Cystic fibrosis liver disease (CFLD), which results from progressive hepatobiliary fibrosis, is an important cause of morbidity and mortality, but it is difficult to identify before portal hypertension (PHT) ensues. Clinical signs, serum alanine aminotransferase (ALT) levels, and ultrasound (US) are widely applied, but their value in predicting the presence of cirrhosis, the development of PHT, or adverse outcomes is undetermined. The potential gold standard, liver biopsy, is not standard practice and, notwithstanding sampling error considerations, has not been systematically evaluated. Forty patients with cystic fibrosis (median age 5 10.6 years) with abnormal clinical, biochemical, and US findings were subjected to dual-pass percutaneous liver biopsy. Clinical outcomes were recorded over 12 years of follow-up (median 5 9.5 years for survivors). Logistic regression and receiver operating characteristic analyses were applied to predict hepatic fibrosis (which was assessed by fibrosis staging and quantitative immunohistochemistry) and the occurrence of PHT. PHT occurred in 17 of 40 patients (42%), including 6 of 7 (17%) who died during follow-up. Clinical examination, serum ALT levels, and US findings failed to predict either the presence of liver fibrosis or the development of PHT. Fibrosis staging on liver biopsy, where the accuracy was improved by dual passes (P 5 0.002, nonconcordance 5 38%), predicted the development of PHT (P < 0.001), which occurred more frequently and at a younger age in those with severe fibrosis. Conclusion: Clinical modalities currently employed to evaluate suspected CFLD help to identify a cohort of children at risk for liver disease and adverse outcomes but do not predict an individual's risk of liver fibrosis or PHT development. Liver fibrosis on biopsy predicts the development of clinically significant liver disease. Dual passes help to address sampling concerns. Liver biopsy has a relevant role in the management of patients with suspected CFLD and deserves more widespread application.

Murine dendritic cells (DCs) can be classified into at least 2 subsets, “myeloid-related” (CD11bbright, CD8α−) and “lymphoid-related” (CD11bdull, CD8α+), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)–dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c+ population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro–derived DCs, when treated with interferon-α or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8α, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti–interleukin-6 (IL-6) antibody, but not anti–GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.

Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholine ring. PMO antisense agents have been reported to effectively interfere with the replication of several positive-strand RNA viruses in cell culture. The filoviruses, Marburg virus and Ebola virus (EBOV), are negative-strand RNA viruses that cause up to 90% lethality in human outbreaks. There is currently no commercially available vaccine or efficacious therapeutic for any filovirus. In this study, PMO conjugated to arginine-rich cell-penetrating peptide (P-PMO) and nonconjugated PMO were assayed for the ability to inhibit EBOV infection in cell culture and in a mouse model of lethal EBOV infection. A 22-mer P-PMO designed to base pair with the translation start site region of EBOV VP35 positive-sense RNA generated sequence-specific and time-and dose-dependent inhibition of EBOV amplification in cell culture. The same oligomer provided complete protection to mice when administered before or after an otherwise lethal infection of EBOV. A corresponding nonconjugated PMO, as well as nonconjugated truncated versions of 16 and 19 base residues, provided length-dependent protection to mice when administered prophylactically. Together, these data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates.The Filoviridae family consists of only two genera, Ebola virus (EBOV) and Marburg virus (MARV), and belongs to the order Mononegavirales. Filoviruses possess a nonsegmented, single-stranded RNA genome of negative polarity that contains seven genes. The nontranscribed genomic ends, termed "leader" (3Ј end) and "trailer" (5Ј end), harbor cis-acting signals important for viral transcription, replication, and encapsidation. Transcription and replication of the viral genome requires viral proteins (VP) VP30 and VP35, the nucleoprotein (NP), and viral polymerase L (27, 28). Along with its role in viral RNA synthesis, VP35 has also been shown to act as a type I interferon antagonist (2). As the only viral surface protein, the glycoprotein (GP) is involved in binding and entry of the virion into host cells. The matrix protein VP40 functions in virus assembly and budding, and it has been suggested that the minor matrix protein VP24 also plays a role in these processes (7,14).Most MARV and EBOV species cause a severe hemorrhagic fever associated with fatality rates up to 90% in humans and nonhuman primates (8, 32). Due to the high mortality rates and the lack of any approved effective human therapeutic or vaccine (5), filoviruses have been classified as biological safety level 4 (BSL-4) agents. Many approaches have been employed in attempts to develop effective therapies for EBOV, including the administration of nucleoside analogues (i.e., ribavirin), immune globulins, type I interferons and other cytokines, anticoagulants, and therapeutic vaccines (10,13,19,20,24,37,39,40). To date, th...

Background and Aims: Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non‐alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade. Methods: Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic stellate cell activation (α‐smooth muscle actin and TGF‐β expression) and collagen type I synthesis (procollagen α1 (I) mRNA). Results: Lipid peroxidation occurred in and adjacent to fat‐laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). α‐Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them. Conclusions: These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes.