Cholestasis alters polarization and suppressor function of hepatic regulatory T cells.

Fibrosing cholangiopathies, including biliary atresia and primary sclerosing cholangitis, involve immune-mediated bile duct epithelial injury and hepatic bile acid (BA) retention (cholestasis). Regulatory T-cells (Tregs) can prevent auto-reactive lymphocyte activation, yet the effects of BA on this CD4 lymphocyte subset are unknown. Gene regulatory networks for hepatic CD4 lymphocytes in a murine cholestasis model revealed Tregs are polarized to Th17 during cholestasis. Following bile duct ligation, Stat3 deletion in CD4 lymphocytes preserved hepatic Treg responses. While pharmacological reduction of hepatic BA in MDR2-/- mice prompted Treg expansion and diminished liver injury, this improvement subsided with Treg depletion. A cluster of patients diagnosed with biliary atresia showed both increased hepatic Treg responses and improved 2-year native liver survival, supporting that Tregs might protect against neonatal bile duct obstruction. Together, these findings suggest liver BA determine Treg function and should be considered as a therapeutic target to restore protective hepatic immune responses.

Urinary sphingolipids in adolescents and young adults with youth-onset diabetes.

BACKGROUND: This study evaluated urinary sphingolipids as a marker of diabetic kidney disease (DKD) in adolescents and young adults with youth-onset type 1 and type 2 diabetes. METHODS: A comprehensive panel of urinary sphingolipids, including sphingomyelin (SM), glucosylceramide (GC), ceramide (Cer), and lactosylceramide (LC) species, was performed in patients with youth-onset diabetes from the SEARCH for Diabetes in Youth cohort. Sphingolipid levels, normalized to urine creatinine, were compared in 57 adolescents and young adults with type 1 diabetes, 59 with type 2 diabetes, and 44 healthy controls. The association of sphingolipids with albumin-to-creatinine (ACR) ratio and estimated glomerular filtration rate (eGFR) was evaluated. RESULTS: The median age (interquartile range [IQR]) of participants was 23.1 years (20.9, 24.9) and the median duration of diabetes was 9.3 (8.5, 10.2) years. Urinary sphingolipid concentrations in patients with and without DKD (ACR ≥ 30 mg/g) were significantly elevated compared to healthy controls. There were no significant differences in sphingolipid levels between participants with type 1 and type 2 diabetes. In multivariable analysis, many sphingolipid species were positively correlated with ACR. Most significant associations were evident for the following species: C18 SM, C24:1 SM, C24:1 GC, and C24:1 Cer (all p < 0.001). Sphingolipid levels were not associated with eGFR. However, several interaction terms (diabetes type*sphingolipid) were significant, indicating diabetes type may modify the association of sphingolipids with eGFR. CONCLUSION: Urinary sphingolipids are elevated in adolescents and young adults with youth-onset diabetes and correlate with ACR. Urinary sphingolipids may therefore represent an early biomarker of DKD.

PGRN deficiency exacerbates, whereas a brain penetrant PGRN derivative protects, GBA1 mutation-associated pathologies and diseases.

Mutations in GBA1, encoding glucocerebrosidase (GCase), cause Gaucher disease (GD) and are also genetic risks in developing Parkinson's disease (PD). Currently, the approved therapies are only effective for directly treating visceral symptoms, but not for primary neuronopathic involvement in GD (nGD). Progranulin (PGRN), encoded by GRN, is a novel modifier of GCase, but the impact of PGRN in GBA1 mutation-associated pathologies in vivo remains unknown. Herein, Grn-/- mice crossed into Gba9v/9v mice, a Gba1 mutant line homozygous for the Gba1 D409V mutation, generating Grn-/-Gba9v/9v (PG9V) mice. PG9V mice exhibited neurobehavioral deficits, early onset, and more severe GD phenotypes compared to Grn-/- and Gba9v/9v mice. Moreover, PG9V mice also displayed PD-like phenotype. Mechanistic analysis revealed that PGRN deficiency caused severe neuroinflammation with microgliosis and astrogliosis, along with impaired autophagy associated with the Gba1 mutation. A PGRN-derived peptide, termed ND7, ameliorated the disease phenotype in GD patient fibroblasts ex vivo. Unexpectedly, ND7 penetrated the blood-brain barrier (BBB) and effectively ameliorated the nGD manifestations and PD pathology in Gba9v/null and PG9V mice. Collectively, this study not only provides the first line of in vivo but also ex vivo evidence demonstrating the crucial role of PGRN in GBA1/Gba1 mutation-related pathologies, as well as a clinically relevant mouse model for mechanistic and potential therapeutics studies for nGD and PD. Importantly, a BBB penetrant PGRN-derived biologic was developed that may provide treatment for rare lysosomal storage diseases and common neurodegenerative disorders, particularly nGD and PD.

Comprehensive and long-term outcomes of enzyme replacement therapy followed by stem cell transplantation in children with Gaucher disease type 1 and 3.

BACKGROUND: Gaucher disease (GD) is a lysosomal storage disorder, characterized by hepatosplenomegaly, pancytopenia, bone diseases, with or without neurological symptoms. Plasma glucosylsphingosine (lyso-Gb1), a highly sensitive and specific biomarker for GD, has been used for diagnosis and monitoring the response to treatment. Enzyme replacement therapy (ERT) is an effective treatment for the non-neurologic symptoms of GD. Neuronopathic GD (type 2 and 3) accounts for 60%-70% of the Asian affected population. METHODS: We explored combination therapy of ERT followed by hematopoietic stem cell transplantation (HSCT) and its long-term outcomes in patients with GD type 3 (GD3). RESULTS: Four patients with GD3 and one with GD type 1 (GD1) underwent HSCT. The types of donor were one matched-related, one matched-unrelated, and three haploidentical. The age at disease onset was 6-18 months and the age at HSCT was 3.8-15 years in the patients with GD3. The latest age at follow-up was 8-22 years, with a post-HSCT duration of 3-14 years. All patients had successful HSCT. Chronic graft-versus-host disease occurred in one patient. The enzyme activities were normalized at 2 weeks post HSCT. Lyso-Gb1 concentrations became lower than the pathological value. All of the patients are still alive and physically independent. Most of them (4/5) returned to school. None of the patients with GD3 had seizures or additional neurological symptoms after HSCT, but showed varying degrees of cognitive impairment. CONCLUSIONS: ERT followed by HSCT could be considered as an alternative treatment for patients with GD3 who have a high risk of fatal neurological progression.

Farnesoid X receptor antagonizes macrophage-dependent licensing of effector T lymphocytes and progression of sclerosing cholangitis.

Immune-mediated bile duct epithelial injury and toxicity of retained hydrophobic bile acids drive disease progression in fibrosing cholangiopathies such as biliary atresia or primary sclerosing cholangitis. Emerging therapies include pharmacological agonists to farnesoid X receptor (FXR), the master regulator of hepatic synthesis, excretion, and intestinal reuptake of bile acids. Unraveling the mechanisms of action of pharmacological FXR agonists in the treatment of sclerosing cholangitis (SC), we found that intestinally restricted FXR activation effectively reduced bile acid pool size but did not improve the SC phenotype in MDR2-/- mice. In contrast, systemic FXR activation not only lowered bile acid synthesis but also suppressed proinflammatory cytokine production by liver-infiltrating inflammatory cells and blocked progression of hepatobiliary injury. The hepatoprotective activity was linked to suppressed production of IL1ß and TNFα by hepatic macrophages and inhibition of TH1/TH17 lymphocyte polarization. Deletion of FXR in myeloid cells caused aberrant TH1 and TH17 lymphocyte responses in diethoxycarbonyl-1,4-dihydrocollidine-induced SC and rendered these mice resistant to the anti-inflammatory and liver protective effects of systemic FXR agonist treatment. Pharmacological FXR activation reduced IL1ß and IFNγ production by liver- and blood-derived mononuclear cells from patients with fibrosing cholangiopathies. In conclusion, we demonstrate FXR to control the macrophage-TH1/17 axis, which is critically important for the progression of SC. Hepatic macrophages are cellular targets of systemic FXR agonist therapy for cholestatic liver disease.

Serum Bile Acid Profiling and Mixed Model Analysis Reveal Biomarkers Associated with Pruritus Reduction in Maralixibat-Treated Patients with BSEP Deficiency.

Progressive familial intrahepatic cholestasis (PFIC) is a debilitating disease manifest by severe cholestasis, intractable pruritus and growth delay that ultimately leads to liver failure or transplantation. Maralixibat (MRX) was recently approved for the treatment of cholestatic pruritus in patients with Alagille syndrome. The aim of this study was to determine whether specific changes in the composition of the serum bile acid metabolome could predict pruritus response to treatment. Serum BAs (sBA) and 7α-hydroxy-4-cholesten-3-one (7α-C4), a surrogate marker of BA synthesis, were monitored by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry over 72 weeks in PFIC patients with mild to moderate non-truncating bile salt export pump (BSEP) mutations (n = 19) treated with MRX. The weekly itch reported outcome observer (ItchRO[Obs]) score measured pruritus severity. Linear mixed models (LMM) were applied to explore the effects of individual sBA profiles and their relationship to pruritus response. Changes in the composition of sBA correlated with pruritus improvement. Notably, the trajectory of serum total and individual BA species and 7α-C4 were significantly associated with ItchRO[Obs] score (p < 0.05). These results reveal that beyond simple total sBA concentrations, specific changes to the BA metabolome are associated with pruritus reduction in patients with BSEP deficiency, thus providing further insight into causal relationship of bile acids and pruritus.

Substrate Reduction Therapy Reverses Mitochondrial, mTOR, and Autophagy Alterations in a Cell Model of Gaucher Disease.

Substrate reduction therapy (SRT) in clinic adequately manages the visceral manifestations in Gaucher disease (GD) but has no direct effect on brain disease. To understand the molecular basis of SRT in GD treatment, we evaluated the efficacy and underlying mechanism of SRT in an immortalized neuronal cell line derived from a Gba knockout (Gba-/-) mouse model. Gba-/- neurons accumulated substrates, glucosylceramide, and glucosylsphingosine. Reduced cell proliferation was associated with altered lysosomes and autophagy, decreased mitochondrial function, and activation of the mTORC1 pathway. Treatment of the Gba-/- neurons with venglustat analogue GZ452, a central nervous system-accessible SRT, normalized glucosylceramide levels in these neurons and their isolated mitochondria. Enlarged lysosomes were reduced in the treated Gba-/- neurons, accompanied by decreased autophagic vacuoles. GZ452 treatment improved mitochondrial membrane potential and oxygen consumption rate. Furthermore, GZ452 diminished hyperactivity of selected proteins in the mTORC1 pathway and improved cell proliferation of Gba-/- neurons. These findings reinforce the detrimental effects of substrate accumulation on mitochondria, autophagy, and mTOR in neurons. A novel rescuing mechanism of SRT was revealed on the function of mitochondrial and autophagy-lysosomal pathways in GD. These results point to mitochondria and the mTORC1 complex as potential therapeutic targets for treatment of GD.

Successful treatment of infantile oxysterol 7α-hydroxylase deficiency with oral chenodeoxycholic acid.

BACKGROUND: Deficiency of oxysterol 7α-hydroxylase, encoded by CYP7B1, is associated with fatal infantile progressive intrahepatic cholestasis and hereditary spastic paraplegia type 5. Most reported patients with CYP7B1 mutations presenting with liver disease in infancy have died of liver failure. However, it was recently reported that two patients treated with chenodeoxycholic acid survived. Correlations between the phenotype and genotype of CYP7B1 deficiency have not been clearly established. CASE PRESENTATION: A 5-month-7-day-old Chinese baby from non-consanguineous parents was referred for progressive cholestasis and prolonged prothrombin time from one month of age. Genetic testing revealed compound heterozygous mutations c.187C > T(p.R63X)/c.334C > T(p.R112X) in CYP7B1, and fast atom bombardment mass spectrometry analysis of the urinary bile acid confirmed the presence of atypical hepatotoxic 3ß-hydroxy-Δ5-bile acids. While awaiting liver transplantation she was orally administered chenodeoxycholic acid. Her liver function rapidly improved, urine atypical bile acids normalized, and she thrived well until the last follow-up at 23 months of age. Her 15-year-old brother, with no history of infantile cholestasis but harboring the same mutations in CYP7B1, had gait abnormality from 13 years of age. Neurological examination revealed hyper-reflexia and spasticity of the lower limbs. Brain MRI revealed enlarged perivascular space in the bilateral basal ganglia and white matter of frontal parietal. CONCLUSIONS: In summary, these findings highlight that the phenotype of CYP7B1 deficiency varies widely, even in siblings and that early administration of chenodeoxycholic acid may improve prognosis.

Modeling Human Bile Acid Transport and Synthesis in Stem Cell-Derived Hepatocytes with a Patient-Specific Mutation.

The bile salt export pump (BSEP) is responsible for the export of bile acid from hepatocytes. Impaired transcellular transport of bile acids in hepatocytes with mutations in BSEP causes cholestasis. Compensatory mechanisms to regulate the intracellular bile acid concentration in human hepatocytes with BSEP deficiency remain unclear. To define pathways that prevent cytotoxic accumulation of bile acid in hepatocytes, we developed a human induced pluripotent stem cell-based model of isogenic BSEP-deficient hepatocytes in a Transwell culture system. Induced hepatocytes (i-Heps) exhibited defects in the apical export of bile acids but maintained a low intracellular bile acid concentration by inducing basolateral export. Modeling the autoregulation of bile acids on hepatocytes, we found that BSEP-deficient i-Heps suppressed de novo bile acid synthesis using the FXR pathway via basolateral uptake and export without apical export. These observations inform the development of therapeutic targets to reduce the overall bile acid pool in patients with BSEP deficiency.

Obeticholic acid ameliorates severity of Clostridioides difficile infection in high fat diet-induced obese mice.

Severe Clostridiodes difficile infection (CDI) is life-threatening and responds poorly to treatment. Obesity is associated with development of severe CDI. Therefore, to define the mechanisms that exacerbate disease severity, we examined CDI pathogenesis in high-fat diet (HFD)-fed obese mice. Compared to control mice, HFD-fed mice failed to clear C. difficile bacteria which resulted in protracted diarrhea, weight loss and colonic damage. After infection, HFD-induced obese mice had an intestinal bile acid (BA) pool that was dominated by primary BAs which are known promoters of C. difficile spore germination, and lacked secondary BAs that inhibit C. difficile growth. Concurrently, synthesis of primary BAs from liver was significantly increased in C. difficile-infected HFD-fed mice. A key pathway that regulates hepatic BA synthesis is via feedback inhibition from intestinal Farnesoid X receptors (FXRs). Our data reveal that the proportion of FXR agonist BAs to FXR antagonist BAs in the intestinal lumen was significantly reduced in HFD-fed mice after CDI. Treatment of HFD-fed mice with an FXR agonist Obeticholic acid, resulted in decreased primary BA synthesis, fewer C. difficile bacteria and better CDI outcomes. Thus, OCA treatment holds promise as a therapy for severe CDI.

Assessment of the role of FGF15 in mediating the metabolic outcomes of murine Vertical Sleeve Gastrectomy (VSG).

Vertical sleeve gastrectomy (VSG) is the best current therapy for remission of obesity and its co-morbidities. It is understood to alter the enterohepatic circulation of bile acids in vivo. Fibroblast growth factor 19 (FGF19) in human and its murine orthologue Fgf15 plays a pivotal role in this bile acid driven enterohepatic signaling. The present study evaluated the metabolic outcomes of VSG in Fgf15 deficient mice. 6-8 weeks old male wildtype mice (WT) and Fgf15 deficient mice (KO) were fed a high fat diet (HFD) for 8 weeks. At 8th week of diet, both WT and KO mice were randomly distributed to VSG or sham surgery. Post-surgery, mice were observed for 8 weeks while fed a HFD and then euthanized to collect tissues for experimental analysis. Fgf15 deficient (KO) mice lost weight post VSG, but glucose tolerance in KO mice did not improve post VSG compared to WT mice. Enteroids derived from WT and KO mice proliferated with bile acid exposure in vitro. Post VSG both WT and KO mice had similarly altered bile acid enterohepatic flux, however Fgf15 deficient mice post VSG had increased hepatic accumulation of free and esterified cholesterol leading to lipotoxicity related ER stress, inflammasome activation, and increased Fgf21 expression. Intact Fgf15 mediated enterohepatic bile acid signaling, but not changes in bile acid flux, appear to be important for the metabolic improvements post-murine bariatric surgery. These novel data introduce a potential point of distinction between bile acids acting as ligands compared to their canonical downstream signaling pathways.

Combination of acid ß-glucosidase mutation and Saposin C deficiency in mice reveals Gba1 mutation dependent and tissue-specific disease phenotype.

Gaucher disease is caused by mutations in GBA1 encoding acid ß-glucosidase (GCase). Saposin C enhances GCase activity and protects GCase from intracellular proteolysis. Structure simulations indicated that the mutant GCases, N370S (0 S), V394L (4L) and D409V(9V)/H(9H), had altered function. To investigate the in vivo function of Gba1 mutants, mouse models were generated by backcrossing the above homozygous mutant GCase mice into Saposin C deficient (C*) mice. Without saposin C, the mutant GCase activities in the resultant mouse tissues were reduced by ~50% compared with those in the presence of Saposin C. In contrast to 9H and 4L mice that have normal histology and life span, the 9H;C* and 4L;C* mice had shorter life spans. 9H;C* mice developed significant visceral glucosylceramide (GC) and glucosylsphingosine (GS) accumulation (GC¼GS) and storage macrophages, but lesser GC in the brain, compared to 4L;C* mice that presents with a severe neuronopathic phenotype and accumulated GC and GS primarily in the brain. Unlike 9V mice that developed normally for over a year, 9V;C* pups had a lethal skin defect as did 0S;C* mice resembled that of 0S mice. These variant Gaucher disease mouse models presented a mutation specific phenotype and underscored the in vivo role of Saposin C in the modulation of Gaucher disease.

Serum Unconjugated Bile Acids and Small Bowel Bacterial Overgrowth in Pediatric Intestinal Failure: A Pilot Study.

BACKGROUND: We determined qualitative and quantitative serum unconjugated bile acid (SUBA) levels among children with history of intestinal failure (IF) and suspected small bowel bacterial overgrowth (SBBO). METHODS: This was a single-center, case-control pilot study conducted at Cincinnati Children's Hospital Medical Center. Children with history of IF and suspected SBBO were enrolled as subjects. Age-matched children without IF or suspected SBBO served as controls. All participants underwent small bowel fluid sampling for microbial culture analysis. Additionally, serum fractionated and total bile acids were measured by liquid chromatography-mass spectrometry at enrollment and following treatment for SBBO. RESULTS: SUBA concentrations were elevated in IF subjects (median 1.16 µM, range 0.43-10.65 µM) compared with controls (median 0.10 µM, range 0.05-0.18 µM, P = 0.001). Among SUBA, chenodeoxycholic acid (CDCA) was significantly elevated in subjects (median 0.8 µM, range 0-7.08 µM) compared with controls (median 0 µM, range 0-0.03 µM, P = 0.012). When controls were excluded from analysis, IF subjects with positive aspirates for SBBO demonstrated higher concentration of CDCA (median 7.36 µM, range 1.1-8.28 µM) compared with IF subjects with negative aspirates (median 0.18 µM, range 0-1.06 µM, P = 0.017). Treatment for SBBO did not alter SUBA concentration. CONCLUSIONS: SUBA concentrations are elevated in children with history of IF and presumed SBBO compared with non-IF controls. CDCA was more prevalent in IF subjects with positive aspirates for SBBO compared with IF subjects with negative aspirates. The determination of SUBA concentration may be a useful surrogate to small bowel fluid aspiration in the diagnosis of SBBO in children with history of IF.

Plasma glucosylceramides and cardiovascular risk in incident hemodialysis patients.

BACKGROUND: Recent population-based studies identified plasma sphingolipids as independent predictors of increased cardiovascular disease (CVD) morbidity and mortality. Understanding the impact of sphingolipids on CVD outcomes in patients on dialysis, who suffer from higher risk of these conditions, is important for risk assessment and treatment. OBJECTIVE: To measure plasma sphingolipid levels and determine their associations with CVD in adults initiating maintenance hemodialysis. METHODS: To evaluate associations of plasma sphingolipids with intermediate cardiovascular outcomes (hypertension, left ventricular hypertrophy, and decreased ejection fraction), cardiovascular mortality, and all-cause mortality in patients with end-stage renal disease, we measured plasma levels of ceramides, glucosylceramides, and lactosylceramides from the family of sphingolipids in 368 incident hemodialysis patients enrolled in the Predictors of Arrhythmic and Cardiovascular Risk in End-Stage Renal Disease study. RESULTS: Glucosylceramide C16GC (per 1 log µM increase) was associated with higher odds of having uncontrolled hypertension (odds ratio [OR]: 1.34; 95% confidential interval [CI]: 1.01-1.76), left ventricular hypertrophy (OR: 1.53; 95% CI: 1.11-2.13), and reduced ejection fraction (OR: 1.05; 95% CI: 1.00-1.11) in fully adjusted models. During a median 2.5 years of follow-up, there were 78 deaths from all causes, of which 33 were from CVD. Mortality was higher among those in the highest tertile of C16GC for all causes (HR: 1.81; 95% CI: 1.02-3.22) and CVD (HR: 2.63, 95% CI: 1.08-6.55). CONCLUSIONS: These results suggest that abnormal glycosphingolipid metabolism might contribute to increased CVD risk in end-stage renal disease.

Lipidomic Profiling Links the Fanconi Anemia Pathway to Glycosphingolipid Metabolism in Head and Neck Cancer Cells.

Purpose: Mutations in Fanconi anemia (FA) genes are common in sporadic squamous cell carcinoma of the head and neck (HNSCC), and we have previously demonstrated that FA pathway depletion in HNSCC cell lines stimulates invasion. The goal of our studies was to use a systems approach in order to define FA pathway-dependent lipid metabolism and to extract lipid-based signatures and effectors of invasion in FA-deficient cells.Experimental Design: We subjected FA-isogenic HNSCC keratinocyte cell lines to untargeted and targeted lipidomics analyses to discover novel biomarkers and candidate therapeutic targets in FA-deficient cells. Cellular invasion assays were carried out in the presence and absence of N-butyldeoxynojirimycin (NB-DNJ), a biosynthetic inhibitor of the newly identified class of gangliosides, to investigate the requirement of ganglioside upregulation in FA-deficient HNSCC cells.Results: The most notable element of the lipid profiling results was a consistent elevation of glycosphingolipids, and particularly the accumulation of gangliosides. Conversely, repression of this same class of lipids was observed upon genetic correction of FA patient-derived HNSCC cells. Functional studies demonstrate that ganglioside upregulation is required for HNSCC cell invasion driven by FA pathway loss. The motility of nontransformed keratinocytes in response to FA loss displayed a similar dependence, thus supporting early and late roles for the FA pathway in controlling keratinocyte invasion through lipid regulation.Conclusions: Elevation of glycosphingolipids including the ganglioside GM3 in response to FA loss stimulates invasive characteristics of immortalized and transformed keratinocytes. An inhibitor of glycosphingolipid biosynthesis NB-DNJ attenuates invasive characteristics of FA-deficient HNSCC cells. Clin Cancer Res; 24(11); 2700-9. ©2018 AACR.

Effect of bariatric surgery on urinary sphingolipids in adolescents with severe obesity.

BACKGROUND: Untreated severe obesity of adolescents is associated with abnormal kidney function and development of chronic kidney disease. Lipotoxicity due to lipid accumulation in glomeruli might be an important mechanism in the progression of kidney disease in obesity. OBJECTIVE: To assess subclinical glomerular injury by measuring urinary sphingolipids in adolescents with severe obesity before and after weight loss surgery. We hypothesized that the levels of urinary sphingolipids would be elevated at baseline and improve after weight reduction. SETTING: Cincinnati Children's Hospital Medical Center, University of Cincinnati. METHODS: Ten adolescents undergoing bariatric surgery with no microalbuminuria and normal kidney function were selected. Urinary sphingolipids (ceramides, glycosphingolipids, and sphingomyelins) were quantified using ultra performance liquid chromatography electrospray ionization tandem mass spectrometry at baseline and 1 year postoperatively. The levels of sphingolipids were compared with lean and moderately obese controls. RESULTS: Participants with severe obesity had a mean baseline body mass index of 50 kg/m2 that decreased to 36 kg/m2 at 1 year postsurgery (28% reduction). Almost all urinary ceramides, glycosphingolipids, and sphingomyelin species were significantly elevated in participants with severe obesity compared with controls at baseline (P<.01). One year after weight loss surgery, levels of urinary sphingolipids improved but were still significantly elevated compared with controls. CONCLUSIONS: Our study indicates that severe obesity is associated with increased urinary excretion of sphingolipids despite the absence of microalbuminuria or decreased kidney function. Urinary sphingolipids may therefore represent a marker of early (subclinical) glomerular injury in adolescents with severe obesity.

Severe Neonatal Cholestasis in Cerebrotendinous Xanthomatosis: Genetics, Immunostaining, Mass Spectrometry.

OBJECTIVES: Cerebrotendinous xanthomatosis (CTX) is caused by defects in sterol 27-hydroxylase (CYP27A1, encoded by CYP27A1), a key enzyme in the bile acid synthesis pathway. CTX usually presents as neurologic disease in adults or older children. The rare reports of CTX manifest as neonatal cholestasis assess the cholestasis as transient, with patient survival. Our experience differs. METHODS: Homozygous or compound heterozygous CYP27A1 mutations were detected in 8 neonatal cholestasis patients by whole exome sequencing, panel sequencing, or Sanger sequencing. Their clinical and biochemical data were retrospectively reviewed. Immunostaining for CYP27A1 was conducted in liver of 4 patients. Mass spectrometry was used to analyze patients' urine samples. RESULTS: All 8 infants had severe cholestasis. Five died from, or were transplanted for, liver failure; 3 cleared their jaundice eventually. Marking for CYP27A1 was weak or absent in 3 of the 4 patient specimens. Mass spectrometry of urine revealed a predominance of sulfated and doubly conjugated (sulfated-glucuronidated) bile alcohols. No patient harbored a putatively pathogenic mutation in genes other than CYP27A1 that have been implicated in cholestatic liver disease. CONCLUSIONS: CTX manifest as neonatal cholestasis has a bile acid profile different from CTX manifest in later life, and thus may be overlooked. Immunostaining, mass spectrometry of urine, and genetic studies can support one another in making the diagnosis. A substantial proportion of CTX patients with severe neonatal cholestasis may die or need liver transplantation. CTX manifest in infancy as severe cholestasis warrants further investigation of biochemical diagnostic criteria and best management.

A convenient approach to facilitate monitoring Gaucher disease progression and therapeutic response.

Gaucher disease (GD) is caused by mutations on the GBA1 gene leading to deficiency in acid ß-glucosidase (GCase) and subsequent accumulation of its substrates, glucosylceramide (GlcC) and glucosylsphingosine (GlcS). GlcS in plasma has been proposed as a highly sensitive and specific biomarker for the diagnosis of GD and for monitoring disease progression and response to therapy. Here we report a novel robust and accurate hydrophilic interaction liquid chromatography tandem mass spectrometric method (HILIC-MS/MS) for the direct measurement of glucosylsphingosine (GlcS) in dried plasma spots (DPS). The method was also capable of resolving the isomeric pair, glucosylsphingosine and galactosylsphingosine, the latter of which was proposed as a promising biomarker for Krabbe disease. The method was fully validated and applied to the analysis of 19 GD patients and carriers. The GlcS levels in 9 GD type I patients who have been on enzyme replacement therapy (ERT) were reduced to a mean of 31.0 nM, much lower compared to a pre-treated specimen at a level of 85.8 nM, but still significantly elevated compared to healthy controls. GlcS concentrations in three treated type III GD patients were much lower compared to an untreated patient. In our preclinical GD studies, 4L;C* mice (subacute nGD model) exhibited comparable levels of plasma GlcS, but had much higher GlcS accumulation in the brain than those of 9V/null mice (chronic neuropathic GD model). Our method for the measurement of GlcS in DPS proved to be a very convenient approach for sample collection, storage and shipping nationwide and internationally.

Tissue Localization of Glycosphingolipid Accumulation in a Gaucher Disease Mouse Brain by LC-ESI-MS/MS and High-Resolution MALDI Imaging Mass Spectrometry.

To better understand regional brain glycosphingolipid (GSL) accumulation in Gaucher disease (GD) and its relationship to neuropathology, a feasibility study using mass spectrometry and immunohistochemistry was conducted using brains derived from a GD mouse model (4L/PS/NA) homozygous for a mutant GCase (V394L [4L]) and expressing a prosaposin hypomorphic (PS-NA) transgene. Whole brains from GD and control animals were collected using one hemisphere for MALDI FTICR IMS analysis and the other for quantitation by LC-ESI-MS/MS. MALDI IMS detected several HexCers across the brains. Comparison with the brain hematoxylin and eosin (H&E) revealed differential signal distributions in the midbrain, brain stem, and CB of the GD brain versus the control. Quantitation of serial brain sections with LC-ESI-MS/MS supported the imaging results, finding the overall HexCer levels in the 4L/PS-NA brains to be four times higher than the control. LC-ESI-MS/MS also confirmed that the elevated hexosyl isomers were glucosylceramides rather than galactosylceramides. MALDI imaging also detected differential analyte distributions of lactosylceramide species and gangliosides in the 4L/PS-NA brain, which was validated by LC-ESI-MS/MS. Immunohistochemistry revealed regional inflammation, altered autophagy, and defective protein degradation correlating with regions of GSL accumulation, suggesting that specific GSLs may have distinct neuropathological effects.

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.