and morphine milligram equivalent per day in the post-CDC group were consistent with guideline recommendations. Concurrent naloxone prescribing increased throughout all time periods. Implementation of guidelines impacted opioid-prescribing trends, ultimately lessening potential for misuse and abuse. However, there is still need for improvement with reducing concurrent benzodiazepine prescriptions.

Structural and biochemical analysis of phosphoethanolamine methyltransferase from the pine wilt nematode Bursaphelenchus xylophilus.

In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the ß1d-α6 loop (W123F, M126I, and Y127F) and ß1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.

Child Life Specialists' Experiences Addressing Social Determinants of Health: A Web-Based Survey.

We used Google Forms to survey 110 child life specialists on their experiences addressing family psychosocial issues. Most respondents were female (99%) and white (95%), with mean age 34 ± 10 years. Ninety-five percent reported addressing family psychosocial issues during the previous 3 months, including parental separation/divorce (71%), poverty/financial needs (64%), parental mental illness (59%), substance abuse at home (54%), homelessness/housing problems (54%), bullying (49%), physical neglect (46%), physical abuse (46%), unemployment (46%), emotional neglect (45%), and hunger/food insecurity (42%). Eighty-five percent of respondents reported addressing family psychosocial issues once per month or more often, with 80% providing coping strategies, 76% providing family support, 66% providing therapeutic play, and 66% providing psychological preparation. These findings indicate that child life specialists frequently address a range of family psychosocial issues. Further research is needed to clarify the role and impact of child life services on social determinants of health.

Role of POMC and AgRP neuronal activities on glycaemia in mice.

Leptin regulates both feeding and glycaemia primarily through its receptors expressed on agouti-related peptide (AgRP) and pro-opiomelanocortin-expressing (POMC) neurons; however, it is unknown whether activity of these neuronal populations mediates the regulation of these processes. To determine this, we injected Cre-dependent designer receptors exclusively activated by designer drugs (DREADD) viruses into the hypothalamus of normoglycaemic and diabetic AgRP-ires-cre and POMC-cre mice to chemogenetically activate or inhibit these neuronal populations. Despite robust changes in food intake, activation or inhibition of AgRP neurons did not affect glycaemia, while activation caused significant (P = 0.014) impairment in insulin sensitivity. Stimulation of AgRP neurons in diabetic mice reversed leptin's ability to inhibit feeding but did not counter leptin's ability to lower blood glucose levels. Notably, the inhibition of POMC neurons stimulated feeding while decreasing glucose levels in normoglycaemic mice. The findings suggest that leptin's effects on feeding by AgRP neurons are mediated by changes in neuronal firing, while the control of glucose balance by these cells is independent of chemogenetic activation or inhibition. The firing-dependent glucose lowering mechanism within POMC neurons is a potential target for the development of novel anti-diabetic medicines.

Patient-Centered Medical Home Recognition and Clinical Performance in U.S. Community Health Centers.

INTRODUCTION: America's community health centers (HCs) are uniquely poised to implement the patient-centered medical home (PCMH) model, as they are effective in providing comprehensive, accessible, and continuous primary care. This study aims to evaluate the relationship between PCMH recognition in HCs and clinical performance. METHODS: Data for this study came from the 2012 Uniform Data System (UDS) as well as a survey of HCs' PCMH recognition achievement. The dependent variables included all 16 measures of clinical performance collected through UDS. Control measures included HC patient, provider, and practice characteristics. Bivariate analyses and multiple logistic regressions were conducted to compare clinical performance between HCs with and without PCMH recognition. FINDINGS: Health centers that receive PCMH recognition generally performed better on clinical measures than HCs without PCMH recognition. After controlling for HC patient, provider, and practice characteristics, HCs with PCMH recognition reported significantly better performance on asthma-related pharmacologic therapy, diabetes control, pap testing, prenatal care, and tobacco cessation intervention. CONCLUSION: This study establishes a positive association between PCMH recognition and clinical performance in HCs. If borne out in future longitudinal studies, policy makers and practices should advance the PCMH model as a strategy to further enhance the quality of primary care.