A one-shot double-slice selection NMR method for biphasic systems.

We propose a new and robust one-shot double-slice selection experiment to detect 1H NMR signals of biphasic systems simultaneously. The resultant spectrum contains opposite-phased peaks representing the chemical species from the two phases, respectively.

Interleukin 2 Promotes Hepatic Regulatory T Cell Responses and Protects From Biliary Fibrosis in Murine Sclerosing Cholangitis.

In the multidrug resistance protein 2 (Mdr2)-/- mouse model, low phospholipid bile instigates biliary epithelial injury, sterile inflammation, and fibrosis, thereby recapitulating disease mechanisms implicated in biliary atresia (BA) and primary sclerosing cholangitis. We hypothesize that T lymphocytes contribute to the biliary injury and fibrosis in murine sclerosing cholangitis (SC) and that they are susceptible to suppression by regulatory T cells (Tregs). In juvenile Mdr2-/- mice, intrahepatic CD8+ lymphocytes were expanded, and contraction of intrahepatic Tregs coincided with rising serum alanine transferase and alkaline phosphatase (ALP) levels between days 14-30 of life. Antibody-mediated depletion of intrahepatic CD8+ lymphocytes during that time reduced ALP levels and the expression of osteopontin (Opn), a pro-fibrogenic cytokine. Depletion of intrahepatic Tregs with anti-CD25 antibody between days 7-30 increased intrahepatic CD8+ T cells, Opn expression, and fibrosis. Conversely, expansion of intrahepatic Tregs with interleukin 2/anti-interleukin 2 immune complexes (IL-2c) downregulated hepatic expression of Opn and Tnf, reduced frequency of intrahepatic CD8+ lymphocytes, and diminished biliary injury and fibrosis. Treatment with IL-2c upregulated hepatic Treg expression of CD39, an ectonucleotidase capable of hydrolyzing pro-inflammatory adenosine triphosphate. In vitro, Tregs expressing CD39 suppressed the proliferation of hepatic CD8+ lymphocytes from Mdr2-/- mice more efficiently than those lacking CD39. In infants with BA, infiltration of interlobular bile ducts with CD8+ cells was associated with biliary expression of Opn and its transcription was negatively correlated with mRNA expression of Treg-associated genes. Conclusion: Hepatic CD8+ T lymphocytes drive biliary injury and fibrosis in murine SC. Their proliferation is controlled by hepatic Tregs through the purinergic pathway, which is responsive to IL-2c, suggesting that Treg-directed low-dose Il-2 treatment may be considered as therapy for SC.

The dendritic cell-T helper 17-macrophage axis controls cholangiocyte injury and disease progression in murine and human biliary atresia.

Biliary atresia (BA) is a fibroinflammatory obstruction of the extrahepatic biliary tree in neonates. While intrahepatic bile duct proliferation is universal at diagnosis, bile duct paucity develops later. We hypothesized that polarized T helper lymphocyte responses orchestrate progression of intrahepatic biliary injury in this disease. Interleukin 17A (IL-17A)-green fluorescent protein, cluster of differentiation 11c (CD11c)/diphtheria toxin receptor, and IL-17 receptor A-/- mice were used to examine T-lymphocyte polarization, inflammatory leukocyte recruitment, and biliary injury in rhesus rotavirus-induced BA. Multiparameter flow cytometry and automated image analysis of immunostaining were applied to liver tissue samples from infants with BA. In the mouse model, activated CD4+ lymphocytes started to emerge in the liver on day 8 after viral challenge, while innate immune responses were waning. Plasma IL-17A levels rose concomitantly with hepatic accumulation of T helper 17 lymphocytes and myeloid dendritic cells. Targeted depletion of CD11c+ dendritic cells diminished hepatic IL-17A production and ameliorated intrahepatic bile duct injury. Recombinant IL-17A induced expression of chemokine (C-C motif) ligand 2 in neonatal cholangiocytes in vitro, and blockade of the corresponding chemokine (C-C motif) receptor 2 reduced recruitment of inflammatory macrophages to the liver in vivo. Genetic disruption of IL-17A signaling was associated with down-regulation of hepatic Ccl2/Ccr2 messenger RNA expression, reduced infiltration of the liver with inflammatory Ly6Chi macrophages, and improved survival. In the liver of infants with BA, cholangiocytes were found to express IL-17 receptor A, and the prevalence of IL-17A+ cells was positively correlated with the degree of CD68+ macrophage infiltration at diagnosis. Hepatic CD4+ lymphocytes were chief producers of IL-17A in patients with progressive disease undergoing liver transplantation. CONCLUSION: These findings identify the dendritic cell-T helper 17-macrophage axis as a target for the development of strategies to block progression of intrahepatic bile duct injury in patients with BA. (Hepatology 2017;65:174-188).

Pharmacological inhibition of apical sodium-dependent bile acid transporter changes bile composition and blocks progression of sclerosing cholangitis in multidrug resistance 2 knockout mice.

UNLABELLED: Deficiency of multidrug resistance 2 (mdr2), a canalicular phospholipid floppase, leads to excretion of low-phospholipid "toxic" bile causing progressive cholestasis. We hypothesize that pharmacological inhibition of the ileal, apical sodium-dependent bile acid transporter (ASBT), blocks progression of sclerosing cholangitis in mdr2(-/-) mice. Thirty-day-old, female mdr2(-/-) mice were fed high-fat chow containing 0.006% SC-435, a minimally absorbed, potent inhibitor of ASBT, providing, on average, 11 mg/kg/day of compound. Bile acids (BAs) and phospholipids were measured by mass spectrometry. Compared with untreated mdr2(-/-) mice, SC-435 treatment for 14 days increased fecal BA excretion by 8-fold, lowered total BA concentration in liver by 65%, reduced total BA and individual hydrophobic BA concentrations in serum by >98%, and decreased plasma alanine aminotransferase, total bilirubin, and serum alkaline phosphatase levels by 86%, 93%, and 55%, respectively. Liver histology of sclerosing cholangitis improved, and extent of fibrosis decreased concomitant with reduction of hepatic profibrogenic gene expression. Biliary BA concentrations significantly decreased and phospholipids remained low and unchanged with treatment. The phosphatidylcholine (PC)/BA ratio in treated mice corrected toward a ratio of 0.28 found in wild-type mice, indicating decreased bile toxicity. Hepatic RNA sequencing studies revealed up-regulation of putative anti-inflammatory and antifibrogenic genes, including Ppara and Igf1, and down-regulation of several proinflammatory genes, including Ccl2 and Lcn2, implicated in leukocyte recruitment. Flow cytometric analysis revealed significant reduction of frequencies of hepatic CD11b(+) F4/80(+) Kupffer cells and CD11b(+) Gr1(+) neutrophils, accompanied by expansion of anti-inflammatory Ly6C(-) monocytes in treated mdr2(-/-) mice. CONCLUSION: Inhibition of ASBT reduces BA pool size and retention of hydrophobic BA, favorably alters the biliary PC/BA ratio, profoundly changes the hepatic transcriptome, attenuates recruitment of leukocytes, and abrogates progression of murine sclerosing cholangitis.

Hepatic MDR3 expression impacts lipid homeostasis and susceptibility to inflammatory bile duct obstruction in neonates.

BackgroundHeterozygous mutations in the gene ABCB4, encoding the phospholipid floppase MDR3 (Mdr2 in mice), are associated with various chronic liver diseases. Here we hypothesize that reduced ABCB4 expression predisposes to extrahepatic biliary atresia (EHBA).MethodsLivers from neonatal wild-type (wt) and heterozygous Mdr2-deficient mice were subjected to mass spectrometry-based lipidomics and RNA sequencing studies. Following postnatal infection with rhesus rotavirus (RRV), liver immune responses and EHBA phenotype were assessed. Hepatic microarray data from 40 infants with EHBA were mined for expression levels of ABCB4.ResultsPhosphatidylcholine (PC) and phosphatidylethanolamine (PE) were increased, whereas the PC/PE ratio was decreased in neonatal Mdr2+/- mice compared with wt mice. Following RRV challenge, hepatic expression of IFNγ and infiltration with CD8+ and NK+ lymphocytes were increased in Mdr2+/- mice. Plasma total bilirubin levels and prevalence of complete ductal obstruction were higher in these mice. In infants with EHBA, hepatic gene expression of ABCB4 was downregulated in those with an inflammatory compared with a fibrosing molecular phenotype.ConclusionDecreased expression of ABCB4 causes dysregulation in (phospho)lipid homeostasis, and predisposes to aberrant pro-inflammatory lymphocyte responses and an aggravated phenotype of EHBA in neonatal mice. Downregulated ABCB4 is associated with an inflammatory transcriptome signature in infants with EHBA.

Regulatory T cells control the CD8 adaptive immune response at the time of ductal obstruction in experimental biliary atresia.

UNLABELLED: CD8 T-lymphocytes are effector cells of cholangiocyte injury in human and in rhesus rotavirus (RRV)-induced experimental biliary atresia (BA). Here we hypothesize that neonatal deficiency in CD25(+) CD4(+) regulatory T cells (Tregs) leads to aberrant activation of hepatic T-lymphocytes in BA. We found that adoptive transfer of total CD4 cells, but not of CD25-depleted CD4 cells, prior to RRV inoculation reduced expansion of CD8 cells, plasma bilirubin levels, ductal inflammation, and bile duct epithelial injury at 7 days postinfection (dpi) compared with age-matched infected controls without adoptive transfer. Searching for mechanisms, we found that in vitro production of interferon-gamma (IFN-γ) by naïve CD8 cells upon polyclonal stimulation was enhanced in coculture with hepatic dendritic cells (DCs) from RRV-infected, but not with DCs from noninfected mice, which was correlated with an increased proportion of CD11b(+) myeloid (m)DCs and up-regulation of the costimulatory molecule CD86 on RRV-primed DCs. Furthermore, DC-dependent T-lymphocyte activation was blocked by anti-CD86 antibody in dose-dependent fashion. Importantly, expression of CD86 on mDCs was down-regulated by Tregs in vitro, and adoptive transfer of Treg-containing CD4 cells decreased expression of CD86 on hepatic mDCs at 7 dpi. On the contrary, in mice resistant to experimental BA, CD25+ cell depletion aggravated bile duct injury at 12 dpi after RRV inoculation, as plasma bilirubin levels were elevated by >20-fold compared with nondepleted infected controls. Increased susceptibility to hepatobiliary injury in Treg-depleted mice was linked to hepatic CD8 expansion and enhanced stimulatory capacity of hepatic DCs. CONCLUSION: Activation of hepatic T-lymphocytes driving biliary obstruction in BA is regulated by mDCs by way of CD86-dependent costimulation and is susceptible to inhibition by Tregs.

sTREM-1 and LBP in central venous catheter-associated bloodstream infections in pediatric intestinal failure.

OBJECTIVE: Central venous catheter-associated bloodstream infections (CVC-BSIs) are a major cause of morbidity and mortality in the pediatric intestinal failure (IF) population. We assessed plasma lipopolysaccharide-binding protein (LBP) and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as biomarkers for CVC-BSI. We hypothesized that sTREM-1 and LBP rise with BSI and decline following treatment, and that baseline LBP is higher in the IF population than in controls. PATIENTS AND METHODS: Patients younger than 4 years were recruited from the IF registry at Cincinnati Children's Hospital. LBP and sTREM-1 levels were measured on 22 patients with IF at baseline, 17 patients with IF with BSIs, and 11 healthy controls. RESULTS: Mean sTREM-1 level (pg/mL) and LBP level (µg/mL) rose with CVC-BSI over baseline (115.0 ±â€Š51.2 vs 85.9 ±â€Š27.6, P = 0.011 and 79.8 ±â€Š45.4 vs 20.5 ±â€Š11.3, P < 0.001, respectively) and declined following antibiotic therapy (115.0 ±â€Š51.2 vs 77.9 ±â€Š29.8, P = 0.003 and 79.8 ±â€Š45.4 vs 26.2 ±â€Š10.8, P < 0.001, respectively). Receiver operating characteristic curves showed that neither sTREM-1 nor LBP is sufficient to predict bacteremia versus fever without bacteremia (area under these curves = 0.57 and 0.82, respectively). Baseline LBP was higher in hospitalized patients than in outpatients (27.5 ±â€Š8.7 vs 13.5 ±â€Š9.2, P = 0.002), patients with previous BSIs versus those without (23.5 ±â€Š10.4 vs 10.1 ±â€Š8.3, P = 0.016), and those listed for transplantation versus those not listed (29.6 ±â€Š9.8 vs 16.2 ±â€Š9.5, P = 0.033). CONCLUSIONS: sTREM-1 and LBP rise with CVC-BSI in IF and decline after treatment; however, neither distinguishes infection from nonbacteremic febrile episodes. Baseline LBP may be a marker of disease severity in IF.

Improving Outpatient Provider Communication for High-Risk Discharges From the Hospitalist Service.

BACKGROUND: Patients are at risk for adverse events during inpatient-to-outpatient transitions of care. Previous improvement work has been targeted at this care transition, but gaps in discharge communication still exist. We aimed to increase documentation of 2-way communication between hospitalists and primary care providers (PCPs) for high-risk discharges from pediatric hospital medicine (PHM) services from 7% to 60% within 30 months. METHODS: A3 improvement methodology was used. A list of high-risk discharge communication criteria was developed through engagement of PCPs and hospitalists. A driver diagram guided interventions. The outcome measure was documentation of successful 2-way communication with the PCP. Any documented 2-way discharge communication attempt was the process measure. Via a survey, hospitalist satisfaction with the discharge communication expectation served as the balancing measure. All patients discharged from PHM services meeting ≥1 high-risk criterion were included. Statistical process control charts were used to assess changes over time. RESULTS: There were 3241 high-risk discharges (442 baseline: November 2017 to January 2018; 2799 intervention and sustain: February 2018 to June 2020). The outcome measure displayed iterative special cause variation from a mean baseline of 7% to peak of 39% but regressed and was sustained at 27%. The process measure displayed iterative special cause variation from a 13% baseline mean to a 64% peak, with regression to 41%. The balancing measure worsened from baseline of 5% dissatisfaction to 13%. Interventions temporally related to special cause improvements were education, division-level performance feedback, standardization of documentation, and offloading the task of communication coordination from hospitalists to support staff. CONCLUSIONS: Improvement methodology resulted in modestly sustained improvements in PCP communication for high-risk discharges from the PHM services.

Readmissions Following Hospitalization for Infection in Children With or Without Medical Complexity.

OBJECTIVE: To describe the prevalence and characteristics of infection-related readmissions in children and to identify opportunities for readmission reduction and estimate associated cost savings. STUDY DESIGN: Retrospective analysis of 380,067 nationally representative index hospitalizations for children using the 2014 Nationwide Readmissions Database. We compared 30-day, all-cause unplanned readmissions and costs across 22 infection categories. We used the Inpatient Essentials database to measure hospital-level readmission rates and to establish readmission benchmarks for individual infections. We then estimated the number of readmissions avoided and costs saved if hospitals achieved the 10th percentile of hospitals' readmission rates (ie, readmission benchmark). All analyses were stratified by the presence/absence of a complex chronic condition (CCC). RESULTS: The overall 30-day readmission rate was 4.9%. Readmission rates varied substantially across infections and by presence/absence of a CCC (CCC: range, 0%-21.6%; no CCC: range, 1.5%-8.6%). Approximately 42.6% of readmissions (n = 3,576) for children with a CCC and 54.7% of readmissions (n = 5,507) for children without a CCC could have been potentially avoided if hospitals achieved infection-specific benchmark readmission rates, which could result in an estimated savings of $70.8 million and $44.5 million, respectively. Bronchiolitis, pneumonia, and upper respiratory tract infections were among infections with the greatest number of potentially avoidable readmissions and cost savings for children with and without a CCC. CONCLUSION: Readmissions following hospitalizations for infection in children vary significantly by infection type. To improve hospital resource use for infections, future preventative measures may prioritize children with complex chronic conditions and those with specific diagnoses (eg, respiratory illnesses).

Post-natal paucity of regulatory T cells and control of NK cell activation in experimental biliary atresia.

BACKGROUND & AIMS: Although recent studies have identified important roles for T and NK cells in the pathogenesis of biliary atresia (BA), the mechanisms by which susceptibility to bile duct injury is restricted to the neonatal period are unknown. METHODS: We characterised hepatic regulatory T cells (Tregs) by flow cytometry in two groups of neonatal mice challenged with rhesus rotavirus (RRV) at day 7 (no ductal injury) or day 1 of life (resulting in BA), determined the functional interaction with effector cells in co-culture assays, and examined the effect of adoptive transfer of CD4+ cells on the BA phenotype. RESULTS: While day 7 RRV infection increased hepatic Tregs (Foxp3+ CD4+ CD25+) by 10-fold within 3 days, no increase in Tregs occurred at this time point following infection on day 1. In vitro, Tregs effectively suppressed NK cell activation by hepatic dendritic cells and decreased the production of pro-inflammatory cytokines, including TNFalpha and IL-15, following RRV infection. In vivo, adoptive transfer of CD4+ cells prior to RRV inoculation led to increased survival, improved weight gain, decreased population of hepatic NK cells, and persistence of donor Tregs in the liver. CONCLUSIONS: (1) The liver is devoid of Tregs early after perinatal RRV infection; (2) Tregs suppress DC-dependent activation of naive NK cells in vitro, and Treg-containing CD4+ cells inhibit hepatic NK cell expansion in vivo. Thus, the post-natal absence of Tregs may be a key factor that allows hepatic DCs to act unopposed in NK cell activation during the initiation of neonatal bile duct injury.

Health Care Utilization and Spending for Children With Mental Health Conditions in Medicaid.

OBJECTIVE: To examine how characteristics vary between children with any mental health (MH) diagnosis who have typical spending and the highest spending; to identify independent predictors of highest spending; and to examine drivers of spending groups. METHODS: This retrospective analysis utilized 2016 Medicaid claims from 11 states and included 775,945 children ages 3 to 17 years with any MH diagnosis and at least 11 months of continuous coverage. We compared demographic characteristics and Medicaid expenditures based on total health care spending: the top 1% (highest-spending) and remaining 99% (typical-spending). We used chi-squared tests to compare the 2 groups and adjusted logistic regression to identify independent predictors of being in the top 1% highest-spending group. RESULTS: Children with MH conditions accounted for 55% of Medicaid spending among 3- to 17-year olds. Patients in the highest-spending group were more likely to be older, have multiple MH conditions, and have complex chronic physical health conditions (P <.001). The highest-spending group had $164,003 per-member-per-year (PMPY) in total health care spending, compared to $6097 PMPY in the typical-spending group. Ambulatory MH services contributed the largest proportion (40%) of expenditures ($2455 PMPY) in the typical-spending group; general health hospitalizations contributed the largest proportion (36%) of expenditures ($58,363 PMPY) in the highest-spending group. CONCLUSIONS: Among children with MH conditions, mental and physical health comorbidities were common and spending for general health care outpaced spending for MH care. Future research and quality initiatives should focus on integrating MH and physical health care services and investigate whether current spending on MH services supports high-quality MH care.

Outpatient Prescription Opioid Use in Pediatric Medicaid Enrollees With Special Health Care Needs.

BACKGROUND AND OBJECTIVES: Although potentially dangerous, little is known about outpatient opioid exposure (OE) in children and youth with special health care needs (CYSHCN). We assessed the prevalence and types of OE and the diagnoses and health care encounters proximal to OE in CYSHCN. METHODS: This is a retrospective cohort study of 2 597 987 CYSHCN aged 0-to-18 years from 11 states, continuously enrolled in Medicaid in 2016, with ≥1 chronic condition. OE included any filled prescription (single or multiple) for opioids. Health care encounters were assessed within 7 days before and 7 and 30 days after OE. RESULTS: Among CYSHCN, 7.4% had OE. CYSHCN with OE versus without OE were older (ages 10-18 years: 69.4% vs 47.7%), had more chronic conditions (≥3 conditions: 49.1% vs 30.6%), and had more polypharmacy (≥5 other medication classes: 54.7% vs 31.2%), P < .001 for all. Most (76.7%) OEs were single fills with a median duration of 4 days (interquartile range: 3-6). The most common OEs were acetaminophen-hydrocodone (47.5%), acetaminophen-codeine (21.5%), and oxycodone (9.5%). Emergency department visits preceded 28.8% of OEs, followed by outpatient surgery (28.8%) and outpatient specialty care (19.1%). Most OEs were preceded by a diagnosis of infection (25.9%) or injury (22.3%). Only 35.1% and 62.2% of OEs were associated with follow-up visits within 7 and 30 days, respectively. CONCLUSIONS: OE in CYSHCN is common, especially with multiple chronic conditions and polypharmacy. In subsequent studies, researchers should examine the appropriateness of opioid prescribing, particularly in emergency departments, as well as assess for drug interactions with chronic medications and reasons for insufficient follow-up.

Morel-lavalée lesions treated with debridement and meticulous dead space closure: surgical technique.

Morel-Lavalée lesions (M-L lesion) can be successfully treated with debridement and meticulous dead space closure on an acute or delayed basis. The technique is simple, reproducible, and involves only one trip to the operating room in all but the open infected lesions. Our series of 24 Morel-Lavalée lesions in 22 patients resulted in no infections or recurrence of fluid collection. Two patients had superficial skin loss treated by local wound management.

Multiple Behavior Change Intervention to Improve Detection of Unmet Social Needs and Resulting Resource Referrals.

OBJECTIVE: It is critical that pediatric residents learn to effectively screen families for active and addressable social needs (ie, negative social determinants of health). We sought to determine 1) whether a brief intervention teaching residents about IHELP, a social needs screening tool, could improve resident screening, and 2) how accurately IHELP could detect needs in the inpatient setting. METHODS: During an 18-month period, interns rotating on 1 of 2 otherwise identical inpatient general pediatrics teams were trained in IHELP. Interns on the other team served as the comparison group. Every admission history and physical examination (H&P) was reviewed for IHELP screening. Social work evaluations were used to establish the sensitivity and specificity of IHELP and document resources provided to families with active needs. During a 21-month postintervention period, every third H&P was reviewed to determine median duration of continued IHELP use. RESULTS: A total of 619 admissions met inclusion criteria. Over 80% of intervention team H&Ps documented use of IHELP. The percentage of social work consults was nearly 3 times greater on the intervention team than on the comparison team (P < .001). Among H&Ps with documented use of IHELP, specificity was 0.96 (95% confidence interval 0.87-0.99) and sensitivity was 0.63 (95% confidence interval 0.50-0.73). Social work provided resources for 78% of positively screened families. The median duration of screening use by residents after the intervention was 8.1 months (interquartile range 1-10 months). CONCLUSIONS: A brief intervention increased resident screening and detection of social needs, leading to important referrals to address those needs.

Deep Sequencing Reveals Novel Genetic Variants in Children with Acute Liver Failure and Tissue Evidence of Impaired Energy Metabolism.

BACKGROUND & AIMS: The etiology of acute liver failure (ALF) remains elusive in almost half of affected children. We hypothesized that inherited mitochondrial and fatty acid oxidation disorders were occult etiological factors in patients with idiopathic ALF and impaired energy metabolism. METHODS: Twelve patients with elevated blood molar lactate/pyruvate ratio and indeterminate etiology were selected from a retrospective cohort of 74 subjects with ALF because their fixed and frozen liver samples were available for histological, ultrastructural, molecular and biochemical analysis. RESULTS: A customized next-generation sequencing panel for 26 genes associated with mitochondrial and fatty acid oxidation defects revealed mutations and sequence variants in five subjects. Variants involved the genes ACAD9, POLG, POLG2, DGUOK, and RRM2B; the latter not previously reported in subjects with ALF. The explanted livers of the patients with heterozygous, truncating insertion mutations in RRM2B showed patchy micro- and macrovesicular steatosis, decreased mitochondrial DNA (mtDNA) content <30% of controls, and reduced respiratory chain complex activity; both patients had good post-transplant outcome. One infant with severe lactic acidosis was found to carry two heterozygous variants in ACAD9, which was associated with isolated complex I deficiency and diffuse hypergranular hepatocytes. The two subjects with heterozygous variants of unknown clinical significance in POLG and DGUOK developed ALF following drug exposure. Their hepatocytes displayed abnormal mitochondria by electron microscopy. CONCLUSION: Targeted next generation sequencing and correlation with histological, ultrastructural and functional studies on liver tissue in children with elevated lactate/pyruvate ratio expand the spectrum of genes associated with pediatric ALF.