Current practices in lipid emulsion utilization in the prevention and treatment of intestinal failure-associated liver disease: A survey of pediatric intestinal rehabilitation and transplant centers.

BACKGROUND: Newer intravenous lipid emulsions (ILEs), such as fish oil-based intravenous lipid emulsions (FO-ILEs) and soybean oil, medium-chain triglycerides, olive oil, and fish oil-based intravenous lipid emulsions (SMOF-ILEs), provide alternatives to soybean oil-based intravenous lipid emulsions (SO-ILEs). We explored current ILE practice patterns among intestinal rehabilitation and transplant centers. METHODS: A survey was developed addressing ILE availability, ILE preference in clinical scenarios, and factors influencing ILE choice. This survey was reviewed locally and by the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Intestinal Rehabilitation Special Interest Group, the Intestinal Rehabilitation and Transplant Association scientific committee, and the American Society of Parenteral and Enteral Nutrition pediatric intestinal failure section research committee. We recruited providers nationally and internationally from centers with and without intestinal transplant programs. RESULTS: We included 34 complete responses, 29 from the United States. Sixteen centers performed intestinal transplants. All centers had access to SMOF-ILEs, 85% had access to FO-ILEs, and 91% had access to SO-ILEs. In new patients, 85% use SMOF-ILEs as the first choice ILE. In those with new intestinal failure-associated liver disease (IFALD), FO-ILE was preferred to SMOF-ILE (56% vs 38%). In those developing IFALD on SMOF-ILE, 65% switched to FO-ILE, whereas 24% remained on SMOF-ILE. CONCLUSIONS: Centers have routine access to alternative ILEs, and these are quickly replacing SO-ILEs in all circumstances. Future work should focus on how this shift in practice affects outcomes to provide decision support in specific clinical scenarios.

Megacystis Microcolon Intestinal Hypoperistalsis Syndrome: A Case Series With Long-term Follow-up and Prolonged Survival.

OBJECTIVES: Describe clinical characteristics, management, and outcome in a cohort of megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) patients. METHODS: We conducted a retrospective chart review of MMIHS patients followed at a large transplant and intestinal rehabilitation center over a period of 17 years. RESULTS: We identified 25 patients with MMIHS (68% girls, 13 transplanted). One transplanted and 1 nontransplanted patient were lost to follow-up. We estimated 100, 100, and 86% for 5-, 10-, and 20-year survival, respectively, with only 1 death. Of the 22 patients alive at the time of study (11 transplanted, 11 nontransplanted), median age was 9.2 years (range 2.7-22.9 years). Longest posttransplant follow-up was 16 years. Seventeen patients had available prenatal imaging reports; all showed distended bladder. Eight had genetic testing (5, ACTG2; 2, MYH11; 1, MYL9). Almost all patients had normal growth with median weight z-score -0.77 (interquartile range -1.39 to 0.26), height z score -1.2 (-2.04 to -0.48) and body mass index z-score 0.23 (-0.37 to 0.93) with no statistical difference between transplanted and nontransplanted patients. All nontransplanted patients were on parenteral nutrition with minimal/no feeds, and all except 1 of the transplanted patients were on full enteral feeds. Recent average bilirubin, INR, albumin, and creatinine fell within the reference ranges. CONCLUSIONS: This is the largest single-center case series with the longest duration of follow-up for MMIHS patients. In the current era of improved intestinal rehabilitation and transplantation, MMIHS patients have excellent outcomes in survival, growth, and liver function. This observation contradicts previous reports and should alter counselling and management decisions in these patients at diagnosis.

Deficient and Null Variants of SERPINA1 Are Proteotoxic in a Caenorhabditis elegans Model of α1-Antitrypsin Deficiency.

α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles. The deficient alleles, like ATZ, misfold in the ER where they accumulate as toxic monomers, oligomers and aggregates. Thus, deficient alleles may predispose to both gain- and loss-of-function phenotypes. Null variants, if translated, typically yield truncated proteins that are efficiently degraded after being transiently retained in the ER. Clinically, null alleles are only associated with the loss-of-function phenotype. We recently developed a C. elegans model of ATD in order to further elucidate the mechanisms of proteotoxicity (gain-of-function phenotype) induced by the aggregation-prone deficient allele, ATZ. The goal of this study was to use this C. elegans model to determine whether different types of deficient and null alleles, which differentially affect polymerization and secretion rates, correlated to any extent with proteotoxicity. Animals expressing the deficient alleles, Mmalton, Siiyama and S (ATS), showed overall toxicity comparable to that observed in patients. Interestingly, Siiyama expressing animals had smaller intracellular inclusions than ATZ yet appeared to have a greater negative effect on animal fitness. Surprisingly, the null mutants, although efficiently degraded, showed a relatively mild gain-of-function proteotoxic phenotype. However, since null variant proteins are degraded differently and do not appear to accumulate, their mechanism of proteotoxicity is likely to be different to that of polymerizing, deficient mutants. Taken together, these studies showed that C. elegans is an inexpensive tool to assess the proteotoxicity of different AT variants using a transgenic approach.

Acute DSS colitis alters EphB6 receptor expression in neurons of the spinal dorsal horn.

The ephrin family of receptors (Eph) and their ephrin ligands are involved in pain associated hyperalgesia, but the underlying mechanisms involved have not been fully elucidated. The EphB6 receptor is a distinctive member of the EphB subclass in that its kinase domain contains several alterations in the conserved amino acids and thus lacks catalytic activity. We sought to identify a role for EphB6 in inflammatory pain, with the murine dextran sulfate sodium (DSS) colitis model of inflammatory bowel disease (IBD). Colitis, induced with the administration of 4% (wt./vol.) DSS in the drinking water, significantly decreased EphB6 protein expression levels in neurons of the lower thoracic superficial layers of spinal dorsal horns, the location of neurons that receive the majority of nociceptive information from the colon, via the primary afferents. A shift towards increased EphB/ephrinB forward signaling, mediated by EphB6 down-regulation in neurons of the dorsal horn, may play a role in inflammatory pain caused by IBD.

Using Caenorhabditis elegans to study serpinopathies.

Protein misfolding, polymerization, and/or aggregation are hallmarks of serpinopathies and many other human genetic disorders including Alzheimer's, Huntington's, and Parkinson's disease. While higher organism models have helped shape our understanding of these diseases, simpler model systems, like Caenorhabditis elegans, offer great versatility for elucidating complex genetic mechanisms underlying these diseases. Moreover, recent advances in automated high-throughput methodologies have promoted C. elegans as a useful tool for drug discovery. In this chapter, we describe how one could model serpinopathies in C. elegans and how one could exploit this model to identify small molecule compounds that can be developed into effective therapeutic drugs.

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z.

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.

Transcriptome profiling of the small intestinal epithelium in germfree versus conventional piglets.

BACKGROUND: To gain insight into host-microbe interactions in a piglet model, a functional genomics approach was used to address the working hypothesis that transcriptionally regulated genes associated with promoting epithelial barrier function are activated as a defensive response to the intestinal microbiota. Cesarean-derived germfree (GF) newborn piglets were colonized with adult swine feces, and villus and crypt epithelial cell transcriptomes from colonized and GF neonatal piglets were compared using laser-capture microdissection and high-density porcine oligonucleotide microarray technology. RESULTS: Consistent with our hypothesis, resident microbiota induced the expression of genes contributing to intestinal epithelial cell turnover, mucus biosynthesis, and priming of the immune system. Furthermore, differential expression of genes associated with antigen presentation (pan SLA class I, B2M, TAP1 and TAPBP) demonstrated that microbiota induced immune responses using a distinct regulatory mechanism common for these genes. Specifically, gene network analysis revealed that microbial colonization activated both type I (IFNAR) and type II (IFNGR) interferon receptor mediated signaling cascades leading to enhanced expression of signal transducer and activator of transcription 1 (STAT1), STAT2 and IFN regulatory factor 7 (IRF7) transcription factors and the induction of IFN-inducible genes as a reflection of intestinal epithelial inflammation. In addition, activated RNA expression of NF-kappa-B inhibitor alpha (NFkappaBIA; a.k.a I-kappa-B-alpha, IKBalpha) and toll interacting protein (TOLLIP), both inhibitors of inflammation, along with downregulated expression of the immunoregulatory transcription factor GATA binding protein-1 (GATA1) is consistent with the maintenance of intestinal homeostasis. CONCLUSION: This study supports the concept that the intestinal epithelium has evolved to maintain a physiological state of inflammation with respect to continuous microbial exposure, which serves to sustain a tight intestinal barrier while preventing overt inflammatory responses that would compromise barrier function.

Measurement of erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus.

OBJECTIVE: C4-derived activation fragments are the only complement ligands present on the surfaces of normal erythrocytes. The significance of this observation is unknown, and the role of erythrocyte-bound C4 (E-C4) in human disease has not been explored. More than any other human disease, the pathogenesis of systemic lupus erythematosus (SLE) has been characterized by defects in clearance of complement-bearing immune complexes via erythrocytes expressing complement receptor 1 (CR1). This study was undertaken to determine whether these functional defects might be reflected by abnormal patterns of E-C4 and E-CR1 expression on erythrocytes of patients with SLE. METHODS: We conducted a cross-sectional study of 100 patients with SLE, 133 patients with other diseases, and 84 healthy controls. Erythrocytes were characterized by indirect immunofluorescence and by flow cytometry for determination of levels of C4d and CR1. RESULTS: Patients with SLE had higher levels of E-C4d and lower levels of E-CR1 than did patients with other diseases (P < or = 0.001) or healthy controls (P < or = 0.001). The test was 81% sensitive and 91% specific for SLE versus healthy controls and 72% sensitive and 79% specific for SLE versus other diseases, and it had an overall negative predictive value of 92%. CONCLUSION: This is the first report of abnormal levels of E-C4d in human disease. We found that abnormally high levels of E-C4d and low levels of E-CR1 are characteristic of SLE, and combined measurement of the 2 molecules has high diagnostic sensitivity and specificity for lupus. Determination of E-C4d/E-CR1 levels may be a useful addition to current tests and criteria for SLE diagnosis.