Once daily ganciclovir as initial pre-emptive therapy delayed until threshold CMV load > or =10000 copies/ml: a safe and effective strategy for allogeneic stem cell transplant patients.

Quantitative polymerase chain reaction (QPCR) for cytomegalovirus (CMV) is emerging as the preferred screening method for detection of CMV viremia in patients following allogeneic bone marrow and peripheral blood stem cell transplant. However, there are currently no universally accepted QPCR treatment thresholds at which to start pre-emptive therapy. We report here results of a pre-emptive therapy strategy using ganciclovir (GCV) 5 mg/kg initiated once daily (ODG) delayed till a threshold CMV load of > or =10 000 copies/ml whole blood in clinically stable patients. Sixty-nine at risk patients underwent allogeneic stem cell transplant. 48/69 (70%) patients had an initial episode of CMV viremia. 5/48 (10%) cleared viremia without requiring treatment. 28/43 (65%) patients requiring treatment initiated treatment with ODG. 17/28 (61%) patients successfully cleared CMV viremia on ODG, 10/28 (36%) patients required dose escalation to twice daily GCV for increasing viral loads. There were two cases of CMV disease (colitis) and no deaths due to CMV disease in patients initiating treatment with ODG. We conclude delaying pre-emptive therapy with ODG until whole blood QPCR> or =10 000 copies/ml is a safe and effective strategy for CMV viremia after allogeneic stem cell transplant in clinically stable patients.

Palmitoyl-protein thioesterase deficiency in fibroblasts of individuals with infantile neuronal ceroid lipofuscinosis and I-cell disease.

Mutations in the gene encoding a recently described lysosomal enzyme, palmitoyl-protein thioesterase (PPT), have recently been shown to result in the neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis (INCL). Reduced palmitoyl-protein thioesterase enzyme has been demonstrated previously in INCL brain and immortalized lymphoblasts. In the current paper, we demonstrate that: (1) PPT can be detected by immunoblotting and enzyme activity assays in normal human skin fibroblasts; (2) INCL fibroblasts are deficient in PPT activity; (3) I-cell disease fibroblasts show markedly reduced intracellular levels of PPT but markedly increased levels of PPT in cell culture medium. These data establish that PPT is transported to lysosomes via the lysosomal enzyme:lysosomal enzyme receptor phosphomannosyl recognition system under normal physiological conditions and provide the basis for a useful clinical assay for INCL.

Palmitoyl-protein thioesterase and the molecular pathogenesis of infantile neuronal ceroid lipofuscinosis.

Palmitoyl-protein thioesterase (PPT) has recently been shown to be the defective enzyme underlying the infantile form of neuronal ceroid lipofuscinosis (INCL). In this paper, we review the enzymology of PPT, evidence for its localization in lysosomes, and recent advances in understanding the metabolic defect caused by PPT deficiency. Absence of PPT activity in lysosomes isolated from INCL lymphoblasts is demonstrated. A model for the formation of the storage bodies in INCL involving defective autophagocytic proteolysis is proposed.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

Lysosomal targeting of palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a newly described long chain fatty-acid hydrolase that removes fatty acyl groups from modified cysteines in proteins. We have recently identified palmitoyl-protein thioesterase as the defective enzyme in the recessive hereditary neurological degenerative disorder infantile neuronal ceroid lipofuscinosis (Vesa, J., Hellsten, E., Verkruyse, L. A., Camp, L. A. , Rapola, J., Santavuori, P., Hofmann, S. L., and Peltonen, L. (1995) Nature 376, 584-587). A defect in a lysosomal enzyme had been postulated for the disease, but until recently, the relevant defective lysosomal enzyme had not been identified. In this paper, we present evidence for the lysosomal localization of palmitoyl-protein thioesterase. We show that COS cells take up exogenously supplied palmitoyl-protein thioesterase intracellularly and that the cellular uptake is blocked by mannose 6-phosphate, a hallmark of lysosomal enzyme trafficking. The enzyme contains endoglycosidase H-sensitive oligosaccharides that contain phosphate groups. Furthermore, palmitoyl-protein thioesterase cosediments with lysosomal enzyme markers by Percoll density gradient centrifugation. Interestingly, the pH optimum for the enzyme is in the neutral range, a property shared by two other lysosomal enzymes that remove post-translational protein modifications. These findings suggest that palmitoyl-protein thioesterase is a lysosomal enzyme and that infantile neuronal ceroid lipofuscinosis is properly classified as a lysosomal storage disorder.

Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCL) represent a group of common progressive encephalopathies of children which have a global incidence of 1 in 12,500. These severe brain diseases are divided into three autosomal recessive subtypes, assigned to different chromosomal loci. The infantile subtype of NCL (INCL), linked to chromosome 1p32, is characterized by early visual loss and rapidly progressing mental deterioration, resulting in a flat electroencephalogram by 3 years of age; death occurs at 8 to 11 years, and characteristic storage bodies are found in brain and other tissues at autopsy. The molecular pathogenesis underlying the selective loss of neurons of neocortical origin has remained unknown. Here we report the identification, by positional candidate methods, of defects in the palmitoyl-protein thioesterase gene in all 42 Finnish INCL patients and several non-Finnish patients. The most common mutation results in intracellular accumulation of the polypeptide and undetectable enzyme activity in the brain of patients.

Molecular cloning and expression of palmitoyl-protein thioesterase.

We have previously reported the purification of a palmitoyl-protein thioesterase (PPT) from bovine brain that removes palmitate from Ha-Ras (Camp, L. A., and Hofmann, S. L. (1993) J. Biol. Chem. 268, 22566-22574). In the current paper, we have isolated bovine and rat cDNA clones encoding PPT. The deduced amino acid sequence of PPT predicts a protein of 306 amino acids that contains amino acid motifs characteristic of thioesterases: "Gly-X-Ser-X-Gly" positioned near the NH2 terminus and "Gly-Asp-His" positioned near the COOH terminus of the protein. The identity of the PPT cDNA was further confirmed by expression in simian COS cells and insect Sf9 cells. Comparison of the DNA and protein sequence data suggests that a hydrophobic NH2-terminal sequence of 27 amino acid residues is removed from the primary translation product. Furthermore, the recombinant protein and the native protein purified from bovine brain contain complex asparagine-linked oligosaccharides and a large proportion of the expressed PPT is secreted from COS and Sf9 cells. Thus, while the palmitoyl-protein thioesterase will deacylate intracellular palmitoylated proteins such as Ha-Ras and the alpha subunits of heterotrimeric G proteins, the physiologic substrates are likely to be externally oriented or secreted proteins.