Emergency Planning as Part of Healthcare Transition Preparation for Patients with Duchenne Muscular Dystrophy.

BACKGROUND: Emergency care planning is an important component of healthcare transition, particularly for patients with medical complexity. Duchenne muscular dystrophy (DMD) is a complex, progressive pediatric-onset disease affecting multiple organ systems including impairment of cardiac and pulmonary function, high risk for fractures, fat embolism, adrenal crisis and malignant hyperthermia. Appropriate interdisciplinary emergency management is critical for survival for these patients. The purpose of this quality improvement project was to develop a process to reliably share an individualized emergency care plan (ECP) with patients and their families as part of a larger plan to develop an integrated transition program. METHODS: An interdisciplinary team of nurses and clinicians used the principles of quality improvement to develop a reliable process to assure patients with DMD received an individualized, multidisciplinary ECP at routine interdisciplinary clinic visits. Additionally, the project used surveys to assess patient and family satisfaction with the letter and whether it improved their knowledge of emergency care. RESULTS: Sixty-two patients were seen during the study timeframe. All received an ECP. Sixty-two surveys were sent and twenty-three surveys were returned. Of those that responded, the majority stated the ECP increased their knowledge of emergency care. CONCLUSION: ECPs can be developed and disseminated to patients with DMD and their caregivers. This tool can potentially promote timely and appropriate emergency care for these patients with unique and complex medical needs.

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.

Obstruction of extrahepatic bile ducts by lymphocytes is regulated by IFN-gamma in experimental biliary atresia.

The etiology and pathogenesis of bile duct obstruction in children with biliary atresia are largely unknown. We have previously reported that, despite phenotypic heterogeneity, genomic signatures of livers from patients display a proinflammatory phenotype. Here, we address the hypothesis that production of IFN-gamma is a key pathogenic mechanism of disease using a mouse model of rotavirus-induced biliary atresia. We found that rotavirus infection of neonatal mice has a unique tropism to bile duct cells, and it triggers a hepatobiliary inflammation by IFN-gamma-producing CD4(+) and CD8(+) lymphocytes. The inflammation is tissue specific, resulting in progressive jaundice, growth failure, and greater than 90% mortality due to obstruction of extrahepatic bile ducts. In this model, the genetic loss of IFN-gamma did not alter the onset of jaundice, but it remarkably suppressed the tissue-specific targeting of T lymphocytes and completely prevented the inflammatory and fibrosing obstruction of extrahepatic bile ducts. As a consequence, jaundice resolved, and long-term survival improved to greater than 80%. Notably, administration of recombinant IFN-gamma led to recurrence of bile duct obstruction following rotavirus infection of IFN-gamma-deficient mice. Thus, IFN-gamma-driven obstruction of bile ducts is a key pathogenic mechanism of disease and may constitute a therapeutic target to block disease progression in patients with biliary atresia.

Urokinase-type plasminogen activator supports liver repair independent of its cellular receptor.

BACKGROUND: The urokinase-type (uPA) and tissue-type (tPA) plasminogen activators regulate liver matrix remodelling through the conversion of plasminogen (Plg) to the active protease plasmin. Based on the efficient activation of plasminogen when uPA is bound to its receptor (uPAR) and on the role of uPA in plasmin-mediated liver repair, we hypothesized that uPA requires uPAR for efficient liver repair. METHODS: To test this hypothesis, we administered one dose of carbon tetrachloride (CCl4) to mice with single or combined deficiencies of uPA, uPAR and tPA, and examined hepatic morphology, cellular proliferation, fibrin clearance, and hepatic proteolysis 2-14 days later. RESULTS: Absence of uPAR alone or the combined absence of uPAR and tPA had no impact on the resolution of centrilobular injury, but the loss of receptor-free uPA significantly impaired the clearance of necrotic hepatocytes up to 14 days after CCl4. In response to the injury, hepatocyte proliferation was normal in mice of all genotypes, except for uPAR-deficient (uPAR degrees) mice, which had a reproducible but mild decrease by 33% at day 2, with an appropriate restoration of liver mass by 7 days similar to experimental controls. Immunostaining and zymographic analysis demonstrated that uPA alone promoted fibrin clearance from centrilobular regions and efficiently activated plasminogen. CONCLUSION: uPA activates plasminogen and promotes liver matrix proteolysis during repair via a process that neither requires its receptor uPAR nor requires a contribution from its functional counterpart tPA.

Neonatal NK cells target the mouse duct epithelium via Nkg2d and drive tissue-specific injury in experimental biliary atresia.

Biliary atresia is a neonatal obstructive cholangiopathy that progresses to end-stage liver disease. Although the etiology is unknown, a neonatal adaptive immune signature has been mechanistically linked to obstruction of the extrahepatic bile ducts. Here, we investigated the role of the innate immune response in the pathogenesis of biliary atresia. Analysis of livers of infants at diagnosis revealed that NK cells populate the vicinity of intrahepatic bile ducts and overexpress several genes involved in cytotoxicity. Using a model of rotavirus-induced biliary atresia in newborn mice, we found that activated NK cells also populated murine livers and were the most abundant cells in extrahepatic bile ducts at the time of obstruction. Rotavirus-primed hepatic NK cells lysed cholangiocytes in a contact- and Nkg2d-dependent fashion. Depletion of NK cells and blockade of Nkg2d each prevented injury of the duct epithelium after rotavirus infection, maintained continuity of duct lumen between the liver and duodenum, and enabled bile flow, despite the presence of virus in the tissue and the overexpression of proinflammatory cytokines. These findings identify NK cells as key initiators of cholangiocyte injury via Nkg2d and demonstrate that injury to the duct epithelium drives the phenotype of experimental biliary atresia.

Hepatic to pancreatic switch defines a role for hemostatic factors in cellular plasticity in mice.

In multiple systems, impaired proteolysis associated with the loss of the hemostatic factor plasminogen (Plg) results in fibrin-dependent defects in tissue repair. However, repair within the liver is known to be defective in Plg-deficient (Plg(o)) mice independent of fibrin clearance and appears to be compromised in part by the poor clearance of necrotic cells. Based on these findings, we examined the hepatic transcriptome after injury in search of transcriptional programs that are sensitive to the Plg/fibrinogen system. To this end, we generated biotinylated cRNA pools from livers of Plg(o) mice and controls before and after a single dose of the hepatotoxin carbon tetrachloride and hybridized them against high-density oligonucleotide arrays. Analysis of the gene expression platform identified an unexpected transcriptional signature within challenged livers of Plg(o) mice for pancreatic gene products, including trypsinogen-2, amylase-2, elastase-1, elastase-2, and cholesteryl-ester lipase. Validation studies found that this transcriptional program also contained products of the endocrine pancreas (Reg-1 and insulin genes) and the expression of the pancreatic transcription factors p48 and PDX-1. By using a LacZ transgene to trace the cellular source of pancreatic gene expression, we found that PDX-1 was expressed in albumin-positive cells that were morphologically indistinguishable from hepatocytes, and in albumin-negative epithelioid cells within zones of pericentral injury. More detailed studies revealed that the mechanisms of heterotopic gene expression in Plg(o) mice required fibrin(ogen). Collectively, these data reveal a regulatory role for the hemostatic factors plasmin(ogen) and fibrin(ogen) in cellular plasticity within adult tissues of the digestive system.

Transcriptional reprogramming in murine liver defines the physiologic consequences of biliary obstruction.

BACKGROUND/AIMS: While the metabolic and histological responses to cholestasis are recognized, the consequences of impaired biliary flow on liver gene expression are largely undefined. We hypothesized that biliary obstruction results in transcriptional reprogramming that dictates the physiologic response. METHODS: We determined global gene expression in murine livers 1-21 days following bile duct ligation. Total hepatic cRNA from experimental and sham mice was hybridized to Affymetrix gene chips. Gene expression data was analyzed by GeneSpring software and validated by Northern analysis. RESULTS: We found 92 genes over-expressed > or =2-fold at one or more time points following bile duct ligation. Functional classification of these genes revealed the activation of three main biological processes in a sequential and time-restricted fashion. At day 1, genes involved in sterol metabolism were uniquely over-expressed, including HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. This was followed by an increased expression of growth-promoting genes at day 7, the time point coinciding with peak cholangiocyte proliferation. In later phases (days 14-21), the liver over-expressed genes encoding structural proteins and proteases. CONCLUSIONS: Transcriptional reprogramming in the liver following biliary obstruction favors the activation of genes regulating metabolism, cell proliferation, and matrix remodeling in a time-restricted and sequential fashion.

Independent and overlapping transcriptional activation during liver development and regeneration in mice.

Liver development and regeneration share the requirement for simultaneous proliferation and acquisition of highly specialized cellular functions. However, little is known about molecules with regulatory roles in both processes. We hypothesized that transcriptional reprogramming induced by regeneration recapitulates that of developing liver. To address this hypothesis, we determined global hepatic gene expression at embryonic day 14.5, postnatal day 14, and 6 to 24 hours following partial hepatectomy using microarrays containing 8,635 cDNAs. Analysis of genes overexpressed during these conditions revealed 3 unique expression patterns. The first was predominantly signature gene clusters specific for each growth phase. Major groups were hematopoiesis-related genes in embryonic livers, metabolic genes during postnatal liver development, and growth/inflammation and metabolic genes during regeneration. The second pattern consisted of dual overexpression during regeneration and at least one phase of development. Consistent with potential regulatory roles in liver growth, most of these transcripts control cell-cell contact, membrane trafficking, cell growth, metabolism, and inflammatory response. The third pattern, revealed by surveying their expression across 76 hepatic and extra-hepatic tissues, uncovered a restricted temporospatial pattern of liver overexpression for CD14, orosomucoid 1, hepcidin, Spi 2.1, Ith3, and Tim-44. In conclusion, these results provide a basis for the identification of gene and gene groups that play critical roles at different phases of liver development and regeneration, and underscore the importance of maintaining metabolic demands during organ growth.

Genetic induction of proinflammatory immunity in children with biliary atresia.

BACKGROUND: Biliary atresia is the commonest cause of pathological jaundice in infants and the leading indication for liver transplantation in children worldwide. The cause and pathogenesis remain largely unknown. Because of clinical heterogeneity and experimental difficulties in addressing molecular mechanisms underlying multifactorial disorders in human beings, we searched for genomic signatures of biliary atresia in affected infants. METHODS: We generated pools of biotinylated cRNA from livers of 14 infants with biliary atresia and six with neonatal intrahepatic cholestasis (diseased controls) and hybridised the cRNA against oligonucleotide-based gene chips. Immunohistochemistry and reverse transcriptase (RT)-PCR were used to assess the specificity of the findings and functional commitment of lymphocytes in affected livers. FINDINGS: Data filtering, to identify genes that are differentially expressed, and cluster analysis revealed a predominant and coordinated activation of immunity/inflammation genes within the livers of infants with biliary atresia. Most of the genes showed differential lymphocyte function, with activation of osteopontin, a regulator of cell-mediated (T-helper 1 [Th-1]) immunity in T-helper lymphocytes, and suppression of immunoglobulin genes in early stages of disease. These findings were associated with production of interferon gamma in 65% of infants with biliary atresia and no diseased control. However, histologically similar inflammatory infiltrates were present in livers of both groups, implying differential activation states of similar cell types. INTERPRETATION: Livers of infants with biliary atresia have a coordinated activation of genes involved in lymphocyte differentiation. Among these genes, the overexpression of osteopontin and interferon gamma points to a potential role of Th-1-like cytokines in disease pathogenesis.