Detection of elemental mercury in abdominal wall soft tissue.

CASE REPORT: A 19-year-old male with an unremarkable medical history presented with his father, who requested an evaluation of a pathology specimen from a reported "lump" under the skin in the middle of the son's lower abdomen. The lump had been excised by a surgeon approximately 3 months prior, per parental request. Upon gross inspection, the specimen appeared to contain small metallic droplets. The patient denied self-injection of any metals, including mercury, despite the results of a 24-hour urine heavy-metal screen (without chelation) that revealed an elevated concentration of mercury (87.6 microg/g creatinine; reference range for nonexposed adults: <4 microg/g creatinine). Confirmatory analysis of the tissue sample included gross and microscopic examination, electron microscopy using secondary and backscattered electron imaging modes, and energy dispersive x-ray spectrum analysis of isolated tissue particles. Grossly, the tissue had small silver spherules suggestive of elemental mercury; these droplets were identified histologically with associated foreign body reaction. Numerous smooth, round-to-oval particles scattered randomly throughout the tissue were identified ultrastructurally, which produced an x-ray energy spectrum corresponding to mercury. DISCUSSION: Elemental mercury is liquid at room temperature and may be injected into the body for recreational, psychiatric, and other purposes. Isolated cases of mercury injection following accidents with broken thermometers have been reported, as well as cases of elevated metallothionein concentration following human gingival amalgam tattoos. CONCLUSION: In cases of surreptitious injection, histology and ultrastructural evidence may be used to confirm the presence of mercury.

Chemically modified beta-glucuronidase crosses blood-brain barrier and clears neuronal storage in murine mucopolysaccharidosis VII.

Enzyme replacement therapy has been used successfully in many lysosomal storage diseases. However, correction of brain storage has been limited by the inability of infused enzyme to cross the blood-brain barrier. The newborn mouse is an exception because recombinant enzyme is delivered to neonatal brain after mannose 6-phosphate receptor-mediated transcytosis. Access to this route is very limited after 2 weeks of age. Recently, several studies showed that multiple infusions of high doses of enzyme partially cleared storage in adult brain. These results raised the question of whether correction of brain storage by repeated high doses of enzyme depends on mannose 6-phosphate receptor-mediated uptake or whether enzyme gains access to brain storage by another route when brain capillaries are exposed to prolonged, high levels of circulating enzyme. To address this question, we used an enzyme whose carbohydrate-dependent receptor-mediated uptake was inactivated by chemical modification. Treatment of human beta-glucuronidase (GUS) with sodium metaperiodate followed by sodium borohydride reduction (PerT-GUS) eliminated uptake by mannose 6-phosphate and mannose receptors in cultured cells and dramatically slowed its plasma clearance from a t(1/2) of <10 min to 18 h. Surprisingly, PerT-GUS infused weekly for 12 weeks was more effective in clearing central nervous system storage than native GUS at the same dose. In fact, PerT-GUS resulted in almost complete reversal of storage in neocortical and hippocampal neurons. This enhanced correction of neuronal storage by long-circulating enzyme, which targets no known receptor, suggests a delivery system across the blood-brain barrier that might be exploited therapeutically.

A murine model of infantile neuronal ceroid lipofuscinosis-ultrastructural evaluation of storage in the central nervous system and viscera.

Infantile neuronal ceroid lipofuscinosis (INCL), also known as Santavuori-Haltia disease, is an inherited neurodegenerative disorder caused by a mutation in the gene encoding the lysosomal enzyme palmitoyl-protein-thioesterase-1 (PPT1). Fatty acid-modified proteins are not degraded and accumulate as granular osmiophilic deposits in cells in the central nervous system; patients have blindness, seizures, progressive psychomotor deterioration, and die in early childhood. Although the disease manifests clinically primarily with neurological symptoms, visceral storage also accumulates. A murine model of INCL due to PPT1 deficiency exhibits clinical findings and pathology similar to those seen in patients with INCL. Homozygous PPT1-deficient mice have a shortened life span and neurological abnormalities including seizures, blindness, and mental and motor deficits. Widespread granular osmiophilic deposits (GRODs) accumulate in lysosomes in neurons and glia in the brain, retinal cells, kidney glomerular cells, aortic smooth muscle cells, and, in lesser amounts, in the fixed-tissue macrophage system. Accumulation of GRODs in aortic smooth muscle cells is accompanied by abnormalities in cardiac function and aortic root dilatation. This PPT1-deficient murine model is a well-defined genetic system that can be used to test potential therapies for lysosomal storage disease and to study the pathophysiology of INCL.

Enzyme therapy in mannose receptor-null mucopolysaccharidosis VII mice defines roles for the mannose 6-phosphate and mannose receptors.

Enzyme replacement therapy (ERT) is available for several lysosomal storage diseases. Except for Gaucher disease, for which an enzyme with exposed mannosyl residues targets mannose receptors (MR) on macrophages, ERT targets primarily the mannose 6-phosphate receptor (MPR). Most recombinant lysosomal enzymes contain oligosaccharides with both terminal mannosyl and mannose 6-phosphate residues. Effective MPR-mediated delivery may be compromised by rapid clearance of infused enzyme by the MR on fixed tissue macrophages, especially Kupffer cells. To evaluate the impact of this obstacle to ERT, we introduced the MR-null mutation onto the mucopolysaccharidosis type VII (MPS VII) background and produced doubly deficient MR-/- MPS VII mice. The availability of both MR+/+ and MR-/- mice allowed us to study the effects of eliminating the MR on MR- and MPR-mediated plasma clearance and tissue distribution of infused phosphorylated (P) and nonphosphorylated (NP) forms of human beta-glucuronidase (GUS). In MR+/+ MPS VII mice, the MR clearance system predominated at doses up to 6.4 mg/kg P-GUS. Genetically eliminating the MR slowed plasma clearance of both P- and NP-GUS and enhanced the effectiveness of P-GUS in clearing storage in kidney, bone, and retina. Saturating the MR clearance system by high doses of enzyme also improved targeting to MPR-containing tissues such as muscle, kidney, heart, and hepatocytes. Although ablating the MR clearance system genetically is not practical clinically, blocking the MR-mediated clearance system with high doses of enzyme is feasible. This approach delivers a larger fraction of enzyme to MPR-expressing tissues, thus enhancing the effectiveness of MPR-targeted ERT.

Attenuation of murine lysosomal storage disease by allogeneic neonatal bone marrow transplantation using costimulatory blockade and donor lymphocyte infusion without myeloablation.

Treatment of nonmalignant childhood disorders by bone marrow transplantation (BMT) is limited by toxicity from preparatory regimens and immune consequences associated with engraftment of allogeneic donor cells. Using costimulatory blockade (anti-CD40L mAb and CTLA-4Ig) combined with high-dose BMT in nonablated neonates, we obtained engraftment and established tolerance using both partially MHC mismatched (H2g7 into H2b) and fully mismatched BM (H2s into H2b). Recipients were mucopolysaccharidosis type VII (MPS VII) mice with lysosomal storage disease in order to assess therapeutic outcome. Recipients treated with donor lymphocyte infusion (DLI) amplified microchimerism to full donor. Recipients without DLI maintained long-term engraftment, tolerance, and had extended life spans. DLI increased donor cell mediated replacement of beta-glucuronidase (GUSB) activity in all tissues and maintained clearance of lysosomes better than in non-DLI-treated mice. DLI amplification of partially mismatched BM and fully mismatched BM caused late onset chronic GvHD in 56% and 100% of recipients, respectively.

Early onset of lysosomal storage disease in a murine model of mucopolysaccharidosis type VII: undegraded substrate accumulates in many tissues in the fetus and very young MPS VII mouse.

Lysosomal storage diseases (LSDs), due to deficiency of a lysosomal enzyme, are inherited, progressive disorders that are often fatal during childhood. The mucopolysaccharidoses (MPS) are LSDs caused by deficiency of a lysosomal enzyme needed for the stepwise degradation of glycosaminoglycans. A murine model of MPS VII shares many clinical, biochemical, and pathologic features with human MPS and has proved valuable for the study of the pathophysiology of MPS and for evaluation of therapies for LSDs. Early therapy of MPS VII mice, initiated in the first weeks of life, is much more effective in decreasing clinical and morphologic evidence of disease than treatment begun in mature animals. Whether such early therapy decreases existing storage or prevents its accumulation is incompletely investigated. We performed an analysis of storage in very young MPS VII mice to define the extent of disease at and before the time of initiation of early treatments. MPS VII pups from 12 days postcoitus (dpc) to 31 days postnatal (dpn) were studied. Storage accumulated in fixed tissue macrophages in the liver and cartilage as soon as 12 dpc and was present in central nervous system glia, leptomeninges, and perivascular cells by 15 dpc. Osteoblast and primitive neocortical cell storage was apparent at 18 to 19 dpc. At 2 dpn, lysosomal distention appeared in circulating leukocytes. Abundant lysosomal storage was present in many sites by 14 dpn. Secondary accumulation of beta-hexosaminidase paralleled increasing glycosaminoglycan storage. These results confirm the presence of widespread storage even in utero and in the very young MPS VII mouse and highlight the importance of early treatment to prevent storage accumulation.

Overcoming the blood-brain barrier with high-dose enzyme replacement therapy in murine mucopolysaccharidosis VII.

Enzyme replacement therapy (ERT) effectively reverses storage in several lysosomal storage diseases. However, improvement in brain is limited by the blood-brain barrier except in the newborn period. In this study, we asked whether this barrier could be overcome by higher doses of enzyme than are used in conventional trials. We measured the distribution of recombinant human beta-glucuronidase (hGUS) and reduction in storage by weekly doses of 0.3-40 mg/kg administered i.v. over 1-13 weeks to mucopolysaccharidosis type VII mice immunotolerant to recombinant hGUS. Mice given up to 5 mg/kg enzyme weekly over 3 weeks had moderate reduction in meningeal storage but no change in neo-cortical neurons. Mice given 20-40 mg/kg three times over 1 week showed no reduction in storage in any area of the CNS except the meninges. In contrast, mice receiving 4 mg/kg per week for 13 weeks showed clearance not only in meninges but also in parietal neocortical and hippocampal neurons and glia. Mice given 20 mg/kg once weekly for 4 weeks also had decreased neuronal, glial, and meningeal storage and averaged 2.5% of wild-type hGUS activity in brain. These results indicate that therapeutic enzyme can be delivered across the blood-brain barrier in the adult mucopolysaccharidosis type VII mouse if administered at higher doses than are used in conventional ERT trials and if the larger dose of enzyme is administered over a sufficient period. These results may have important implications for ERT for lysosomal storage diseases with CNS involvement.

Intravitreal gene therapy reduces lysosomal storage in specific areas of the CNS in mucopolysaccharidosis VII mice.

The mucopolysaccharidoses (MPSs) are lysosomal storage diseases resulting from impaired catabolism of sulfated glycosaminoglycans. MPS VII mice lack lysosomal beta-glucuronidase (GUSB) activity, leading to the accumulation of partially degraded chondroitin, dermatan, and heparan sulfates in most tissues. Consequently, these mice develop most of the symptoms exhibited by human MPS VII patients, including progressive visual and cognitive deficits. To investigate the effects of reducing lysosomal storage in nervous tissues, we injected recombinant adeno-associated virus encoding GUSB directly into the vitreous humor of young adult mice. Interestingly, GUSB activity was subsequently detected in the brains of the recipients. At 8-12 weeks after treatment, increased GUSB activity and reduced lysosomal distension were found in regions of the thalamus and tectum that received inputs from the injected eye. Lysosomal storage was also reduced in adjacent nonvisual regions, including the hippocampus, as well as in the visual cortex. The findings suggest that both diffusion and trans-synaptic transfer contribute to the dissemination of enzyme activity within the CNS. Intravitreal injection may thus provide a means of delivering certain therapeutic gene products to specific areas within the CNS.

Transgene produces massive overexpression of human beta -glucuronidase in mice, lysosomal storage of enzyme, and strain-dependent tumors.

beta-Glucuronidase (GUSB) is a lysosomal enzyme important in the normal step-wise degradation of glycosaminoglycans. Deficiency of GUSB causes the lysosomal storage disease mucopolysaccharidosis VII (MPS VII, Sly disease). Affected patients have widespread progressive accumulation of beta-glucuronide-containing glycosaminoglycans in lysosomes. Enzyme replacement, bone marrow transplantation, and gene therapy can correct lysosomal storage in the MPS VII mouse model. Gene therapy in MPS VII patients and animals may result in massive overexpression of GUSB in individual tissues, and the toxicity of such overexpression is incompletely investigated. To gain insight into the effect of massive overexpression of GUSB, we established 19 transgenic mouse lines, two of which expressed very high levels of human GUSB in many tissues. The founder overexpressing mice had from >100- to several thousand-fold increases in tissue and serum GUSB. The enzyme expression in most tissues decreased in subsequent generations in one line, and expression in liver and marrow fell in subsequent generations of the other. Both lines had morphologically similar widespread lysosomal storage of GUSB and secondary elevations of other lysosomal enzymes, a finding characteristic of lysosomal storage disease. One line developed tumors, and one did not. These transgenic models show that massive overexpression of a lysosomal enzyme can be associated with dramatic morphological alterations, which, at least in one of the two lines, had little clinical consequence. For the other transgenic line, the high frequency of tumor development in F(2) FVB progeny suggests that the vector used to generate the transgenic lines has an integration site-dependent potential to be oncogenic, at least in this strain background.