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Fabry disease is caused by reduced α-GAL A activity and accumulation of globotriaosylceramide (Gb 3 ). Here, we describe a microplate Gb 3 assay using fluorophore-tagged antibody and crude cellular lipid extracts. The assay is able to detect higher Gb 3 concentrations in human Fabry cells compared to non-diseased cells. This result was verified by immunofluorescence staining that revealed large amounts of Gb 3 deposits in Fabry cell lines, demonstrating the accuracy of this method. This assay may provide the basis for detecting Fabry disease by quantifying Gb 3 deposits from human biological samples, for example, from urine and blood., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Substitution of the defective lysosomal enzyme in lysosomal storage disorders (LSDs) often elicits antibody formation towards the infused protein. Aside from Gaucher disease, antibodies often lead to infusion associated reactions and a reduced biochemical response. In Pompe disease, antibody titer is predictive of clinical outcome, but this is less apparent in other LSDs and warrants further study. Few laboratories are capable of enzyme-antibody determination: often physicians need to rely on the enzyme manufacturer for analysis. Currently, laboratories employ different antibody assays which hamper comparisons between cohorts or treatment regimens. Assay standardisation, including measurement of antibody-related enzyme inhibition, is therefore urgently needed. Successful immunomodulation has been reported in Pompe and in Gaucher disease, with variable success. Immunomodulation regimens that contain temporary depletion of B-cells (anti-CD20) are most used. Bone marrow transplantation in MPS-I results in disappearance of antibodies. No other clinical studies have been conducted in humans with immunomodulation in other LSDs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Fabry disease is an X-linked inherited disorder that is caused by excessive lysosomal globotriaosylceramide (CTH) storage due to a deficiency in alpha-galactosidase A (alpha-Gal A). Two recombinant enzyme preparations have been approved as treatment modality. We studied emergence and properties of alpha-Gal A antibodies in treated patients., Methods: During the first 6 to 12 months of intravenous administration of recombinant enzymes (rh-alpha-Gal A) formation of antibodies was studied in 18 adult Fabry patients (two females)., Results: The female patients did not develop detectable amounts of antibodies following enzyme therapy. After 6 months of treatment with either agalsidase alpha or beta, 11/16 male patients showed high titers of immunoglobulin G (IgG) antibodies that cross-react in vitro similarly with both recombinant enzymes. The anti-rh-alpha-Gal A IgG neutralizes rh-alpha-Gal A activity in vitro for 65% to 95%. During infusion with rh-alpha-Gal A, circulating enzyme-antibody complexes are formed and these complexes are taken up by leukocytes in the peripheral blood. After 6 months of treatment all IgG-negative patients showed a significant (P < 0.01) reduction of urinary CTH (1890 +/- 797 to 603 +/- 291 nmol CTH/24hr urine), compared to IgG-positive patients (mean increase from 2535 +/- 988 to 2723 +/- 1212), suggesting a negative effect of circulating antibodies on renal tubular CTH clearance., Conclusion: Emergence of antibodies with in vivo neutralizing capacities is frequently encountered in treated Fabry disease patients. Complete cross-reactivity of these antibodies suggests that it is unlikely that switching from one to the other recombinant protein prevents the immune response and related effects. Further studies on the clinical implications of alpha-Gal A antibodies are essential.

Background: Fabry disease is an inherited X-linked disorder resulting in the loss of activity of the lysosomal hydrolase alpha-galactosidase A and causing the clinical manifestations of renal failure, cerebral vascular disease, and myocardial infarction. The phenotypic expression of this disorder is manifest by the accumulation of glycosphingolipids containing alpha-galactosyl linkages, most prominently globotriaosylceramide., Methods: Based on quantitative structure activity studies, we recently reported two newly designed glucosylceramide synthase inhibitors based on 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4). These inhibitors, 4'-hydroxy-P4 and ethylenedioxy-P4, were evaluated for their ability to deplete globotriaosylceramide and other glucosylceramide-based lipids in Fabry lymphocytes and were compared with N-butyldeoxynojirimycin, another reported glucosylceramide synthase inhibitor., Results: Concentrations as low as 10 nmol/L of 4'-hydroxy-P4 and ethylenedioxy-P4 resulted in 70 and 80% depletion, respectively, of globotriaosylceramide, with maximal depletion occurring at three days of treatment. There was no impairment of cell growth. In contrast, N-butyldeoxynojirimycin only minimally lowered globotriaosylceramide levels, even at concentrations as high as 10 micromol/L. Globotriaosylceramide depletion was confirmed by the loss of binding of FITC-conjugated verotoxin B subunit to the lymphoblasts., Conclusions: These findings suggest that selective glucosylceramide synthase inhibitors are highly effective in the depletion of globotriaosylceramide from Fabry cell lines. We suggest that these compounds have potential therapeutic utility in the treatment of Fabry disease.

The kinetics of ATP release from Burkitt lymphoma target cells following complement activation by rat monoclonal IgM antibody have been used to develop a standard assay for antibody-complement mediated cytotoxicity. This assay permitted the study of the reactivity of the monoclonal IgM with Burkitt and non-Burkitt cell lines and to perform IgM-complement mediated cytotoxicity inhibition experiments with oligosaccharides. This led to a precise definition of the antigenic determinant recognized by the monoclonal IgM on globotriaosylceramide.

A simple liposome immunoassay, liposome immune-lysis inhibition (LILI) assay, is described for quantitative determination of individual glycolipid antigens. Liposomes containing fluorogenic marker, 4-methylumbelliferyl phosphate, were prepared from sphingomyelin, cholesterol, dicetylphosphate and standard glycolipid. Release of trapped markers from these liposomes by antibody and complement (liposome lysis) was inhibited by preincubating the antibody with test glycolipid incorporated into inhibitor liposomes. Based on the competitive inhibition, it was possible to quantitate each glycolipid antigen in less than picomolar amounts. The sensitivity and specificity of the assay were examined with purified glycolipid standards. LILI assay has been applied for the determination of Forssman glycolipid and blood group A-active glycolipid in human gastric mucosa and cancer tissues.