Impaired Bile Acid Metabolism and Gut Dysbiosis in Mice Lacking Lysosomal Acid Lipase.

Lysosomal acid lipase (LAL) is the sole enzyme known to be responsible for the hydrolysis of cholesteryl esters and triglycerides at an acidic pH in lysosomes, resulting in the release of unesterified cholesterol and free fatty acids. However, the role of LAL in diet-induced adaptations is largely unexplored. In this study, we demonstrate that feeding a Western-type diet to Lal-deficient (LAL-KO) mice triggers metabolic reprogramming that modulates gut-liver cholesterol homeostasis. Induction of ileal fibroblast growth factor 15 (three-fold), absence of hepatic cholesterol 7α-hydroxylase expression, and activation of the ERK phosphorylation cascade results in altered bile acid composition, substantial changes in the gut microbiome, reduced nutrient absorption by 40%, and two-fold increased fecal lipid excretion in LAL-KO mice. These metabolic adaptations lead to impaired bile acid synthesis, lipoprotein uptake, and cholesterol absorption and ultimately to the resistance of LAL-KO mice to diet-induced obesity. Our results indicate that LAL-derived lipolytic products might be important metabolic effectors in the maintenance of whole-body lipid homeostasis.

Hepatic synthesis of triacylglycerols containing medium-chain fatty acids is dominated by diacylglycerol acyltransferase 1 and efficiently inhibited by etomoxir.

OBJECTIVE: Medium-chain fatty acids (MCFAs) play an increasing role in human nutrition. In the liver, one fraction is used for synthesis of MCFA-containing triacylglycerol (MCFA-TG), and the rest is used for oxidative energy production or ketogenesis. We investigated which enzymes catalyse the synthesis of MCFA-TG and how inhibition of MCFA-TG synthesis or fatty acid (FA) oxidation influences the metabolic fate of the MCFAs. METHODS: FA metabolism was followed by time-resolved tracing of alkyne-labelled FAs in freshly isolated mouse hepatocytes. Quantitative data were obtained by mass spectrometry of several hundred labelled lipid species. Wild-type hepatocytes and cells from diacylglycerol acyltransferase (DGAT)1-/- mice were treated with inhibitors against DGAT1, DGAT2, or FA ß-oxidation. RESULTS: Inhibition or deletion of DGAT1 resulted in a reduction of MCFA-TG synthesis by 70%, while long-chain (LC)FA-TG synthesis was reduced by 20%. In contrast, DGAT2 inhibition increased MCFA-TG formation by 50%, while LCFA-TG synthesis was reduced by 5-25%. Inhibition of ß-oxidation by the specific inhibitor teglicar strongly increased MCFA-TG synthesis. In contrast, the widely used ß-oxidation inhibitor etomoxir blocked MCFA-TG synthesis, phenocopying DGAT1 inhibition. CONCLUSIONS: DGAT1 is the major enzyme for hepatic MCFA-TG synthesis. Its loss can only partially be compensated by DGAT2. Specific inhibition of ß-oxidation leads to a compensatory increase in MCFA-TG synthesis, whereas etomoxir blocks both ß-oxidation and MCFA-TG synthesis, indicating a strong off-target effect on DGAT1.

Myeloperoxidase and Septic Conditions Disrupt Sphingolipid Homeostasis in Murine Brain Capillaries In Vivo and Immortalized Human Brain Endothelial Cells In Vitro.

During inflammation, activated leukocytes release cytotoxic mediators that compromise blood-brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO-/- compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.

Facilitating adaptation to climate change while restoring a montane plant community.

Montane plant communities throughout the world have responded to changes in temperature regimes by shifting ranges upward in elevation, and made downslope movements to track shifts in climatic water balance. Organisms that cannot disperse or adapt biologically to projected climate scenarios in situ may decrease in distributional range and abundance over time. Restoration strategies will need to incorporate the habitat suitability of future predicted conditions to ensure long-term persistence. We propagated seedlings of three native Hawaiian montane plant species from high- (~2,500 m asl) and low-elevation (~1,900 m asl) sources, planted them in 8 common plots along a 500 m elevation gradient, and monitored microclimate at each plot for 20 weeks. We explored how temperature and precipitation influenced survival and growth differently among high- and low-elevation origin seedlings. Significantly more seedlings of only one species, Dodonaea viscosa, from high-elevation origin (75.2%) survived than seedlings from low-elevation origin (58.7%) across the entire elevation gradient. Origin also influenced survival in generalized linear mixed models that controlled for temperature, precipitation, and elevation in D. viscosa and Chenopodium oahuense. Survival increased with elevation and soil moisture for Sophora chrysophylla, while it decreased for the other two species. Responses to microclimate varied between the three montane plant species; there were no common patterns of growth or survival. Although limited in temporal scope, our experiment represents one of the few attempts to examine local adaptation to prospective climate scenarios and addresses challenges to restoration efforts within species' current ranges.

Intestine-Specific Overexpression of Carboxylesterase 2c Protects Mice From Diet-Induced Liver Steatosis and Obesity.

Murine hepatic carboxylesterase 2c (Ces2c) and the presumed human ortholog carboxylesterase 2 (CES2) have been implicated in the development of nonalcoholic fatty liver disease (NAFLD) in mice and obese humans. These studies demonstrated that Ces2c hydrolyzes triglycerides (TGs) in hepatocytes. Interestingly, Ces2c/CES2 is most abundantly expressed in the intestine, indicating a role of Ces2c/CES2 in intestinal TG metabolism. Here we show that Ces2c is an important enzyme in intestinal lipid metabolism in mice. Intestine-specific Ces2c overexpression (Ces2cint) provoked increased fatty acid oxidation (FAO) in the small intestine accompanied by enhanced chylomicron clearance from the circulation. As a consequence, high-fat diet-fed Ces2cint mice were resistant to excessive diet-induced weight gain and adipose tissue expansion. Notably, intestinal Ces2c overexpression increased hepatic insulin sensitivity and protected mice from NAFLD development. Although lipid absorption was not affected in Ces2cint mice, fecal energy content was significantly increased. Mechanistically, we demonstrate that Ces2c is a potent neutral lipase, which efficiently hydrolyzes TGs and diglycerides (DGs) in the small intestine, thereby generating fatty acids (FAs) for FAO and monoglycerides (MGs) and DGs for potential re-esterification. Consequently, the increased availability of MGs and DGs for re-esterification and primordial apolipoprotein B48 particle lipidation may increase chylomicron size, ultimately mediating more efficient chylomicron clearance from the circulation. Conclusion: This study suggests a critical role for Ces2c in intestinal lipid metabolism and highlights the importance of intestinal lipolysis to protect mice from the development of hepatic insulin resistance, NAFLD, and excessive diet-induced weight gain during metabolic stress.

Hepatocyte-specific lysosomal acid lipase deficiency protects mice from diet-induced obesity but promotes hepatic inflammation.

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa-/-) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa-/- mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa-/- mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa-/- mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.

Myristic acid induces proteomic and secretomic changes associated with steatosis, cytoskeleton remodeling, endoplasmic reticulum stress, protein turnover and exosome release in HepG2 cells.

Myristic acid, the 14-carbon saturated fatty acid (C14:0), is associated to an increased cardiovascular disease risk. Since it is found in low concentration in cells, its specific properties have not been fully analyzed. The aim of this study was to explore the cell response to this fatty acid to help explaining clinical findings on the relationship between C14:0 and cardiovascular disease. The human liver HepG2 cell line was used to investigate the hepatic response to C14:0 in a combined proteomic and secretomic approach. A total of 47 intracellular and 32 secreted proteins were deregulated after treatments with different concentrations of C14:0. Data are available via ProteomeXchange (PXD007902). In addition, C14:0 treatment of primary murine hepatocytes confirmed that C14:0 induces lipid droplet accumulation and elevates perilipin-2 levels. Functional enrichment analysis revealed that C14:0 modulates lipid droplet formation and cytoskeleton organization, induce ER stress, changes in exosome and extracellular miRNA sorting in HepG2cells. Our data provide for the first time a proteomic profiling of the effects of C14:0 in human hepatoma cells and contribute to the elucidation of molecular mechanisms through which this fatty acid may cause adverse health effects. BIOLOGICAL SIGNIFICANCE: Myristic acid is correlated with an increase in plasma cholesterol and mortality due to cardiovascular diseases. This study is the first example of an integration of proteomic and secretomic analysis of HepG2 cells to investigate the specific properties and functional roles of myristic acid on hepatic cells. Our analyses will lead to a better understanding of the myristic acid induced effects and can elicit new diagnostic and treatment strategies based on altered proteins.

Lysosomal acid lipase regulates fatty acid channeling in brown adipose tissue to maintain thermogenesis.

Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal-/-) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal-/- mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal-/- mice.

Monoglyceride lipase deficiency affects hepatic cholesterol metabolism and lipid-dependent gut transit in ApoE-/- mice.

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (-/-) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)-/- mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO mice is likely not a consequence of potentiated CB1R signaling but rather dependent on the activation of alternative signaling pathways. We conclude that MGL deficiency causes complex metabolic changes including cholesterol metabolism and regulation of gut transit independent of the endocannabinoid system.

Lysosomal lipid hydrolysis provides substrates for lipid mediator synthesis in murine macrophages.

Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipase known to be active at acidic pH. We found LAL to be expressed in murine immune cells with highest mRNA expression in macrophages and neutrophils. Furthermore, we observed that loss of LAL in mice caused lipid accumulation in white blood cells in the peripheral circulation, which increased in response to an acute inflammatory stimulus. Lal-deficient (-/-) macrophages accumulate neutral lipids, mainly cholesteryl esters, within lysosomes. The cholesteryl ester fraction is particularly enriched in the PUFAs 18:2 and 20:4, important precursor molecules for lipid mediator synthesis. To investigate whether loss of LAL activity affects the generation of lipid mediators and to eliminate potential systemic effects from other cells and tissues involved in the pronounced phenotype of Lal-/- mice, we treated macrophages from Wt mice with the LAL-specific inhibitor LAListat-2. Acute inhibition of LAL resulted in reduced release of 18:2- and 20:4-derived mediators from macrophages, indicating that lipid hydrolysis by LAL is an important source for lipid mediator synthesis in macrophages. We conclude that lysosomes should be considered as organelles that provide precursor molecules for lipid mediators such as eicosanoids.

Lysosomal Acid Lipase Hydrolyzes Retinyl Ester and Affects Retinoid Turnover.

Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of cholesteryl esters and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study, we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with RE gavage exhibited largely reduced post-prandial circulating RE content, indicating that LAL is required for efficient nutritional vitamin A availability. In summary, our results indicate that LAL is the major acid RE hydrolase and required for functional retinoid homeostasis.

Novel role of a triglyceride-synthesizing enzyme: DGAT1 at the crossroad between triglyceride and cholesterol metabolism.

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1(-/-)) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1(-/-) and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia.

Lysosomal acid lipase regulates VLDL synthesis and insulin sensitivity in mice.

AIMS/HYPOTHESIS: Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal (-/-) ) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). METHODS: We studied metabolic adaptations in Lal (-/-) mice. RESULTS: Despite loss of adipose tissue, Lal (-/-) mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [(3)H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal (-/-) mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal (-/-) mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal (-/-) mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal (-/-) mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal (-/-) mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. CONCLUSIONS/INTERPRETATION: Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal (-/-) mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply.

Active autophagy but not lipophagy in macrophages with defective lipolysis.

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.

Synthesis and evaluation of novel amide amino-ß-lactam derivatives as cholesterol absorption inhibitors.

The ß-lactam cholesterol absorption inhibitor ezetimibe is so far the only representative of this class of compounds on the market today. The goal of this work was to synthesize new amide ezetimibe analogs from trans-3-amino-(3R,4R)-ß-lactam and to test their cytotoxicity and activity as cholesterol absorption inhibitors. We synthesized six new amide ezetimibe analogs. All new compounds exhibited low toxicity in MDCKIIwt, hNPC1L1/MDCKII and HepG2 cell lines and showed significant inhibition of cholesterol uptake in hNPC1L1/MDCKII cells. In addition, we determined the activity of the three compounds to inhibit cholesterol absorption in vivo. Our results demonstrate that these compounds considerably reduce cholesterol concentrations in liver and small intestine of mice. Thus, our newly synthesized amide ezetimibe analogs are cholesterol absorption inhibitors in vitro and in vivo.

Outcome of allogeneic stem cell transplantation in myelodysplastic syndrome patients: prognostic implication of monosomal karyotype.

OBJECTIVE: Monosomal karyotype (MK) is defined as the presence of two or more autosomal monosomies or a single monosomy associated with a structural abnormality. It was first described as a high-risk cytogenetic abnormality for acute myeloid leukemia and more recently in myelodysplastic syndromes (MDS). However, allotransplant outcome in MDS with MK has not been described. PATIENTS AND METHODS: We retrospectively reviewed data of 79 patients with MDS who underwent allotransplant at the University of Iowa from 1990 to 2009. We recorded patients' cytogenetic data, clinical characteristics and evaluated outcome following allogeneic stem cell transplant stratified by cytogenetic classification. RESULTS: Of 79 patients, 37 (47%) had unfavorable karyotypes (23 complex karyotype, 25 abnormal chromosome 7). Twenty-four patients (30%) had MK. Twenty-four patients (30%) relapsed and 59 (74.7%) died during study period. Patients with MK had higher 2-yr relapse incidence (RI) (51% vs. 29%; P = 0.01), lower 2-yr event-free survival (EFS) (8% vs. 40%; P = 0.02), and lower 2-yr overall survival (OS)(6% vs. 41%; P = 0.02) than patients without MK. We further analyzed the effect of MK in each unfavorable karyotype composite. Although the outcome was not statistically different, unfavorable karyotypes with patients with MK showed a trend toward higher 2-yr RI [hazard ratio (HR), 1.7; P = 0.34], lower 2-yr EFS (HR, 1.5; P = 0.29), and lower 2-yr OS (HR, 1.5; P = 0.28) compared to unfavorable karyotypes without MK. CONCLUSION: Cytogenetic abnormalities remain an important prognostic factor for allotransplant outcome of MDS. Our results suggested poor allotransplant outcomes with high RI and low OS in MDS with MK.

Interim results from a national multicenter phase II trial of combination bacillus Calmette-Guerin plus interferon alfa-2b for superficial bladder cancer.

PURPOSE: Interim results are provided from a large multicenter trial of combination bacillus Calmette-Guerin (BCG) plus interferon (IFN) alfa-2b for BCG naive (BCG-N) and previous BCG failure (BCG-F) cases of superficial bladder cancer. MATERIALS AND METHODS: A total of 490 patients enrolled from May 1999 to May 2000 with a median of 24 months of followup were analyzed. The BCG-N group (259) was treated with a 6-week induction course of standard dose BCG plus 50 million units of IFN followed by 3, 3-week maintenance cycles of reduced dose BCG (1/3 to 1/10) plus 50 million units IFN at 3, 9 and 15 months after induction. The BCG-F group (231) was treated similarly except induction therapy began at a decreased (1/3 to 1/10) BCG dose. RESULTS: The simple tumor recurrence rates for BCG-N and BCG-F groups were 40% and 52%, and the Kaplan-Meier estimates for disease freedom at 24 months were 57% and 42%, respectively. Progression to muscle invasion occurred in 5% and 4.3% while metastasis occurred in 2.3% and 2.6%, respectively. In each group 3.9% of patients underwent cystectomy and 2 patients in each group died of bladder cancer. Serious adverse events occurred in 5.5% with infection related serious adverse events being less prevalent in the BCG-F group (2.6% vs 5.4%). Toxicity related dropout, treatment delay and/or further BCG dose reduction, and need for symptomatic drugs were similar. Moderate to severe local side effects during induction were higher in the BCG-F group (6.2% vs 16.9%) but equilibrated during maintenance therapy. Systemic reactions were rare. CONCLUSIONS: This multicenter trial provides a benchmark for the efficacy and safety of combination BCG and IFN as up front and salvage therapy. The incremental value of IFN cannot be determined from this study.