Human Molecular Genetics, 2001, Vol. 10, No. 16 1639-1648
© 2001 Oxford University Press
Enzyme therapy for lysosomal acid lipase deficiency in the mouse
The Children’s Hospital Research Foundation, Division of Human Genetics, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA and 1Genzyme Corporation, Cambridge, MA, USA
Received March 23, 2001; Revised and Accepted June 11, 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
|---|
Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of the triglycerides (TG) and cholesteryl esters (CE) delivered to lysosomes. Its deficiency produces two human phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD). A targeted disruption of the LAL locus produced a null (lal –/–) mouse model that mimics human WD/CESD. The potential for enzyme therapy was tested using mannose terminated human LAL expressed in Pichia pastoris (phLAL), purified, and administered by tail vein injections to lal –/– mice. Mannose receptor (MR)-dependent uptake and lysosomal targeting of phLAL were evidenced ex vivo using competitive assays with MR-positive J774E cells, a murine monocyte/macrophage line, immunofluorescence and western blots. Following (bolus) IV injection, phLAL was detected in Kupffer cells, lung macrophages and intestinal macrophages in lal –/– mice. Two-month-old lal –/– mice received phLAL (1.5 U/dose) or saline injections once every 3 days for 30 days (10 doses). The treated lal –/– mice showed nearly complete resolution of hepatic yellow coloration; hepatic weight decreased by
36% compared to PBS-treated lal –/– mice. Histologic analyses of numerous tissues from phLAL-treated mice showed reductions in macrophage lipid storage. TG and cholesterol levels decreased by 50% in liver, 69% in spleen and 50% in small intestine. These studies provide feasibility for LAL enzyme therapy in human WD and CESD. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
|---|
Lysosomal acid lipase (LAL) is the critical enzyme for the cleavage of cholesteryl esters and triglycerides that are delivered to the lysosomes through numerous cell-specific receptor systems. The deficiency of LAL in humans causes two distinct phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD) (1). WD is an early-onset fulminant disorder of infancy with massive infiltration of the liver, spleen and other organs by macrophages filled with cholesteryl esters and triglycerides. In addition, accumulation of cholesteryl esters in the zona reticularis of the adrenal gland leads to adrenal calcification and cortical insufficiency (1). Cachexia and steatorrhea result from malabsorption due to accumulation of macrophages filled with cholesteryl esters and triglycerides in the villi of the small intestine. Death occurs early in life from inanition, possibly contributed to by adrenal cortical insufficiency. WD is very rare with an incidence of less than one in 100 000 live births.
CESD is a milder, later-onset disorder with primary hepatic involvement by macrophages engorged with cholesteryl esters. This slowly progressive visceral disease has a very wide spectrum of involvement ranging from early onset with severe cirrhosis to later onset of more slowly progressive hepatic disease with survival into adulthood (1).
A naturally occurring lal–/– model in rats (2) and a targeted gene disruption in mice (3) provide phenotypes that are biochemically similar to WD, but with survival of the animals into early adulthood. Death results from massively enlarged livers and spleens and, possibly, malabsorption due to intestinal infiltration by macrophages laden with cholesteryl esters and triglycerides. Both murine analogs are similar biochemically and histologically to the human WD, particularly in the tissue distribution of involvement including the adrenal glands. Calcification of the adrenal glands is not observed in the murine models of LAL deficiency (2,3). The long-term sequelae (5–8 months) in the lal–/– mouse include massive hepatosplenomegaly, severe infiltration of the small intestine, and lymph nodes that are engorged with lipid-filled macrophages. This is accompanied by nearly complete loss of brown and white adipose tissues and foamy enlargement of the adrenal glands (4).
The human disease results from numerous mutations, missense or nonsense, at the LAL locus on chromosome 10. The milder phenotypes may correlate with the presence of increased amounts of residual enzyme activity due to missense mutations (1,5). Although diagnostically useful, the molecular mutations are not currently useful for prognostication in CESD and WD. Therapy for WD has been attempted by bone marrow transplantation with inconsistent success (6). In CESD, therapeutic efforts have been focused on diminishing the accumulation of cholesteryl esters within the liver and spleen by use of HMG CoA reductase inhibitors. Some biochemical effects are observed on lipoprotein metabolism, but no demonstrable effects are noted on the phenotype (7). However, reports are scarce and biochemical documentation is still insufficient.
The availability of the mouse analog of human WD/CESD provides an excellent model to test new therapeutic strategies including enzyme therapy. Here the initial results of enzyme infusions in LAL-deficient mice using mannose-terminated human LAL produced in yeast are presented. These results indicate potential utility of enzyme therapy in WD and CESD in humans.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
|---|
Mouse macrophage mannose receptor (MR)-dependent Pichia-derived human LAL (phLAL) incorporation and lysosomal targeting
J774E cells are a mouse macrophage sub-line that express the MR. The parental cell line, J774A.1, does not express this receptor. Immunofluorescence staining of cells that had been incubated with phLAL showed strong positive staining only with J774E cells (Fig. 1B and C). J774A.1 cells were nearly negative (Fig. 1D). The weak signals detected in J774A.1 cells represent cross reactivity of the anti-human LAL antibody with endogenous mouse LAL protein. At 5 h post-incubation, the positive staining for phLAL was mostly in a plasma membrane (Fig. 1B). An intracellular granular pattern (lysosomal localization pattern) was present at 18 h post-incubation (Fig. 1C).
|
To confirm the lysosomal localization of phLAL in J774E cells, double immunofluorescence staining was performed using rabbit anti-hLAL and rat anti-LAMP-1. LAMP-1 is a lysosomal membrane protein that is commonly used as lysosomal marker. The results show that phLAL (Fig. 2A) had the same immunofluorescence patterns as LAMP-1 (Fig. 2B) by merger of the images (Fig. 2C) after incubation (18 h) with media containing phLAL.
|
Additional evidence for MR-dependent incorporation of phLAL into J774E cells was obtained in competition studies. Incubation of these cells with media containing either phLAL (Fig. 3A) or Ceredase (Fig. 3B) showed a dosage-dependent uptake of proteins. Varying amounts of phLAL or Ceredase were co-incubated with J774E cells. Intracellular phLAL and Ceredase were analyzed by western blots using both rabbit anti-hLAL and rabbit anti-GCase. With increased amounts of Ceredase in the media, the uptake of phLAL decreased (Fig. 3C). In the reverse experiment, with fixed amounts of Ceredase and different amounts of phLAL, similar competition for intracellular uptake was obtained (Fig. 3D). These data support MR-dependent uptake of phLAL into these cells.
|
Cellular and subcellular targeting of p-LAL
Following tail vein bolus injections of phLAL, hepatic, splenic and small intestine samples were obtained and analyzed by immunohistochemistry with anti-hLAL. Typical results in liver (Fig. 4) and spleen showed sinusoidal cell accumulation of the administered enzyme detected as brown patches in these areas. In addition, but not as well visualized, was a generalized dark granular appearance to many of the storage cells indicating uptake of enzyme into these cells. The large cytoplasmic volume of the storage cells diluted this signal. Similar results were obtained with Ceredase infusions using human-specific acid ß-glucosidase antibody (data not shown). These results indicate that the distribution spaces in these LAL-deficient mice for phLAL and mannose-terminated acid ß-glucosidase were similar.
|
Phenotypic and gross pathologic changes
In lal–/– mice, treatment with phLAL enzyme resulted in significant correction of lipid storage phenotypes in various organs (Table 1). At 3 months of age, untreated lal–/– mice developed a yellow/white creamy color to the liver and significant hepatosplenomegaly was present. In comparison, the phLAL-treated mice had livers and spleens with much more normal colors (Fig. 5). The normal livers in age-matched controls were
5% of body weight, whereas the livers were 14% of body weight in the untreated lal–/– mice (Table 1). phLAL administration decreased liver and spleen size by 30% (P = 0.0029) in males. In females these differences were not present. However, the spleens reverted to nearly normal color in the treated group (Fig. 5). The duodenum of untreated lal–/– mice was yellow and the jejunum was creamy white. In the treated group, the small intestine reverted to nearly normal color (Fig. 5). Grossly, the adrenal glands were unchanged.
|
|
Histologic studies
Hematoxylin and eosin (H&E) or Oil-Red-O staining of liver, spleen and small intestine from untreated and treated mice showed clear differences. H&E sections of the liver showed that the phLAL-treated lal–/– mice had significant reductions in the size and number of lipid-filled Kupffer cells (Fig. 6A and B). Hepatocytes had less lipid storage than Kupffer cells in untreated lal–/– mice and this hepatocyte storage appeared unchanged in the treated group. Using Oil-Red-O staining for neutral lipids, a significant difference between the livers of the treated and untreated mice was apparent (Fig. 6C and D). In the spleen, the treated group showed a reduction of lipid storage cells (Fig. 6E and F) compared with untreated mice. In the small intestine, the Oil-Red-O staining of phLAL-treated and untreated mice showed substantial differences. The sections of intestine from untreated mice were full of Oil-Red-O staining cells (macrophages) in lamina propria while comparable sections from treated mice were almost completely negative for Oil-Red-O staining cells (Fig. 6I and J).
|
Biochemical findings
Tissue cholesterol (both free and esterified) and triglycerides from liver, spleen and small intestine were determined by chemical analyses. Compared to age matched wild-type mice, the lal–/– mice have elevated CE and TG in several tissues. Average total cholesteryl ester per organ at 3.5 months of age was increased 31-fold in liver and 19-fold in spleen of lal–/– mice compared to wild-type (Fig. 7A and B). phLAL administration to lal–/– mice led to reductions of total cholesterol of 47% in the liver [267.22 ± 8.22 mg (untreated) versus 144.23 ± 7.99 mg (treated); P = 0.0003, n = 3] and 69% in the spleen [8.73 ± 0.43 mg (untreated) versus 2.63 ± 0.50 mg (treated); P = 0.0008, n = 3]. Similar decreases of TGs were also observed: 58% in liver (96.52 ± 17.93 mg versus 39.79 ± 6.38 mg; P = 0.047, n = 4) and 45% in spleen (8.23 ± 0.68 mg versus 4.55 ± 1.26 mg; P = 0.042, n = 4) (Fig. 7A and B). Although no change (P = 0.67) was observed in the concentration of cholesterol in small intestine, the TG concentration of the treated group was reduced by 65% (49.52 ± 2.40 µg/mg versus 17.09 ± 4.8 µg/mg; P = 0.042) (Fig. 7C).
|
Plasma chemistries and lipid levels
No differences of plasma glucose levels were observed in treated or untreated lal–/– mice (data not shown). Plasma non-esterified fatty acids (NEFA) levels were increased (162%) in lal–/– mice compared to the wild-type controls. phLAL administration was associated with increases (32.6%) of the NEFA in lal–/– mice. Plasma triglyceride levels decreased in treated male lal–/– mice, but not in female lal–/– mice. Plasma free and esterified cholesterol levels were also decreased in male lal–/– mice, but not in female lal–/–mice (Table 1).
Survival and antibody development
None of the lal–/– mice died before termination. No untoward effects or abnormal behaviors were noted in either the treated or untreated groups. As assessed by western blot analyses of plasma samples obtained at the time of termination, all mice receiving 10 injections of phLAL showed positive signals against phLAL at 1:100 dilution of serum. One lal–/– mouse developed higher titre antibodies with positive signals at 1:3200 dilution of serum. To evaluate the time-course of anti-phLAL antibody development in phLAL-treated lal–/– mice, serum samples from independent pilot experiments were collected from lal–/– mice with one, two, three or five phLAL injections. None of these sera showed any anti-phLAL activity by western blot analyses (data not shown). These data suggested that lal–/– mice developed the low titre anti-phLAL after five doses. The differential reactivities of these mouse sera were determined with phLAL or unglycosylated hLAL produced in Escherichia coli. No signals were obtained with the unglycosylated hLAL, except in one mouse that produced low level signals on western blots (data not shown). In addition, none of these sera produced inhibition of phLAL activity. To explore the potential that phLAL may form complexes with antibodies and facilitate uptake through the FC receptor, phLAL was mixed with different antibody-positive anti-sera. These mouse anti-phLAL sera were pre-incubated with different amounts of phLAL (0.0, 0.1, 0.5, 1.0, 2.0 and 4.0 µg). These mixtures were then added to the media of J774A.1 and J774E cells which are FC receptor positive. J774E cells are also MR positive. The dose-dependent uptake of phLAL into J774E cells was not changed by the presence of anti-sera, and uptake into MR-negative and FC-positive cells, J774A, did not occur (data not shown).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
|---|
Purified recombinant human LAL produced in Pichia pastoris was shown to have in vivo activity in reducing the pathology of LAL deficiency in the mouse. For several lysosomal storage diseases (LSDs), enzyme therapy is becoming available for the treatment of their complications. In Gaucher disease (acid ß-glucosidase deficiency), enzyme replacement is the standard-of-care for the treatment of the major overt visceral manifestations of this disease (8–11). It has proven to be safe and effective in a large cohort of patients (12). Less advanced, but with demonstrated biochemical efficacy, is enzyme therapy for human and murine Fabry disease (
-galactosidase A deficiency) (13), murine Niemann-Pick A or B (sphingomyelinase deficiency) (14) and murine MPSVII (ß-glucuronidase deficiency) (15,16). These initial studies have indicated the potential for developing effective therapies of lysosomal disease by using the specific receptors and receptor-mediated endocytotic pathways for lysosomal delivery of active molecules. For these studies, stably transfected mammalian cells including CHO cells (17), mouse L cells (15,16) and human foreskin fibroblast (13) have been used as high level sources for the production of the requisite enzymes. Here, the recombinant human LAL was produced in a yeast expression system and was well tolerated in the lal–/– mouse model.
The general theory of enzyme replacement in LSDs centers around the use of receptor-mediated endocytosis for the specific delivery of requisite enzymes to subcellular sites in targeted tissues of pathology (18). For those diseases that primarily involve the macrophage system, e.g. WD and CESD, Pichia pastoris may provide a high level source for the production of mannose-terminated, and therefore macrophage-targeted, enzymes. The Pichia system was chosen since it produces secreted proteins containing 11–12 mannoses in an -mannose termination (19–21). Such oligosaccharides would have the structure needed for mono- or multi-valent recognition by the MR on a variety of cells, including macrophage-derived cells (Kupffer cells and tissue macrophages), endothelial cells and other sinusoidal lining cells in the liver and spleen (22). The use of the MR for targeted delivery exploits the innate immune reaction to mannans that are produced by a large number of yeast and are presented to the macrophages in various organs for processing (22–24). However, unlike the large arborizing mannosyl oligosaccharides produced by many yeast strains, those in P.pastoris are shorter and may have less tendency to induce an immune reaction.
Kupffer cell targeting was shown by immunohistochemical staining and by effects of the administered enzyme, including diminution in the numbers and types of Kupffer cells and other tissue macrophages. However, in addition to the targeting to the liver, the decrease in splenic and small intestinal macrophages indicates that active enzyme was delivered in significant amounts to other organs. This was not the case for other organs, e.g. substantial decreases in the numbers or sizes of macrophages were not appreciated in the lymph nodes. Adrenal pathology had involvement by non-macrophage cells in the zona reticularis and this was unaltered by the enzyme administration. Thus, similar to the experience in human Gaucher disease, and potentially in other LSDs, there may be sequestered or inaccessible sites that cannot be reached by exogenously administered enzyme for the treatment of these diseases. It might be anticipated that continued administration of LAL may affect the primary target sites of pathology in WD and CEDS including the liver, spleen, intestine and bone marrow, but other sites such as the lymph nodes may not be significantly altered and may present long-term evaluation and outcome issues.
In addition to the specific targeting, the administered phLAL clearly had enzymatic effects at the tissue level in the liver, small intestine and spleen. While the Kupffer cell number and progression of involvement of the liver over the 1 month period of enzyme therapy were clearly affected, the total disease pathology from the initiation of therapy was not reduced to normal levels. Consequently, additional amounts of enzyme may be required to completely reverse the pathology in the Kupffer cells rather than just stabilizing the effects from the initiation of therapy. Stopping of disease progression at two months in the lal–/– mice was a significant benefit, but early initiation of therapy with increased amounts of enzyme may be needed to completely reverse the manifestations of the disease in the liver. Complete reversal of the pathology in the small intestine was observed on the dosage of enzyme used here, indicating the potential for tissue-specific requirements of active enzyme for effective and complete treatment. The lack of major and clearly demonstrable decreases in lipid storage of hepatocyte and adrenal zona reticularis indicates that specific targeting to the macrophage alone may not be sufficient for complete resolution of WD or CESD. Thus, the approach of specifically treating the macrophage involvement may lead to a progression of other cell-type involvement. This implies the need to use enzymes either at different doses or with modified carbohydrate side chains for more generalized delivery, including hepatocyte and adrenal gland, e.g. mannose-6-phosphate receptor targeted form. With Gaucher disease, anecdotal evidence indicates that enzyme may not gain access to the lymphoid system in sufficient amounts to affect reversal of macrophage infiltration of the lymphoid tissues.
Clearly, a major cell type involvement in WD and CESD was the macrophage and the use of J774E cells clearly indicates the delivery of phLAL to the lysosomes by the MR. There was no uptake of this enzyme or of mannose-terminated glucocerebrosidase (alglucerase) by J774A.1 cells which lack the MR. Furthermore, the two enzymes, phLAL and alglucerase, competed for uptake with the MR into the J744E cells. Importantly, the enzyme not only gained access to these macrophage-like cells, but also was delivered to LAMP-1 positive compartments, meaning it was delivered to the lysosome. The processing of oligosaccharides following uptake of the enzyme into the lysosomes of the J744E cells may have resulted from the action of lysosomal -mannosidases but this remains to be proven.
These studies demonstrate the feasibility for enzyme therapy in WD and CESD using a yeast expression system such as P.pastoris. The targeted enzyme delivery appears to be fairly specific since the hepatic cellular involvement was not significantly changed, at least histologically, by these enzyme infusions and more enzyme or differently terminated enzyme may be required. Future studies will address these issues. Furthermore, the lack of significant benefit of bone marrow transplantation in severely ill children with WD indicates that adjunctive therapies, i.e. enzyme replacement, may be required for adequate chances of recovery.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
|---|
Animals
The mice were provided care in accordance with institutional guidelines and all procedures received prior approval by the IACUC at the Children’s Hospital Research Foundation. The lal–/– mice originated from mixed genetic backgrounds of 129Sv and CF-1 (3). Mice were housed in micro-isolation, under 12 h dark–12 h light cycles. Water and food, regular chow diets, were available ad libitum. The mice were genotyped by PCR-based screening of tail DNA (3).
Study design
Litter-matched lal–/– mice were divided into two cohorts that received phLAL or PBS injections (100 µl) by tail vein bolus. The injections were given to each mouse every 3 days for 30 days for a total of 10 injections (1.5 U phLAL) in 1x PBS, 10 mM DTT and 2% human serum albumin. All injections were successful. The lal–/– mice were harvested 2 days following the final injection and tissues were processed for histologic, immunohistochemical and biochemical analyses. One unit is 1 µmol of 4-methylumbeliferyl-oleate (4-MUO) cleaved per min under standard assay conditions (25). phLAL was a gift from the Genzyme Corp. The enzyme was estimated to be 96% pure from silver-stained SDS–PAGE.
Enzyme uptake studies in J774E and J774A.1 macrophage cultures
J744E cells express the MR (26) and were obtained from Dr Philip Stahl at Washington University, St Louis, MO. J744A.1 cells were from the ATCC and do not express the MR. J774E and J774A.1 cells were maintained in MEM with 6-thioguanine (60 µM) or in DMEM medium, respectively, supplemented with 10% fetal calf serum, penicillin and streptomycin (37°C; 5% CO2). For the uptake studies, cells were seeded at 2 x 105 per well 1 day before adding phLAL or Ceredase® (mannose-terminated glucocerebrosidase; Genzyme Corp.). The indicated amounts of phLAL, either with or without pre-incubation with mouse anti-sera at room temperature for 30 min, were then added to cells. At designated post-incubation times, cells were washed twice with 1x PBS, collected with a rubber policeman, and centrifuged (13 000 g, 1 min) at room temperature. The intracellular proteins were extracted by cell lysis with 1% taurocholate/1% Triton X-100, freeze–thawed five times (dry ice and 37°C water bath) and centrifuged (13 000 g, 10 min) at 4°C. The protein extracts were analyzed by western blot.
For immunofluorescence staining, cells (1.5 x 105) were seeded on chamber slides, incubated with phLAL for 5, 18 or 24 h, washed with PBS twice, and fixed with 2% paraformaldehyde for 1 h. Immunofluorescence staining was performed as described previously (27). A rat anti-LAMP-1 (mouse) monoclonal antibody developed by J.T.August was obtained from the Development Studies Hybridoma Bank (University of Iowa, Department of Biological Science). Goat anti-rat IgG conjugated with Texas Red and goat anti-rabbit IgG conjugated with FITC (Jackson Immuno Research Laboratory) were used as secondary antibodies.
Immunohistochemical staining
Immunohistochemical analyses were performed with paraffin-embedded liver sections using rabbit anti-hLAL antibody. The endogenous peroxidase activity was saturated by incubation in methanol containing 0.5% H2O2 for 10 min. The primary antibody (1:200) was incubated at 4°C overnight. The sections were then washed with 1x PBS three times (5 min per wash), incubated with biotinylated conjugated IgG as secondary antibody for 30 min at room temperature, and washed with 1x PBS for 5 min. The signal was detected using VECTASTAIN ABC kit (Vector) and counterstained with Nuclear Fast Red.
Plasma lipid analyses
Blood was collected from the inferior vena cava of mice after overnight fast and anesthesia with 200 µl triple sedative (ketamine, acepromazine and xylazine). Plasma was collected by centrifugation (5000 g, 10 min, 4°C) of blood and stored at –20°C. Total plasma free cholesterol was determined colorimetrically with a COD-PAP kit (Wako Chemicals). Plasma TC or cholesterol levels were determined using Triglycerides/GB or Cholesterol/HP kits (Boehringer Mannheim). The results are expressed as the mean ± SD of assays conducted in duplicate.
Tissue lipid analyses
Total lipids were extracted from liver, spleen and small intestine by the Folch method (3,28). Triglyceride concentrations were measured by chemical analysis (29). Briefly, both standards and samples in chloroform were evaporated under vacuum. The lipids were re-suspended into the following reagents in the order: 0.5 ml of isopropanol, 4.5 ml of H2O:isopropanol:40 mM H2SO4 (0.5:3.0:1.0) and 2.0 ml of heptane, and mixed by vigorous agitation at each step. The tubes were left to biphase (5 min). Upper phase (1 ml) from each tube was transferred into a set of new tubes containing florisil (80 mg) and vigorously mixed. Then, the upper phase (0.2 ml) was removed and treated with 28 mM sodium alkoxide (2.0 ml) at 60°C for 5 min. Sodium metaperiodate (3 mM, 1 ml) was added and mixed well. After 45 min, acetyl acetone (73 mM, 1 ml) was added to each tube and incubated (60°C, 20 min). The tubes were cooled to room temperature (25 min) and absorbance (
= 410 nm) was determined spectrophotometrically (Beckman DU640 ).
Total tissue cholesterol concentrations were estimated using O-phthalaldehyde (30). Briefly, cholesterol standards and Folch extracted samples were evaporated under N2. O-phthalaldehyde (3 ml; Sigma) was added and mixed. Concentrated sulfuric acid (1.5 ml) was added slowly, then mixed and cooled for 5–10 min. Absorbance was determined at = 550 nm.
Western blot analysis and LAL activity assay
Immunoblots were conducted with anti-hLAL anti-serum as described previously (27). Mouse serum was also used as a primary antibody. phLAL activities were with 4-MUO (25). To test the inhibitory capacity of mouse anti-sera, standard assays were set up with serial dilutions of mouse sera, pre-immune or immune, and added to the reaction mixtures. All assays were conducted in duplicate. Assays were linear within the time frame used and <10% of substrates was cleaved.
Histologic analyses
Light microscopic examinations of the liver, spleen, intestine, adrenal glands, kidneys, heart, lung, thymus, pancreas and brain were performed. The sections were stained with paraffin-embedded H&E or Oil-red-O (frozen sections) for light microscopic analysis. Representative sections are presented.
|
ACKNOWLEDGEMENTS |
|---|
We thank Lisa Artmayer for the histology analysis, Jill Brannock for mouse antibody testing, Alicia Emley for the color photograph and Pam Groen for immunohistochemical staining. This work was partially supported by grants from NIH (DK 54930 to H.D.), and by a grant to G.A.G. from Genzyme Corporation. Additional support was provided by the Children’s Hospital Research Foundation.
|
NOTE ADDED IN PROOF |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
|---|
Two phase III trials in Fabry disease show promise for enzyme therapy as a safe and effective approach to treatment of this disease (31,32)
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +1 513 636 7290; Fax: +1 513 636 2261; Email: grabg0@chmcc.org
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
|---|
1 Assman, G. and Seedorf, U. (2001) Acid lipase deficiency: Wolman disease and cholesteryl ester storage disease. In Scriver, C.R., Beaudet, A.L., Valle, D. and Sly, W.S. (eds), The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York, pp. 3551–3572.
2 Nakagawa, H., Matsubara, S., Kuriyama, M., Yoshidome, H., Fujiyama, J., Yoshida, H. and Osame, M. (1995) Cloning of rat lysosomal acid lipase cDNA and identification of the mutation in the rat model of Wolman’s disease. J. Lipid Res., 36, 2212–2218.[Abstract]
3 Du, H., Duanmu, M., Witte, D.P. and Grabowski, G. (1998) Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage. Hum. Mol. Genet., 7, 1347–1354.
4 Du, H., Heur, M., Duanmu, M., Grabowski, G.A., Hui, D.Y., Witte, D.P. and Mishra, J. (2001) Lysosomal acid lipase deficient mice: depletion of white and brown fat, severe hepatosplenomegaly and shortened life span. J. Lipid Res., 42, 489–500.
5 Lohse, P., Chahrokh-Zadeh, S. and Seidel, D. (1997) Human lysosomal acid lipase/cholestryl ester hydrolase and human gastric lipase: identification of the catalytically active serine, aspartic acid, and histidine residues. J. Lipid Res., 38, 892–903.[Abstract]
6 Krivit, W., Peters, C., Dusenbery, K., Ben-Yoseph, Y., Ramsay, N.K., Wagner, J.E. and Anderson, R.A. (2000) Wolman disease successfully treated by bone marrow transplantation. Bone Marrow Transplant., 26, 567–570.[ISI][Medline]
7 Ginsberg, H., Le, N.A., Short, M.P., Ramakrishnan, R. and Desnick, R.J. (1987) Suppression of apolipoprotein B production during treatment of cholesteryl ester storage disease with lovastatin. Implications for regulation of apolipoprotein B synthesis. J. Clin. Invest., 80, 1692–1697.
8 Barton, N.W., Brady, R.O., Dambrosia, J.M., Di Bisceglie, A.M., Doppelt, S.H., Hill, S.C., Mankin, H.J., Murray, G.J., Parker, R.I. and Argoff, C.E. (1991) Replacement therapy for inherited enzyme deficiency–macrophage-targeted glucocerebrosidase for Gaucher’s disease. N. Eng. J. Med., 324, 1464–1470.[Abstract]
9 Kay, A.C., Saven, A., Garver, P., Thurston, D.W., Rosenbloom, B.F. and Beutler, E. (1991) Enzyme replacement therapy in type I Gaucher disease. Trans. Assoc. Am. Phys., 104, 258–264.[Medline]
10 Fallet, S., Grace, M.E., Sibille, A., Mendelson, D.S., Shapiro, R.S., Hermann, G. and Grabowski, G.A. (1992) Enzyme augmentation in moderate to life-threatening Gaucher disease. Pediatr. Res., 31, 496–502.[ISI][Medline]
11 Pastores, G.M., Sibille, A.R. and Grabowski, G.A. (1993) Enzyme therapy in Gaucher disease type 1: dosage efficacy and adverse effects in 33 patients treated for 6 to 24 months. Blood, 82, 408–416.
12 Grabowski, G.A., Leslie, N. and Wenstrup, R.J. (1998) Enzyme therapy in Gaucher disease: the first five years. Blood Rev., 12, 115–133.[ISI][Medline]
13 Shiffmann, R., Murray, G.J., Treco, D., Daniel, P., Sellos-Moura, M., Myers, M., Quirk, J.M., Zirzow, G.C., Borowoski, M., Loveday, K. et al. (2000) Infusion of -galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease. Proc. Natl Acad. Sci. USA, 97, 365–370.
14 Miranda, S.R., He, X., Simonaro, C.M., Gatt, S., Dagan, A., Desnick, R.J. and Schuchman, E.H. (2000) Infusion of recombinant human acid sphingomyelinase into Niemann–Pick disease mice leads to visceral, but not neurological, correction of the pathophysiology. FASEB J., 14, 1988–1995.
15 Vogler, C., Levy, B., Galvin, N., Birkenmeier, E.H. and Sly, W.S. (1996) Enzyme replacement with recombinant ß-glucuronidase in murine mucopolysaccharidosis type VII: impact of therapy during the first six weeks of life on subsequent lysosomal storage, growth and survival. Pediatr. Res., 39, 1050–1054.[ISI][Medline]
16 Vogler, C., Higgins, A., Levy, B., Grubb, J., Birkenmeier, E.H. and Sly, W.S. (1993) Enzyme replacement with recombinant ß-glucuronidase in the newborn mucopolysaccharidosis type VII mouse. Pediatr. Res., 34, 837–840.[ISI][Medline]
17 Ioannou, Y.A., Zeidner, K.M., Gordon, R.E. and Desnck, R.J. (2001) Fabry disease: preclinical studies demonstrate the effectiveness of -galactosidase A replacement in enzyme-deficient mice. Am. J. Hum. Genet., 68, 14–25.[ISI][Medline]
18 Desnick, R.J. and Grabowski, G.A. (1981) Advances in the treatment of inherited metabolic diseases. Adv. Hum. Genet., 11, 281–369.
19 Gemmill, T.R. and Trimble, R.B. (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta, 1426, 227–237.[Medline]
20 Montesino, R., Garcia, R., Quintero, O. and Cremata, J.A. (2001) Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris. Protein Expr. Purif., 14, 197–207.
21 Miele, R.G., Castellino, F.J. and Bretthauer, R.K. (1997) Characterization of the acidic oligosaccharides assembled on the Pichia pastoris-expressed recombinant kringle 2 domain of human tissue-type plasminogen activator. Biotechnol. Appl. Biochem., 26, 79–83.
22 Wileman, T.E., Lennartz, M.R. and Stahl, P.D. (1986) Identification of the macrophage mannose receptor as a 175 kDa membrane protein. Proc. Natl Acad. Sci. USA, 83, 2501–2505.
23 Fiete, D., Beranek, M.C. and Baenziger, J.U. (1997) The macrophage/endothelial cell mannose receptor cDNA encodes a protein that binds oligosaccharides terminating with SO4-4-GalNAcß1, 4GlcNAcß or Man at independent sites. Proc. Natl Acad. Sci. USA, 94, 11256–11261.
24 Ezkowitz, A.B., Sastry, K., Bailly, P. and Warner, A. (1990) Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in Cos-1 cells. J. Exp. Med., 172, 1785–1794.
25 Sheriff, S., Du, H. and Grabowski, G. (1995) Characterization of lysosomal acid lipase by site directed mutagenesis and heterologous expression. J. Biol. Chem., 270, 27766–27772.
26 Mukhopadhyay, A. and Stahl, P. (1995) Bee venom phospholipase A2 is recognized by the macrophage mannose receptor. Arch. Biochem. Biophys., 324, 78–84.[ISI][Medline]
27 Du, H., Witte, D.P. and Grabowski, G. (1996) Tissue and cellular specific expression of murine lysosomal acid lipase mRNA and protein. J. Lipid Res., 37, 937–949.[Abstract]
28 Folch, J., Lees, M. and Stanley, G.H.S. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 125, 497–509.
29 Biggs, H.G., Erikson, J.M. and Moorehead, W.R. (1975) A manual colorimetric assay of triglycerides in serum. Clin. Chem., 21, 437–441.[ISI][Medline]
30 Rudel, L.L. and Morris, M.D. (1973) Determination of cholesterol using O-phthalaldehyde. J. Lipid Res., 14, 164–166
31 Eng, C.M., Guffon, N., Wilcox, W.R., Germain, D.P., Lee, P., Waldek, S., Caplan, L., Linthorst, G.E. and Desnick, R.J. (2001) Safety and efficacy of recombinant human -galactosidase A—replacement therapy in Fabry’s disease. N. Engl. J. Med., 345, 9–16.
32 Schiffman, R., Kopp, J.B., Austin, H.A., Sabnis, S., Moore, D.F., Weibel, T., Balow, J.E. and Brady, R.O. (2001) Enzyme replacement therapy in Fabry disease: a randomized control trial. J. Am. Med. Assoc., 285, 2743–2749.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Du, T. L. Cameron, S. J. Garger, G. P. Pogue, L. A. Hamm, E. White, K. M. Hanley, and G. A. Grabowski Wolman disease/cholesteryl ester storage disease: efficacy of plant-produced human lysosomal acid lipase in mice J. Lipid Res., August 1, 2008; 49(8): 1646 - 1657. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Yan, X. Lian, Y. Li, Y. Dai, A. White, Y. Qin, H. Li, D. A. Hume, and H. Du Macrophage-Specific Expression of Human Lysosomal Acid Lipase Corrects Inflammation and Pathogenic Phenotypes in lal-/- Mice Am. J. Pathol., September 1, 2006; 169(3): 916 - 926. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Zschenker, T. Illies, and D. Ameis Overexpression of lysosomal Acid lipase and other proteins in atherosclerosis. J. Biochem., July 1, 2006; 140(1): 23 - 38. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Roces, R. Lullmann-Rauch, J. Peng, C. Balducci, C. Andersson, O. Tollersrud, J. Fogh, A. Orlacchio, T. Beccari, P. Saftig, et al. Efficacy of enzyme replacement therapy in {alpha}-mannosidosis mice: a preclinical animal study Hum. Mol. Genet., September 15, 2004; 13(18): 1979 - 1988. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Lian, C. Yan, L. Yang, Y. Xu, and H. Du Lysosomal acid lipase deficiency causes respiratory inflammation and destruction in the lung Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L801 - L807. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Du, S. Schiavi, N. Wan, M. Levine, D. P. Witte, and G. A. Grabowski Reduction of Atherosclerotic Plaques by Lysosomal Acid Lipase Supplementation Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 147 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Mahtani and H. F. Willard Physical and Genetic Mapping of the Human X Chromosome Centromere: Repression of Recombination Genome Res., February 1, 1998; 8(2): 100 - 110. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||