Lysosomal acid lipase (LAL) gene mutations were identified in three patients with cholesteryl ester storage disease (CESD). Direct sequencing of genomic DNA revealed that: patient 1 was a compound heterozygote for a P181L mutation and an A to G3' splice site substitution that causes skipping of exon 7, with a loss of 49 amino acids from LAL (delta 205-253); patient 2 was a compound heterozygote for a G66V mutation and a 5' splice site mutation (G to A) that leads to skipping of exon 8 (delta 254-277); and patient 3 was a compound heterozygote for a L273S mutation and an unidentified null allele. Furthermore, patients 2 and 3 showed a novel G-2A polymorphism that could be detected by an Xbal restriction fragment length polymorphism. All these mutants and a previously reported H274Y allele were expressed in vitro in HeLa cells using the vaccinia T7 expression system. The resulting recombinant proteins were inactive towards cholesteryl oleate and trioleylglycerol, demonstrating the direct involvement of these mutations in the pathogenesis of CESD. Immunoblotting of normal LAL expressed in HeLa cells revealed four major molecular forms, at least two of high molecular mass (54 and 50-51 kDa) and two of low molecular mass (42 and 43 kDa). L273S and P181L substitutions and delta 254-277 were shown to result in altered LAL molecular forms, some of which suggest that post-translational processing may interfere with the catalytic activity of LAL.
Between June 1988 and May 1991 88 orthotopic liver transplants and 1 liver and pancreas transplant were performed at the Liver Transplantation Department of the Ospedale Maggiore of Milan. All the patients underwent mycological surveillance and received antifungal prophylaxis with oral amphotericin B (6000 mg/day) or oral or intravenous fluconazole (200 mg/day) from the time of their transplant. The incidence of Candida colonization was 67%. Fluconazole was superior to oral amphotericin B in the treatment of C. albicans colonization (9/9 vs 6/15), but less effective in the treatment of colonization by other Candida spp. (0/3 vs 3/3). Deep-seated candidiasis developed in 5 patients, caused by C. albicans in 4 cases and C. krusei in 1. C. albicans infection resolved rapidly with fluconazole in 2 subjects, with intravenous amphotericin B alone in 1, and with amphotericin B plus flucytosine in the other. On the contrary, C. krusei infection did not respond to treatment with amphotericin B combined with flucytosine. Aspergillosis was diagnosed in 11 patients, of whom 4 died from invasive aspergillosis, despite 15 and 26 days of amphotericin B treatment in 2. In another patient invasive aspergillosis, diagnosed a few hours before retransplantation, improved with liposomal amphotericin B, but this man died from cytomegalovirus infection one month later. Aspergillosis was eradicated by itraconazole in 4 other patients and by topical amphotericin B in 2 whose infection was localized to surgical wound.
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