Identification of a Novel Acid Sphingomyelinase Activity Associated with Recombinant Human Acid Ceramidase.

Acid ceramidase (AC) is a lysosomal enzyme required to hydrolyze ceramide to sphingosine by the removal of the fatty acid moiety. An inherited deficiency in this activity results in two disorders, Farber Lipogranulomatosis and spinal muscular atrophy with myoclonic epilepsy, leading to the accumulation of ceramides and other sphingolipids in various cells and tissues. In addition to ceramide hydrolysis, several other activities have been attributed to AC, including a reverse reaction that synthesizes ceramide from free fatty acids and sphingosine, and a deacylase activity that removes fatty acids from complex lipids such as sphingomyelin and glycosphingolipids. A close association of AC with another important enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM), has also been observed. Herein, we used a highly purified recombinant human AC (rhAC) and novel UPLC-based assay methods to investigate the recently described deacylase activity of rhAC against three sphingolipid substrates, sphingomyelin, galactosyl- and glucosylceramide. No deacylase activities were detected using this method, although we did unexpectedly identify a significant ASM activity using natural (C-18) and artificial (Bodipy-C12) sphingomyelin substrates as well as the ASM-specific fluorogenic substrate, hexadecanoylamino-4-methylumbelliferyl phosphorylcholine (HMU-PC). We showed that this ASM activity was not due to contaminating, hamster-derived ASM in the rhAC preparation, and that the treatment of ASM-knockout mice with rhAC significantly reduced sphingomyelin storage in the liver. However, unlike the treatment with rhASM, this did not lead to elevated ceramide or sphingosine levels.

Apolipoprotein-mimetic nanodiscs reduce lipid accumulation and improve liver function in acid sphingomyelinase deficiency.

Acid sphingomyelinase deficiency (ASMD) is a severe lipid storage disorder caused by the diminished activity of the acid sphingomyelinase enzyme. ASMD is characterized by the accumulation of sphingomyelin in late endosomes and lysosomes leading to progressive neurological dysfunction and hepatosplenomegaly. Our objective was to investigate the utility of synthetic apolipoprotein A-I (ApoA-I) mimetics designed to act as lipid scavengers for the treatment of ASMD. We determined the lead peptide, 22A, could reduce sphingomyelin accumulation in ASMD patient skin fibroblasts in a dose dependent manner. Intraperitoneal administration of 22A formulated as a synthetic high-density lipoprotein (sHDL) nanodisc mobilized sphingomyelin from peripheral tissues into circulation and improved liver function in a mouse model of ASMD. Together, our data demonstrates that apolipoprotein mimetics could serve as a novel therapeutic strategy for modulating the pathology observed in ASMD.

The cholesterol transporter NPC1 is essential for epigenetic regulation and maturation of oligodendrocyte lineage cells.

The intracellular cholesterol transporter NPC1 functions in late endosomes and lysosomes to efflux unesterified cholesterol, and its deficiency causes Niemann-Pick disease Type C, an autosomal recessive lysosomal disorder characterized by progressive neurodegeneration and early death. Here, we use single-nucleus RNA-seq on the forebrain of Npc1-/- mice at P16 to identify cell types and pathways affected early in pathogenesis. Our analysis uncovers significant transcriptional changes in the oligodendrocyte lineage during developmental myelination, accompanied by diminished maturation of myelinating oligodendrocytes. We identify upregulation of genes associated with neurogenesis and synapse formation in Npc1-/- oligodendrocyte lineage cells, reflecting diminished gene silencing by H3K27me3. Npc1-/- oligodendrocyte progenitor cells reproduce impaired maturation in vitro, and this phenotype is rescued by treatment with GSK-J4, a small molecule inhibitor of H3K27 demethylases. Moreover, mobilizing stored cholesterol in Npc1-/- mice by a single administration of 2-hydroxypropyl-ß-cyclodextrin at P7 rescues myelination, epigenetic marks, and oligodendrocyte gene expression. Our findings highlight an important role for NPC1 in oligodendrocyte lineage maturation and epigenetic regulation, and identify potential targets for therapeutic intervention.

Modulation of Dietary Choline Uptake in a Mouse Model of Acid Sphingomyelinase Deficiency.

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder caused by mutations in the gene-encoding acid sphingomyelinase (ASM). ASMD impacts peripheral organs in all patients, including the liver and spleen. The infantile and chronic neurovisceral forms of the disease also lead to neuroinflammation and neurodegeneration for which there is no effective treatment. Cellular accumulation of sphingomyelin (SM) is a pathological hallmark in all tissues. SM is the only sphingolipid comprised of a phosphocholine group linked to ceramide. Choline is an essential nutrient that must be obtained from the diet and its deficiency promotes fatty liver disease in a process dependent on ASM activity. We thus hypothesized that choline deprivation could reduce SM production and have beneficial effects in ASMD. Using acid sphingomyelinase knock-out (ASMko) mice, which mimic neurovisceral ASMD, we have assessed the safety of a choline-free diet and its effects on liver and brain pathological features such as altered sphingolipid and glycerophospholipid composition, inflammation and neurodegeneration. We found that the choline-free diet was safe in our experimental conditions and reduced activation of macrophages and microglia in the liver and brain, respectively. However, there was no significant impact on sphingolipid levels and neurodegeneration was not prevented, arguing against the potential of this nutritional strategy to assist in the management of neurovisceral ASMD patients.

Sphingomyelin 16:0 is a therapeutic target for neuronal death in acid sphingomyelinase deficiency.

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder caused by mutations in the SMPD1 gene encoding for the acid sphingomyelinase (ASM). While intravenous infusion of recombinant ASM is an effective treatment for the peripheral disease, the neurological complications of ASMD remain unaddressed. It has been shown that aberrantly high level of total brain sphingomyelin (SM) is a key pathological event leading to neurodegeneration. Using mice lacking ASM (ASMko), which mimic the disease, we here demonstrate that among the SM species, SM16:0 shows the highest accumulation and toxicity in ASMko neurons. By targeting lysosomes, SM16:0 causes permeabilization and exocytosis of these organelles and induces oxidative stress and cell death. We also show that genetic silencing of Ceramide Synthase 5, which is involved in SM16:0 synthesis and overexpressed in the ASMko brain, prevents disease phenotypes in ASMko cultured neurons and mice. The levels of SM16:0 in plasma also show a strong correlation with those in brain that is higher than in liver, even at early stages of the disease. These results identify SM16:0 both as a novel therapeutic target and potential biomarker of brain pathology in ASMD.

Immunotherapy targeting plasma ASM is protective in a mouse model of Alzheimer's disease.

Acid sphingomyelinase (ASM) has been implicated in neurodegenerative disease pathology, including Alzheimer's disease (AD). However, the specific role of plasma ASM in promoting these pathologies is poorly understood. Herein, we explore plasma ASM as a circulating factor that accelerates neuropathological features in AD by exposing young APP/PS1 mice to the blood of mice overexpressing ASM, through parabiotic surgery. Elevated plasma ASM was found to enhance several neuropathological features in the young APP/PS1 mice by mediating the differentiation of blood-derived, pathogenic Th17 cells. Antibody-based immunotherapy targeting plasma ASM showed efficient inhibition of ASM activity in the blood of APP/PS1 mice and, interestingly, led to prophylactic effects on neuropathological features by suppressing pathogenic Th17 cells. Our data reveals insights into the potential pathogenic mechanisms underlying AD and highlights ASM-targeting immunotherapy as a potential strategy for further investigation.

The clinical spectrum of SMA-PME and in vitro normalization of its cellular ceramide profile.

OBJECTIVE: The objectives of this study were to define the clinical and biochemical spectrum of spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) and to determine if aberrant cellular ceramide accumulation could be normalized by enzyme replacement. METHODS: Clinical features of 6 patients with SMA-PME were assessed by retrospective chart review, and a literature review of 24 previously published cases was performed. Leukocyte enzyme activity of acid ceramidase was assessed with a fluorescence-based assay. Skin fibroblast ceramide content and was assessed by high performance liquid chromatography, electrospray ionization tandem mass spectroscopy. Enzyme replacement was assessed using recombinant human acid ceramidase (rhAC) in vitro. RESULTS: The six new patients showed the hallmark features of SMA-PME, with variable initial symptom and age of onset. Five of six patients carried at least one of the recurrent SMA-PME variants observed in two specific codons of ASAH1. A review of 30 total cases revealed that patients who were homozygous for the most common c.125C > T variant presented in the first decade of life with limb-girdle weakness as the initial symptom. Sensorineural hearing loss was associated with the c.456A > C variant. Leukocyte acid ceramidase activity varied from 4.1%-13.1% of controls. Ceramide species in fibroblasts were detected and total cellular ceramide content was elevated by 2 to 9-fold compared to controls. Treatment with rhAC normalized ceramide profiles in cultured fibroblasts to control levels within 48 h. INTERPRETATION: This study details the genotype-phenotype correlations observed in SMA-PME and shows the impact of rhAC to correct the abnormal cellular ceramide profile in cells.

A 51-Year-Old Woman With Interstitial Lung Disease and Subsequent COVID-19 Presenting With Worsening Dyspnea.

CASE PRESENTATION: A 51-year-old Puerto Rican woman, with a known but inconclusive diagnosis of interstitial lung disease (ILD) since 2002 and recent moderate COVID-19, is now presenting with subacute worsening dyspnea on exertion. The patient had sporadic medical care over the years for her ILD (Table 1). Prior workup included chest CT imaging with a "crazy-paving" pattern of lung disease, as defined by ground-glass opacity with superimposed interlobular septal thickening and visible intralobular lines. Bronchoscopy showed normal airway examination, and BAL revealed clear fluid with foamy macrophages and negative cultures. Video-assisted thoracoscopic surgery and transbronchial biopsy specimens both showed foamy macrophages. Results of pulmonary function testing (PFT) revealed an isolated gas transfer defect on diffusing capacity of the lungs for carbon monoxide (Dlco). She had lived with mild yet nonprogressive functional impairment and stable exercise intolerance over these years. She was then hospitalized for COVID-19 in August 2020 and for recurrent shortness of breath in September 2020. She now presented 4 months following her September 2020 hospitalization.

Discovery of a dual-action small molecule that improves neuropathological features of Alzheimer's disease mice.

Alzheimer's disease (AD) is characterized by complex, multifactorial neuropathology, suggesting that small molecules targeting multiple neuropathological factors are likely required to successfully impact clinical progression. Acid sphingomyelinase (ASM) activation has been recognized as an important contributor to these neuropathological features in AD, leading to the concept of using ASM inhibitors for the treatment of this disorder. Here we report the identification of KARI 201, a direct ASM inhibitor evaluated for AD treatment. KARI 201 exhibits highly selective inhibition effects on ASM, with excellent pharmacokinetic properties, especially with regard to brain distribution. Unexpectedly, we found another role of KARI 201 as a ghrelin receptor agonist, which also has therapeutic potential for AD treatment. This dual role of KARI 201 in neurons efficiently rescued neuropathological features in AD mice, including amyloid beta deposition, autophagy dysfunction, neuroinflammation, synaptic loss, and decreased hippocampal neurogenesis and synaptic plasticity, leading to an improvement in memory function. Our data highlight the possibility of potential clinical application of KARI 201 as an innovative and multifaceted drug for AD treatment.

Acid Ceramidase Protects Against Hepatic Ischemia/Reperfusion Injury by Modulating Sphingolipid Metabolism and Reducing Inflammation and Oxidative Stress.

Ceramide is a bioactive signaling lipid involved in the pathogenesis of numerous diseases. It also plays an important role in ischemia reperfusion (IR) injury via activation of inflammatory/oxidative stress-stimulated signaling pathways, resulting in tissue damage. Acid ceramidase is a lipid hydrolase that modulates the levels of ceramide, and as such has a potential therapeutic role in many human diseases where ceramide has been implicated. Here we investigated the therapeutic potential of recombinant acid ceramidase in a murine model of hepatic IR injury. Serum ALT, AST, and LDH activities, as well as oxidative stress (MDA) and inflammatory (MCP-1) markers, were increased in mice subjected to IR compared to a sham group. In contrast, these elevations were significantly lower in an IR group pretreated with a single injection of acid ceramidase. Histological examination by two different assessment criteria also revealed that acid ceramidase pretreatment alleviated IR-induced hepatocyte damage, including reduced evidence of cell death and necrosis. In addition, elevated ceramide and sphingosine levels were observed in the IR group compared to sham, and were markedly reduced when pretreated with acid ceramidase. In contrast, the levels of the protective signaling lipid, sphingosine-1-phosphate (S1P), were reduced following IR and elevated in response to acid ceramidase pretreatment. These changes in sphingolipid levels could be correlated with changes in the activities of several sphingolipid-metabolizing enzymes. Overall, these results indicated that sphingolipid changes were an important pathologic component of hepatic IR injury, and that acid ceramidase administration ameliorated these lipid changes and other downstream pathologic changes.

New paradigms for the treatment of lysosomal storage diseases: targeting the endocannabinoid system as a therapeutic strategy.

Over the past three decades the lysosomal storage diseases have served as model for rare disease treatment development. While these efforts have led to considerable success, important challenges remain. For example, no treatments are currently approved for nearly two thirds of all lysosomal diseases, and there is limited impact of the existing drugs on the central nervous system. In addition, the costs of these therapies are extremely high, in part due to the fact that drug development has focused on a "single hit" approach - i.e., one drug for one disease. To overcome these obstacles researchers have begun to focus on defining common disease mechanisms in the lysosomal diseases, particularly in the central nervous system, with the hope of identifying drugs that might be used in several lysosomal diseases rather than an individual disease. With this concept in mind, herein we review a new potential treatment approach for the lysosomal storage diseases that focuses on modulation of the endocannabinoid system. We provide a short introduction to lysosomal storage diseases and the endocannabinoid system, followed by a brief review of data supporting this concept.

SiO2 stimulates macrophage stress to induce the transformation of lung fibroblasts into myofibroblasts and its relationship with the sphingomyelin metabolic pathway.

Silicosis is a serious occupational disease with the highest incidence in China. However, its pathogenesis has not been fully elucidated. Studies have shown that the sphingomyelin signaling pathway may play an important role in different fibrotic diseases but its role in silicosis-mediated fibrosis is still unclear. In this study, the supernatant of human peripheral blood mononuclear cell line (THP-1)-derived macrophages exposed to silica (SiO2 ) was used to stimulate the transformation of human embryonic lung fibroblast cell line (HFL-1) into myofibroblasts, and the intervention effect of recombinant human acid ceramidase (rAC) was observed. The results showed that SiO2 stimulated the production of reactive oxygen species and malondialdehyde in the supernatant of THP-1-derived macrophages and increased the secretion of TGF-ß1, TNF-α, and IL-8. In addition, we found that the expression levels of α-SMA, FN, Col I, and Col III in HFL-1 cells increased. Meanwhile, the activities of ASMase and ACase and the expression levels of Cer, Sph, and S1P were increased. Intervention by rAC can suppress these changes to different degrees. In conclusion, the present study shows that SiO2 dust poisoning may stimulate HFL-1 cell differentiation into myofibroblasts by inducing oxidative stress in THP-1-derived macrophages, thereby promoting the secretion of a variety of inflammatory factors and activating the sphingolipid signaling pathway in HFL-1 cells. Exogenous rAC can effectively interfere with the stimulation of HFL-1 cells by silica in vitro.

Ceramides are necessary and sufficient for diet-induced impairment of thermogenic adipocytes.

OBJECTIVE: Aging and weight gain lead to a decline in brown and beige adipocyte functionality that exacerbates obesity and insulin resistance. We sought to determine whether sphingolipids, such as ceramides, a class of lipid metabolites that accumulate in aging and overnutrition, are sufficient or necessary for the metabolic impairment of these thermogenic adipocytes. METHODS: We generated new mouse models allowing for the conditional ablation of genes required for ceramide synthesis (i.e., serine palmitoyltransferase subunit 2, Sptlc2) or degradation (i.e., acid ceramidase 1, Asah1) from mature, thermogenic adipocytes (i.e., from cells expressing uncoupling protein-1). Mice underwent a comprehensive suite of phenotyping protocols to assess energy expenditure and glucose and lipid homeostasis. Complementary studies were conducted in primary brown adipocytes to dissect the mechanisms controlling ceramide synthesis or action. RESULTS: Depletion of Sptlc2 increased energy expenditure, improved glucose homeostasis, and prevented diet-induced obesity. Conversely, depletion of Asah1 led to ceramide accumulation, diminution of energy expenditure, and exacerbation of insulin resistance and obesity. Mechanistically, ceramides slowed lipolysis, inhibited glucose uptake, and decreased mitochondrial respiration. Moreover, ß-adrenergic receptor agonists, which activate thermogenesis in brown adipocytes, decreased transcription of enzymes required for ceramide synthesis. CONCLUSIONS: These studies support our hypothesis that ceramides are necessary and sufficient for the impairment in thermogenic adipocyte function that accompanies obesity. Moreover, they suggest that implementation of therapeutic strategies to block ceramide synthesis in thermogenic adipocytes may serve as a means of improving adipose health and combating obesity and cardiometabolic disease.

A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots

Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the method.

Inhibition of fatty acid amide hydrolase prevents pathology in neurovisceral acid sphingomyelinase deficiency by rescuing defective endocannabinoid signaling.

Acid sphingomyelinase deficiency (ASMD) leads to cellular accumulation of sphingomyelin (SM), neurodegeneration, and early death. Here, we describe the downregulation of the endocannabinoid (eCB) system in neurons of ASM knockout (ASM-KO) mice and a ASMD patient. High SM reduced expression of the eCB receptor CB1 in neuronal processes and induced its accumulation in lysosomes. Activation of CB1 receptor signaling, through inhibition of the eCB-degrading enzyme fatty acid amide hydrolase (FAAH), reduced SM levels in ASM-KO neurons. Oral treatment of ASM-KO mice with a FAAH inhibitor prevented SM buildup; alleviated inflammation, neurodegeneration, and behavioral alterations; and extended lifespan. This treatment showed benefits even after a single administration at advanced disease stages. We also found CB1 receptor downregulation in neurons of a mouse model and a patient of another sphingolipid storage disorder, Niemann-Pick disease type C (NPC). We showed the efficacy of FAAH inhibition to reduce SM and cholesterol levels in NPC patient-derived cells and in the brain of a NPC mouse model. Our findings reveal a pathophysiological crosstalk between neuronal SM and the eCB system and offer a new treatment for ASMD and other sphingolipidoses.

Apolipoprotein D-mediated preservation of lysosomal function promotes cell survival and delays motor impairment in Niemann-Pick type A disease.

Lysosomal Storage Diseases (LSD) are genetic diseases causing systemic and nervous system dysfunction. The glia-derived lipid binding protein Apolipoprotein D (ApoD) is required for lysosomal functional integrity in glial and neuronal cells, ensuring cell survival upon oxidative stress or injury. Here we test whether ApoD counteracts the pathogenic consequences of a LSD, Niemann Pick-type-A disease (NPA), where mutations in the acid sphingomyelinase gene result in sphingomyelin accumulation, lysosomal permeabilization and early-onset neurodegeneration. We performed a multivariable analysis of behavioral, cellular and molecular outputs in 12 and 24 week-old male and female NPA model mice, combined with ApoD loss-of-function mutation. Lack of ApoD in NPA mice accelerates cerebellar-dependent motor deficits, enhancing loss of Purkinje neurons. We studied ApoD expression in brain sections from a NPA patient and age-matched control, and the functional consequences of ApoD supplementation in primary human fibroblasts from two independent NPA patients and two control subjects. Cell viability, lipid peroxidation, and lysosomal functional integrity (pH, Cathepsin B activity, Galectin-3 exclusion) were examined. ApoD is endogenously overexpressed in NPA patients and NPA mouse brains and targeted to lysosomes of NPA patient cells, including Purkinje neurons and cultured fibroblasts. The accelerated lysosomal targeting of ApoD by oxidative stress is hindered in NPA fibroblasts, contributing to NPA lysosomes vulnerability. Exogenously added ApoD reduces NPA-prompted lysosomal permeabilization and alkalinization, reverts lipid peroxides accumulation, and significantly increases NPA cell survival. ApoD administered simultaneously to sphingomyelin overload results in complete rescue of cell survival. Our results reveal that ApoD protection of lysosomal integrity counteracts NPA pathology. ApoD supplementation could significantly delay not only the progression of NPA disease, but also of other LSDs through its beneficial effects in lysosomal functional maintenance.

Recombinant Acid Ceramidase Reduces Inflammation and Infection in Cystic Fibrosis.

Rationale: In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems.Objectives: To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection.Methods: Sphingolipids were measured using mass spectrometry in fully differentiated cultures of primary human airway epithelial cells and cocultures with Pseudomonas aeruginosa. In situ activity assays, Western blotting, and quantitative PCR were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models.Measurements and Main Results: Ceramide is increased in cystic fibrosis airway epithelium owing to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to P. aeruginosa by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection.Conclusions: Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration.

N-AS-triggered SPMs are direct regulators of microglia in a model of Alzheimer's disease.

Sphingosine kinase1 (SphK1) is an acetyl-CoA dependent acetyltransferase which acts on cyclooxygenase2 (COX2) in neurons in a model of Alzheimer's disease (AD). However, the mechanism underlying this activity was unexplored. Here we show that N-acetyl sphingosine (N-AS) is first generated by acetyl-CoA and sphingosine through SphK1. N-AS then acetylates serine 565 (S565) of COX2, and the N-AS-acetylated COX2 induces the production of specialized pro-resolving mediators (SPMs). In a mouse model of AD, microglia show a reduction in N-AS generation, leading to decreased acetyl-S565 COX2 and SPM production. Treatment with N-AS increases acetylated COX2 and N-AS-triggered SPMs in microglia of AD mice, leading to resolution of neuroinflammation, an increase in microglial phagocytosis, and improved memory. Taken together, these results identify a role of N-AS in the dysfunction of microglia in AD.

Safety Study of Sodium Pentosan Polysulfate for Adult Patients with Mucopolysaccharidosis Type II.

Current therapies for the mucopolysaccharidoses (MPS) do not effectively address skeletal and neurological manifestations. Pentosan polysulfate (PPS) is an alternative treatment strategy that has been shown to improve bone architecture, mobility, and neuroinflammation in MPS animals. The aims of this study were to a) primarily establish the safety of weekly PPS injections in attenuated MPS II, b) assess the efficacy of treatment on MPS pathology, and c) define appropriate clinical endpoints and biomarkers for future clinical trials. Subcutaneous injections were administered to three male Japanese patients for 12 weeks. Enzyme replacement therapy was continued in two of the patients while they received PPS and halted for two months in one patient before starting PPS. During treatment, one patient experienced an elevation of alanine transaminase, and another patient experienced convulsions; however, these incidences were non-cumulative and unrelated to PPS administration, respectively. Overall, the drug was well-tolerated in all patients, and no serious drug-related adverse events were noted. Generally, PPS treatment led to an increase in several parameters of shoulder range of motion and decrease of the inflammatory cytokines, MIF and TNF-α, which are potential clinical endpoints and biomarkers, respectively. Changes in urine and serum glycosaminoglycans were inconclusive. Overall, this study demonstrates the safety of using PPS in adults with MPS II and suggests the efficacy of PPS on MPS pathology with the identification of potential clinical endpoints and biomarkers.

Control of CD1d-restricted antigen presentation and inflammation by sphingomyelin.

Invariant natural killer T (iNKT) cells recognize activating self and microbial lipids presented by CD1d. CD1d can also bind non-activating lipids, such as sphingomyelin. We hypothesized that these serve as endogenous regulators and investigated humans and mice deficient in acid sphingomyelinase (ASM), an enzyme that degrades sphingomyelin. We show that ASM absence in mice leads to diminished CD1d-restricted antigen presentation and iNKT cell selection in the thymus, resulting in decreased iNKT cell levels and resistance to iNKT cell-mediated inflammatory conditions. Defective antigen presentation and decreased iNKT cells are also observed in ASM-deficient humans with Niemann-Pick disease, and ASM activity in healthy humans correlates with iNKT cell phenotype. Pharmacological ASM administration facilitates antigen presentation and restores the levels of iNKT cells in ASM-deficient mice. Together, these results demonstrate that control of non-agonistic CD1d-associated lipids is critical for iNKT cell development and function in vivo and represents a tight link between cellular sphingolipid metabolism and immunity.