Analysis of Globotriaosylceramide (Gb3) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry.

Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine-D3, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curve Support Protocol 3: Preparation of the Gb3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.

Usefulness of antibody-drug conjugate as preconditioning for hematopoietic stem cell-targeted gene therapy in wild-type and Fabry disease mouse models.

BACKGROUND: Fabry disease (FD) is characterized by deficient activity of α-galactosidase A (GLA). Consequently, globotriaosylceramide (Gb3) accumulates in various organs, causing cardiac, renal, and cerebrovascular damage. Gene therapies for FD have been investigated in humans. Strong conditioning is required for hematopoietic stem cell-targeted gene therapy (HSC-GT). However, strong conditioning leads to various side effects and should be avoided. In this study, we tested antibody-based conditioning for HSC-GT in wild-type and FD model mice. METHODS: After preconditioning with an antibody-drug conjugate, HSC-GT using a lentiviral vector was performed in wild-type and Fabry model mice. In the wild-type experiment, the EGFP gene was introduced into HSCs and transplanted into preconditioned mice, and donor chimerism and EGFP expression were analyzed. In the FD mouse model, the GLA gene was introduced into HSCs and transplanted into preconditioned Fabry mice. GLA activity and Gb3 accumulation in the organs were analyzed. RESULTS: In the wild-type mouse experiment, when anti-CD45 antibody-drug conjugate was used, the percentage of donor cells at 6 months was 64.5%, and 69.6% of engrafted donor peripheral blood expressed EGFP. When anti-CD117 antibody-drug conjugate and ATG were used, the percentage of donor cells at 6 months was 80.7%, and 73.4% of engrafted donor peripheral blood expressed EGFP. Although large variations in GLA activity among mice were observed in the FD mouse experiment for both preconditioning regimens, Gb3 was significantly reduced in many organs. CONCLUSIONS: Antibody-based preconditioning may be an alternative preconditioning strategy for HSC-GT for treating FD.

Mass cytometry reveals atypical immune profile notably impaired maturation of memory CD4 T with Gb3-related CD27 expression in CD4 T cells in Fabry disease.

Fabry disease (FD) is an X-linked disease characterized by an accumulation of glycosphingolipids, notably of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3) leading to renal failure, cardiomyopathy, and cerebral strokes. Inflammatory processes are involved in the pathophysiology. We investigated the immunological phenotype of peripheral blood mononuclear cells in Fabry patients depending on the clinical phenotype, treatment, Gb3, and lysoGb3 levels and the presence of anti-drug antibodies (ADA). Leucocytes from 41 male patients and 20 controls were analyzed with mass cytometry using both unsupervised and supervised algorithms. FD patients had an increased expression of CD27 and CD28 in memory CD45- and CD45 + CCR7-CD4 T cells (respectively p < 0.014 and p < 0.02). Percentage of CD45RA-CCR7-CD27 + CD28+ cells in CD4 T cells was correlated with plasma lysoGb3 (r = 0.60; p = 0.0036) and phenotype (p < 0.003). The correlation between Gb3 and CD27 in CD4 T cells almost reached significance (r = 0.33; p = 0.058). There was no immune profile associated with the presence of ADA. Treatment with agalsidase beta was associated with an increased proportion of Natural Killer cells. These findings provide valuable insights for understanding FD, linking Gb3 accumulation to inflammation, and proposing new prognostic biomarkers.

Flow Cytometry-Based Assay to Detect Alpha Galactosidase Enzymatic Activity at the Cellular Level.

BACKGROUND: Fabry disease is a progressive, X chromosome-linked lysosomal storage disorder with multiple organ dysfunction. Due to the absence or reduced activity of alpha-galactosidase A (AGAL), glycosphingolipids, primarily globotriaosyl-ceramide (Gb3), concentrate in cells. In heterozygous women, symptomatology is heterogenous and currently routinely used fluorometry-based assays measuring mean activity mostly fail to uncover AGAL dysfunction. The aim was the development of a flow cytometry assay to measure AGAL activity in individual cells. METHODS: Conventional and multispectral imaging flow cytometry was used to detect AGAL activity. Specificity was validated using the GLA knockout (KO) Jurkat cell line and AGAL inhibitor 1-deoxygalactonojirimycin. The GLA KO cell line was generated via CRISPR-Cas9-based transfection, validated with exome sequencing, gene expression and substrate accumulation. RESULTS: Flow cytometric detection of specific AGAL activity is feasible with fluorescently labelled Gb3. In the case of Jurkat cells, a substrate concentration of 2.83 nmol/mL and 6 h of incubation are required. Quenching of the aspecific exofacial binding of Gb3 with 20% trypan blue solution is necessary for the specific detection of lysosomal substrate accumulation. CONCLUSION: A flow cytometry-based assay was developed for the quantitative detection of AGAL activity at the single-cell level, which may contribute to the diagnosis of Fabry patients.

Fabry disease in W162C mutation: a case report of two patients and a review of literature.

BACKGROUND: Fabry disease is a multisystemic disorder characterized by deposition of globotriaosylceramide (Gb3) and its deacylated form in multiple organs, sometimes localized in specific systems such as the nervous or cardiovascular system. As disease-modifying therapies are now available, early diagnosis is paramount to improving life quality and clinical outcomes. Despite the widespread use of non-invasive techniques for assessing organ damage, such as cardiac magnetic resonance imaging (MRI) for patients with cardiac disease, organ biopsy remains the gold standard to assess organ involvement. CASE PRESENTATION: The cases of two patients, father and daughter with a W162C mutation, are described. The father presented with late-onset, cardiac Fabry disease, subsequently developing systolic dysfunction and heart failure. His daughter, while asymptomatic and with normal cardiac assessment (except for slightly reduced native T1 values by cardiac MRI), had already initial myocyte Gb3 deposits on the endomyocardial biopsy, allowing her to start therapy precociously and potentially modifying the course of her disease. A review of the literature concerning the W162C mutation is then provided, showing that it is usually associated to classic, multisystemic Fabry disease rather than the cardiac-restricted form as in these two cases. CONCLUSIONS: Three main points can be concluded from this report. First, the W162C mutation can present with a more variegate phenotype than that predicted on a molecular basis. Second, endomyocardial biopsy was shown in this case to precede non-invasive investigation in determining organ involvement, justifying further studies on this potentially reliable technique, Third, difficulties can arise in the management of asymptomatic female carriers.

Phase 1 Healthy Volunteer Study of AL01211, an Oral, Non-brain Penetrant Glucosylceramide Synthase Inhibitor, to Treat Fabry Disease and Type 1 Gaucher Disease.

Glycosphingolipid (GSL) storage diseases are caused by deficiencies in the enzymes that metabolize different GSLs in the lysosome. Glucosylceramide synthase (GCS) inhibitors reduce GSL production and have potential to treat multiple GSL storage diseases. AL01211 is a potent, oral GCS inhibitor being developed for the treatment of Type 1 Gaucher disease and Fabry disease. AL01211 has minimal central nervous system penetration, allowing for treatment of peripheral organs without risking CNS-associated adverse effects. AL01211 was evaluated in a Phase 1 healthy volunteer study with single ascending dose (SAD) and multiple ascending dose (MAD) arms, to determine safety, pharmacokinetics including food effect, and pharmacodynamic effects on associated GSLs. In the SAD arm, AL01211 showed a Tmax of approximately 3.5 hours, mean clearance (CL/F) of 130.1 L/h, and t1/2 of 39.3 hours. Consuming a high-fat meal prior to dose administration reduced exposures 3.5-5.5-fold, indicating a food effect. In the MAD arm, AL01211 had an approximately 2-fold accumulation, reaching steady-state levels by 10 days. Increasing exposure inversely correlated with a decrease in GSL with plasma glucosylceramide and globotriacylceramide reduction from baseline levels, reaching 78% and 52% by day 14, respectively. AL01211 was generally well-tolerated with no AL01211 associated serious adverse events, thus supporting its further clinical development.

Effects of switching from agalsidase-α to agalsidase-ß on biomarkers, renal and cardiac parameters, and disease severity in fabry disease forming neutralizing antidrug antibodies: a case report.

Fabry disease is an X-linked hereditary disorder caused by deficient α-galactosidase A (GLA) activity. Patients with Fabry disease are often treated with enzyme replacement therapy (ERT). However, ERT often induces the formation of neutralizing antidrug antibodies (ADAs), which may impair the therapeutic efficacy. Here, we report the case of a 32-year-old man with Fabry disease and resultant neutralizing ADAs who was treated by switching from agalsidase-α to agalsidase-ß. We monitored biomarkers, such as plasma globotriaosylsphingosine (lyso-Gb3), urinary globotriaosylceramide (Gb3), urinary mulberry bodies, renal and cardiac parameters, and disease severity during the treatment period. Although plasma lyso-Gb3 and urinary Gb3 levels quickly decreased within two months after the initiation of ERT with agalsidase-α, they gradually increased thereafter. The urinary mulberry bodies continued to appear. Both the ADA titer and serum mediated GLA inhibition rates started to increase after two months. Moreover, 3.5 years after ERT, the vacuolated podocyte area in the renal biopsy decreased slightly from 23.1 to 18.9%. However, plasma lyso-Gb3 levels increased, and urinary Gb3, mulberry body levels, and ADA titers remained high. Therefore, we switched to agalsidase-ß which reduced, but did not normalize, plasma lyso-Gb3 levels and stabilized renal and cardiac parameters. Disease severity was attenuated. However, urinary Gb3 and mulberry body levels did not decrease noticeably in the presence of high ADA titers. The kidneys take up a small amount of the administered recombinant enzyme, and the clearance of Gb3 that has accumulated in the kidney may be limited despite the switching from agalsidase-α to agalsidase-ß.

Impact of GLA Variant Classification on the Estimated Prevalence of Fabry Disease: A Systematic Review and Meta-Analysis of Screening Studies.

BACKGROUND: The diagnosis of Fabry disease (FD) has relevant implications related to the management. Thus, a clear assignment of GLA variant pathogenicity is crucial. This systematic review and meta-analysis aimed to investigate the prevalence of FD in high-risk populations and newborns and evaluate the impact of different GLA variant classifications on the estimated prevalence of FD. METHODS: We searched the EMBASE and PubMed databases on February 21, 2023. Observational studies evaluating the prevalence of FD and reporting the identified GLA variants were included. GLA variants were re-evaluated for their pathogenicity significance using the American College of Medical Genetics and Genomics criteria and the ClinVar database. The pooled prevalence of FD among different settings was calculated. The study was registered on PROSPERO (CRD42023401663) and followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. RESULTS: Of the 3941 studies identified, 110 met the inclusion criteria. The pooled prevalence of FD was significantly different according to the clinical setting and criteria used for the pathogenicity assessment. Using the American College of Medical Genetics and Genomics criteria, the pooled prevalence was 1.2% in patients with left ventricular hypertrophy/hypertrophic cardiomyopathy (26 studies; 10 080 patients screened), 0.3% in end-stage renal disease/chronic kidney disease (38 studies; 62 050 patients screened), 0.7% in stroke (25 studies; 15 295 patients screened), 0.7% in cardiac conduction disturbance requiring pacemaker (3 studies; 1033 patients screened), 1.0% in small-fiber neuropathy (3 studies; 904 patients screened), and 0.01% in newborns (15 studies; 11 108 793 newborns screened). The pooled prevalence was different if the GLA variants were assessed using the ClinVar database, and most patients with a discrepancy in the pathogenicity assignment carried 1 of the following variants: p.A143T, p.D313Y, and p.E66Q. CONCLUSIONS: This systematic review and meta-analysis describe the prevalence of FD among newborns and high-risk populations, highlighting the need for a periodic reassessment of the GLA variants in the context of recent clinical, biochemical, and histological data. REGISTRATION: URL: https://crd.york.ac.uk/PROSPERO/; Unique identifier: CRD42023401663.

Fabry disease biomarkers in patients switched from enzyme-replacement therapy to migalastat oral chaperone therapy.

Background: A biomarker profile was evaluated longitudinally in patients with Fabry disease switched from enzyme-replacement therapy (ERT) to migalastat. Methods: 16 Gb3 isoforms and eight lyso-Gb3 analogues were analyzed in plasma and urine by LC-MS/MS at baseline and at three different time points in naive participants and participants switching from either agalsidase α or ß to migalastat. Results: 29 adult participants were recruited internationally (seven centers). The Mainz Severity Score Index and mean biomarker levels remained stable (p ≥ 0.05) over a minimum of 12 months compared with baseline following the treatment switch. Conclusion: In this cohort of patients with Fabry disease with amenable mutations, in the short term, a switch from ERT to migalastat did not have a marked effect on the average biomarker profile.

Prevalence of Kidney and Urinary Tract Complications in Fabry Disease from 2000 to 2020: A Global Cohort Study Including 10,637 Patients.

BACKGROUND/AIM: Fabry disease, an X-linked lysosomal storage disorder, causes progressive globotriaosylceramide accumulation in cells throughout the body. Characteristic multiorgan manifestations include renal dysfunction (Fabry nephropathy) and associated urinary tract complications. Enzyme replacement therapy (ERT) has been available since 2001, but contemporary real-world data are lacking regarding Fabry nephropathy risks and treatment outcomes. PATIENTS AND METHODS: This retrospective cohort study analyzed electronic medical records data for 10,637 Fabry disease patients from the TriNetX research database. Kidney and urinary tract outcomes were evaluated over two decades, 2000-2010 and 2011-2020. Outcomes assessed included chronic kidney disease (CKD), urinary tract infections, urinary incontinence, obstruction, renal insufficiency, and end-stage renal disease (ESRD). RESULTS: The prevalence of stage 4-5 CKD nearly doubled between 2000-2010 and 2011-2020, while ESRD prevalence rose over 4-fold. Incidence rates showed similar marked elevations across renal and urologic complications. Females and Black patients experienced disproportionate escalations in kidney and urinary tract morbidity. CONCLUSION: This large cohort study revealed significantly increased Fabry nephropathy and associated urologic complications over the past two decades, contradicting expectations of reduced morbidity with ERT availability. The findings highlight needs to optimize screening, treatment strategies, monitoring practices, and address disparities to curb rising disease burden and improve patient outcomes.

Systematic gene therapy derived from an investigative study of AAV2/8 vector gene therapy for Fabry disease.

BACKGROUND: Fabry disease (FD) is a progressive multisystemic disease characterized by a lysosomal enzyme deficiency. A lack of α-galactosidase A (α-Gal A) activity results in the progressive systemic accumulation of its substrates, including globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), which results in renal, cardiac, and/or cerebrovascular disease and early death. Enzyme replacement therapy (ERT) is the current standard of care for FD; however, it has important limitations, including a low half-life, limited distribution, and requirement of lifelong biweekly infusions of recombinant enzymes. METHODS: Herein, we evaluated a gene therapy approach using an episomal adeno-associated viral 2/8 (AAV2/8) vector that encodes the human GLA cDNA driven by a liver-specific expression cassette in a mouse model of FD that lacks α-Gal A activity and progressively accumulates Gb3 and Lyso-Gb3 in plasma and tissues. RESULTS: A pharmacology and toxicology study showed that administration of AAV2/8-hGLA vectors (AAV2/8-hGLA) in FD mice without immunosuppression resulted in significantly increased plasma and tissue α-Gal A activity and substantially normalized Gb3 and Lyso-Gb3 content. CONCLUSIONS: Moreover, the plasma enzymatic activity of α-Gal A continued to be stably expressed for up to 38 weeks and sometimes even longer, indicating that AAV2/8-hGLA is effective in treating FD mice, and that α-Gal A is continuously and highly expressed in the liver, secreted into plasma, and absorbed by various tissues. These findings provide a basis for the clinical development of AAV2/8-hGLA.

Catfish Egg Lectin Enhances the Cytotoxicity of Sunitinib on Gb3-Expressing Renal Cancer Cells.

Metastatic renal cell carcinoma (RCC) is not sufficiently responsive to anticancer drugs, and thus, developing new drugs for advanced RCC remains vital. We previously reported that the treatment of globotriaosylceramide (Gb3)-expressing cells with catfish (Silurus asotus) egg lectin (SAL) increased the intracellular uptake of propidium iodide (PI) and sunitinib (SU). Herein, we investigated whether SAL pretreatment affects the intracellular uptake and cytotoxic effects of molecular-targeted drugs in RCC cells. We analyzed Gb3 expression in TOS1, TOS3, TOS3LN, and ACHN human RCC cells. Surface Gb3 expression was higher in TOS1 and TOS3 cells than in TOS3LN and ACHN cells. In the PI uptake assay, 41.5% of TOS1 cells and 21.1% of TOS3 cells treated with SAL were positive for PI. TOS1 cell viability decreased to 70% after treatment with 25 µM SU alone and to 48% after pretreatment with SAL (50 µg/mL). Time-series measurements of the intracellular fluorescence of SU revealed significantly enhanced SU uptake in SAL-treated TOS1 cells compared to control cells. SAL treatment did not increase PI uptake in normal renal cells. Our findings suggest that adequate cytotoxic activity may be achieved even when SU is administered at a sufficiently low dose not to cause side effects in combination with SAL.

A Shiga Toxin B-Subunit-Based Lectibody Boosts T Cell Cytotoxicity towards Gb3-Positive Cancer Cells.

Aberrant glycosylation plays a crucial role in tumour progression and invasiveness. Tumour-associated carbohydrate antigens (TACAs) represent a valuable set of targets for immunotherapeutic approaches. The poor immunogenicity of glycan structures, however, requires a more effective and well-directed way of targeting TACAs on the surface of cancer cells than antibodies. The glycosphingolipid globotriaosylceramide (Gb3) is a well-established TACA present in a multitude of cancer types. Its overexpression has been linked to metastasis, invasiveness, and multidrug resistance. In the present study, we propose to use a dimeric fragment of the Shiga toxin B-subunit (StxB) to selectively target Gb3-positive cancer cells in a StxB-scFv UCHT1 lectibody. The lectibody, comprised of a lectin and the UCHT1 antibody fragment, was produced in E. coli and purified via Ni-NTA affinity chromatography. Specificity of the lectibody towards Gb3-positive cancer cell lines and specificity towards the CD3 receptor on T cells, was assessed using flow cytometry. We evaluated the efficacy of the lectibody in redirecting T cell cytotoxicity towards Gb3-overexpressing cancer cells in luciferase-based cytotoxicity in vitro assays. The StxB-scFv UCHT1 lectibody has proven specific for Gb3 and could induce the killing of up to 80% of Gb3-overexpressing cancer cells in haemorrhagic and solid tumours. The lectibody developed in this study, therefore, highlights the potential that lectibodies and lectins in general have for usage in immunotherapeutic approaches to boost the efficacy of established cancer treatments.

Circulating Anti-GB3 Antibody as a Biomarker of Myocardial Inflammation in Patients with Fabry Disease Cardiomyopathy.

BACKGROUND: Fabry disease cardiomyopathy (FDCM) has manifested some resistance to enzyme replacement therapy (ERT), particularly in its advanced phase. Recently, myocardial inflammation of autoimmune origin has been demonstrated in FDCM. AIMS: The objective of this study was the assessment of circulating anti-globotriaosylceramide (GB3) antibodies as potential biomarkers of myocardial inflammation in FDCM, defined by the additional presence of ≥CD3+ 7 T lymphocytes/low-power field associated with focal necrosis of adjacent myocytes. Its sensitivity was based on the evidence of overlapping myocarditis at left ventricular endomyocardial biopsy. METHODS AND RESULTS: From January 1996 to December 2021, 85 patients received a histological diagnosis of FDCM in our department and 48 (56.5%) of them had an overlapping myocardial inflammation with negative PCR for common cardiotropic viruses, positive antiheart, and antimyosin abs. The presence of anti-GB3 antibodies was evaluated with an in-house ELISA assay (BioGeM scarl Medical Investigational Research, MIR-Ariano Irpino, Italy), along with antiheart and antimyosin abs, in the FDCM patients and compared with control healthy individuals. The correlation between levels of circulating anti-GB3 autoantibody myocardial inflammation and FDCM severity was assessed. Anti-Gb3 antibodies were above the positivity cut-off in 87.5% of FDCM subjects with myocarditis (42 out of 48), while 81.1% of FDCM patients without myocarditis were identified as negative for Gb3 antibodies. Positive anti-Gb3 abs correlated with positive antiheart and antimyosin abs. CONCLUSIONS: The present study suggests a potential positive role of anti-GB3 antibodies as a marker of overlapping cardiac inflammation in patients with FDCM.

Migalastat como monoterapia oral para la enfermedad de Fabry

Contexto: la enfermedad de Fabry es una patología de depósito lisosomal poco frecuente, ligada al cromosoma X y causada por la deficiencia o ausencia de la enzima α-galactosidasa-A. La nefropatía, junto con la cardiopatía y el compromiso neurológico de la enfermedad, conduce a una muerte prematura. Objetivo: esta revisión describe la monoterapia oral con migalastat en pacientes con enfermedad de Fabry y mutaciones "amenables". Metodología: una chaperona farmacológica oral denominada migalastat (Galafold®), estabiliza y favorece el pasaje de formas mutadas "amenables" de la enzima hacia los lisosomas, aumentando así su actividad. Resultados: los estudios de fase III Facets y Attract demostraron seguridad y eficacia en comparación con las terapias de reemplazo enzimático disponibles, alcanzando estabilización de la función renal, reducción de la masa ventricular izquierda y estabilización del biomarcador plasmático Lyso-Gb3. Conclusiones: migalastat fue generalmente bien tolerado en ambos estudios. Publicaciones posteriores de extensión evidenciaron resultados similares, confirmando la seguridad y la eficacia, tanto en pacientes que previamente se encontraban con terapia de reemplazo enzimático y han sido rotados a migalastat, como también en pacientes que han iniciado migalastat como primer tratamiento.

Background: Fabry disease is a rare lysosomal storage disorder, linked to the X chromosome, and caused by the deficiency or absence of the enzyme α-galactosidase-A. Nephropathy together with heart disease and neurological involvement lead to premature death. Purpose: This review describes oral migalastat monotherapy in patients with Fabry disease and "amenable" mutations. Methodology: An oral pharmacological chaperone called Migalastat (Galafold®), stabilizes and facilitates the trafficking of "amenable" mutated forms of the enzyme to the lysosomes, thus increasing its activity. Results: The phase III FACETS and ATTRACT studies have demonstrated safety and efficacy compared to available enzyme replacement therapies; achieving renal function stabilization, reduction of left ventricular mass and maintenance of plasmatic Lyso-Gb3 levels. Conclusions: Migalastat was generally well tolerated in both trials. Subsequent extension publications showed similar results, confirming the safety and efficacy both in patients who were previously on enzyme replacement therapy and have been switched to migalastat, as well as in patients who have started migalastat as their first treatment.

Enzymatic Glyco-Modification of Synthetic Membrane Systems.

The present report assesses the capability of a soluble glycosyltransferase to modify glycolipids organized in two synthetic membrane systems that are attractive models to mimic cell membranes: giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs). The objective was to synthesize the Gb3 antigen (Galα1,4Galß1,4Glcß-Cer), a cancer biomarker, at the surface of these membrane models. A soluble form of LgtC that adds a galactose residue from UDP-Gal to lactose-containing acceptors was selected. Although less efficient than with lactose, the ability of LgtC to utilize lactosyl-ceramide as an acceptor was demonstrated on GUVs and SLBs. The reaction was monitored using the B-subunit of Shiga toxin as Gb3-binding lectin. Quartz crystal microbalance with dissipation analysis showed that transient binding of LgtC at the membrane surface was sufficient for a productive conversion of LacCer to Gb3. Molecular dynamics simulations provided structural elements to help rationalize experimental data.

Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease.

Fabry disease (FD) is an X-linked inherited lysosomal metabolism disorder in which globotriaosylceramide (Gb3) accumulates in various organs resulting from a deficiency in alpha-galactosidase A. The clinical features of FD include progressive impairments of the renal, cardiac, and peripheral nervous systems. In addition, patients with FD often develop neuropsychiatric symptoms, such as depression and dementia, which are believed to be induced by the cellular injury of cerebrovascular and partially neuronal cells due to Gb3 accumulation. Although the analysis of autopsy brain tissue from patients with FD showed no accumulation of Gb3, abnormal deposits of Gb3 were found in the neurons of several brain areas, including the hippocampus. Therefore, in this study, we generated induced pluripotent stem cells (iPSCs) from patients with FD and differentiated them into neuronal cells to investigate pathological and biological changes in the neurons of FD. Neural stem cells (NSCs) and neurons were successfully differentiated from the iPSCs we generated; however, cellular damage and morphological changes were not found in these cells. Immunostaining revealed no Gb3 accumulation in NSCs and neurons. Transmission electron microscopy did not reveal any zebra body-like structures or inclusion bodies, which are characteristic of FD. These results indicated that neuronal cells derived from FD-iPSCs exhibited normal morphology and no Gb3 accumulation. It is likely that more in vivo environment-like cultures are needed for iPSC-derived neurons to reproduce disease-specific features.

Monitoring of anti-drug antibodies and disease-specific biomarkers in three patients from a Japanese Fabry family treated with enzyme replacement therapy.

We monitored anti-drug antibodies and disease-specific biomarkers in three patients with a nonsense mutation from a Japanese Fabry family treated with enzyme replacement therapy (ERT). In two male patients from the family, neutralizing anti-drug antibodies were induced at an early stage of ERT, the antibody titer peak being found at an earlier stage of ERT in the patient treated with 1.0 mg/kg agalsidase beta than in that treated with 0.2 mg/kg agalsidase alfa. Then, the antibody titers decreased with continuation of ERT. The formation of neutralizing anti-drug antibodies adversely affected the plasma globotriaosylsphingosine (Lyso-Gb3) level and urinary globotriaosylceramide (Gb3) excretion in both patients, the impact being greater in the patient treated with 0.2 mg/kg agalsidase alfa than in that treated with 1.0 mg/kg agalsidase beta. The difference might be explained by the different doses of the infused enzymes based on supersaturation of the antibodies. In a heterozygous Fabry female from the family, no sign of antibody formation was found, and both the plasma Lyso-Gb3 level and urinary Gb3 excretion, which were moderately increased at the baseline, decreased gradually. No deterioration of the manifestations or laboratory findings was observed during ERT in either of the patients. Thus, monitoring of anti-drug antibodies and biomarkers in these Fabry patients provided us with important information on their pathological condition during ERT.

Correlation of X chromosome inactivation with clinical presentation of Fabry disease in a case report.

Fabry disease or also called Anderson-Fabry disease (FD) is a rare disease caused by pathogenic variants in the GLA gene, located on the X chromosome. This gene is involved in the metabolism of glycosphingolipids and its pathogenic variants cause a deficit or absence of α-galactosidase A causing the deposition of globotriaosylceramide throughout the body. Females have a variable phenotypic expression and a better prognosis than males. This is due to the X chromosome inactivation phenomenon. We present a clinical case of Fabry disease in a female with predominantly renal involvement and demonstrate how the X chromosome inactivation phenomenon is tissue dependent, showing preferential inactivation of the mutated allele at the renal level.

Shiga Toxin-B Targeted Gold Nanorods for Local Photothermal Treatment in Oral Cancer Clinical Samples.

Introduction: A great challenge in nanomedicine, and more specifically in theranostics, is to improve the specificity, selectivity, and targeting of nanomaterials towards target tissues or cells. The topical use of nanomedicines as adjuvants to systemic chemotherapy can significantly improve the survival of patients affected by localized carcinomas, reducing the side effects of traditional drugs and preventing local recurrences. Methods: Here, we have used the Shiga toxin, to design a safe, high-affinity protein-ligand (ShTxB) to bind the globotriaosylceramide receptor (GB3) that is overexpressed on the surfaces of preneoplastic and malignant cancer cells in the head and neck tumors. Results: We find that ShTxB functionalized gold nanorods are efficiently retrotranslocated to the GB3-positive cell cytoplasms. After 3 minutes of laser radiation with a wavelength resonant with the AuNR longitudinal localized surface plasmon, the death of the targeted cancer cells is activated. Both preclinical murine models and patient biopsy cells show the non-cytotoxic nature of these functionalized nanoparticles before light activation and their treatment selectivity. Discussion: These results show how the use of nanomedicines directed by natural ligands can represent an effective treatment for aggressive localized cancers, such as squamous cell carcinoma of the oral cavity.