Anti-drug antibody formation in Japanese Fabry patients following enzyme replacement therapy.

Enzyme replacement therapy (ERT) for Fabry disease (deficiency of α-galactosidase A, α-Gal) with recombinant α-Gals (agalsidase alfa and agalsidase beta) is widely available and improves some of the clinical manifestations and biochemical findings. However, recent reports suggest that recurrent administration of recombinant enzymes often induces the formation of anti-drug antibodies, which may have a negative impact on the outcome of the therapy. We examined the formation of anti-drug antibodies using blood samples from 97 Japanese Fabry patients following ERT and tried to characterize them by means of enzyme-linked immunosorbent assay (ELISA), serum-mediated α-Gal inhibition, and immunochromatographic (IC) assay, followed by GLA gene analysis and measurement of plasma globotriaosylsphingosine (lyso-Gb3). ELISA revealed that 20/35 (57%) classic Fabry males were antibody (Immunoglobulin G, IgG) -positive (Ab+) at 6 months after the initiation of ERT, although only two of the seventeen (12%) later-onset Fabry males and none of the 45 Fabry females were. The Ab+ state was maintained at least until 24 months after the initiation of ERT in most of the cases, the exceptions being two patients who acquired immune tolerance during ERT. As many Ab+ patients have nonsense mutations, attention should be paid to the formation of anti-drug antibodies in Fabry patients harboring such gene mutations, who hardly produce α-Gal protein. Serum-mediated α-Gal inhibition was seen in most of the Ab+ patients and the antibodies affected the reduction of the plasma lyso-Gb3 level following ERT, suggesting that the antibodies inhibit the enzyme activity. There was a correlation between the results of the IC test and those of the ELISA. As the former is easy and rapid, it should be useful as a bed-side test.

Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis.

Lipophilic Malassezia species may induce catheter-associated sepsis in premature neonates and immunocompromised patients receiving parenteral lipid emulsions. To assess the participation of lipolytic enzymes in the pathogenesis of this yeast, we cloned a gene encoding the enzyme. A lipolytic enzyme in the culture supernatant of Malassezia pachydermatis was purified 210-fold to homogeneity. The enzyme showed high esterase activity toward p-nitrophenyl octanoate. The cDNA encoding the enzyme was cloned using a degenerate oligonucleotide primer constructed from the N-terminal amino acid sequence. The cDNA consisted of 1582 bp, including an open reading frame encoding 470 amino acids. The first 19 amino acids and the following 13 amino-acid sequence were predicted to be the signal peptides for secretion and prosequence, respectively. The predicted molecular mass of the 438-amino acid mature protein was 48 kDa. Analysis of the deduced amino acid sequence revealed that it contains the consensus motif (Gly-X-Ser-X-Gly), which is conserved among lipolytic enzymes. Homology investigations showed that the enzyme has similarities principally with 11 lipases produced by Candida albicans (29-34% identity) and some other yeast lipases.

Total synthesis of (+/-)-trachyspic acid and determination of the relative configuration.

[reaction: see text] The first total synthesis of (+/-)-trachyspic acid, a tumor cell heparanase inhibitor, was accomplished based on Cr(II)/Ni(II)-mediated reaction of the aldehyde containing the citric acid moiety and the long-chain triflate, and the relative configuration of this natural product was determined.