Recombinant macrophage targeted enzyme replacement therapy for Gaucher disease in India.

OBJECTIVE: Gaucher disease in India has been reported only in a few case reports from India. The aim of the study was to assess the response to enzyme replacement therapy in Indian patients with Gaucher disease. DESIGN: Retrospective analysis of patients receiving CHO-derived recombinant macrophage-targetted glucocorebrosidase. SETTING: Five centers from India with experience in treating lysosomal storage disorders. PATIENTS: The diagnosis of Gaucher disease was confirmed by low glucocerebrosidase levels, though it was first made on splenectomy in 8 and on bone marrow examination in 9 patients. Twenty five of 52 patients diagnosed with Gaucher disease (17 Type I, 8 mild Type III) received treatment for >6 months. Indications for treatment included symptomatic anemia, thrombo-cytopenia, organomegaly, bone disease or mild neurological symptoms leading to impairment of quality of life. Patients with significant neurological involvement were excluded. The drug infusions were given intravenously every 15 days. MAIN OUTCOME MEASURES: Hemoglobin, platelet counts, liver and spleen volumes and growth parameters. RESULTS: 22 of the 25 children who survived were analyzed. After 6 months of treatment, the mean (range) increase in hemoglobin was 1.5 (-3.4 to 6.1) g/dL (P=0.01) and in platelet count was 32 x 10(9)/L (-98.5 x 109 to 145.5 x10(9))/L (P=0.02). The mean (range) increase in weight was 3 kg (-5.6 to 10.5) (P=0.04) and in height was 7.1 cm (0 to 26.5) (P=0.0003). Liver size decreased by a mean (range) of 38.5% (- 5.5 to 86.7) (P=0.0003) and the spleen size by 34.8% (0 to 91.7) (P=0.004). All patients had improvement in bone pains and in 2 patients, neurological symptoms improved with others remaining static. CONCLUSIONS: This is the first reported cohort of patients in India reporting our experience with imiglucerase enzyme replacement therapy for treatment of Gaucher Disease in India.

Molecular diagnosis of Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene. The diagnosis is based on clinical and biochemical criteria but these are increasingly recognized to have low sensitivity. Genetic diagnosis is considered impractical due to the large coding region of the ATP7B gene and extreme diversity of mutations. We assessed the feasibility and utility of genetic diagnosis in WD. The coding region of the ATP7B gene was scanned by single-stranded conformation polymorphism (SSCP) analysis in 6 cases in whom the diagnosis of WD was uncertain. In addition, we attempted molecular diagnosis in 26 WD patients of similar ethnicity but variable disease manifestations. In 6 individuals in whom the biochemical/clinical diagnosis was uncertain, DNA analyses were useful for assigning their status with respect to WD. Molecular diagnosis identified presymptomatic individuals in families affected by WD and assigned heterozygote carrier or wild-type status to individuals previously diagnosed as affected. In 26 WD patients, 92% of disease alleles were identified. The most common mutations were H1069Q, L936X, and 2532delA representing 48, 10, and 8% of disease alleles, respectively. Three novel mutations were identified: Q898R, 3061(-1)g --> a, and 3972insC. Genetic diagnosis is feasible for WD. Greater application of molecular diagnosis should enable an appreciation of the full spectrum of WD phenotype that is not possible with currently available diagnostic criteria.

Ulcerative colitis has an aggressive course after orthotopic liver transplantation for primary sclerosing cholangitis.

BACKGROUND: The course of inflammatory bowel disease after liver transplantation has been reported as variable with usually no change or improvement, but there may be an increased risk of early colorectal neoplasms. In many centres steroids are often withdrawn early after transplantation and this may affect inflammatory bowel disease activity. AIMS: To evaluate the course of inflammatory bowel disease in primary sclerosing cholangitis transplant patients who were treated without long term steroids. METHODS: Between 1989 and 1996, there were 30 patients transplanted for primary sclerosing cholangitis who survived more than 12 months. Ulcerative colitis was diagnosed in 18 (60%) patients before transplantation; two had previous colectomy. All patients underwent colonoscopy before and after transplantation and were followed for 38 (12-92) months. All received cyclosporin or tacrolimus with or without azathioprine as maintenance immunosuppression. RESULTS: Ulcerative colitis course after transplantation compared with that up to five years before transplantation was the same in eight (50%) and worse in eight (50%) patients. It remained quiescent in eight and worsened in four of the 12 patients with pretransplant quiescent course, whereas it worsened in all four patients with pretransplant active course (p=0.08). New onset ulcerative colitis developed in three (25%) of the 12 patients without inflammatory bowel disease before transplantation. No colorectal cancer has been diagnosed to date. CONCLUSIONS: Preexisting ulcerative colitis often has an aggressive course, while de novo ulcerative colitis may develop in patients transplanted for primary sclerosing cholangitis and treated without long term steroids.

Outcome of orthotopic liver transplantation in patients with haemophilia.

BACKGROUND: Many patients with haemophilia have developed cirrhosis or hepatocellular carcinoma due to transfusion acquired chronic viral hepatitis. AIMS: To assess the long term outcome of all haemophilic patients reported to have undergone orthotopic liver transplantation. METHODS: Transplant centres of patients identified by medical database search were contacted and survival data assessed by Kaplan-Meier analysis. RESULTS: Twenty six haemophilic men (median age 46 years, range 5-63 years) underwent orthotopic liver transplantation in 16 centres between 1982 and 1996. Indications for transplantation were hepatitis C cirrhosis (69%), hepatitis B with or without C cirrhosis (15%), viral hepatitis related hepatocellular carcinoma (12%), and biliary atresia (4%). Six patients (23%) were infected with human immunodeficiency virus (HIV). Postoperatively, the median time to normal clotting factor levels was 24 hours (range 0-48 hours) and exogenous clotting factors were stopped at a median of 24 hours (range 0-480 hours). Four patients (15%) had bleeding complications. The one and three year survival of HIV positive recipients (67% and 23%) was significantly poorer (p = 0.0003) than that of HIV negative recipients (90% and 83%). Coagulopathy was cured in all patients surviving more than 12 days post-transplant. Six of the 20 patients (30%) with hepatitis C cirrhosis pretransplant had evidence of disease recurrence at a mean of nine months post-transplant. CONCLUSIONS: Hepatitis C cirrhosis is the most common indication for orthotopic liver transplantation in patients with haemophilia. Transplantation results in long term cure of haemophilia but may be complicated by the effects of HIV infection or recurrent viral hepatitis.

Therapeutic delivery of proteins to macrophages: implications for treatment of Gaucher's disease.

BACKGROUND: The primary defect in Gaucher's disease, a lysosomal disorder affecting macrophages, is in the activity of glucocerebrosidase. Treatment with exogenous enzyme (modified to increase its affinity for macrophage glycoprotein receptors) aims to restore this activity. However, the fate of the exogenous enzyme in vivo is unknown. We used radiolabelled enzyme to assess macrophage receptor activity for mannosylated ligands in vivo. METHODS: We examined the uptake and tissue distribution of radiolabelled enzyme molecules by gamma scintigraphy after bolus injection of iodine-123-labelled recombinant or placental enzyme (imiglucerase and alglucerase, respectively) in eight patients with type 1 Gaucher's disease, and in one healthy individual. The metabolism of the tracer enzyme was followed by scintigraphy and by analysis of blood, urine, and faeces. RESULTS: The tracer enzyme was rapidly cleared from blood (half-life 4.7 min [SD 1.0]). Concomitantly, there was avid uptake by the liver (about 30% of the injected dose), the spleen (about 15%), and the bone marrow. 40-55% of the tracer was cleared rapidly from the viscera (half-life 1-2 h) and 45-60% was cleared slowly (half-life 34-42 h). The half-life in the bone marrow was 14.1 h. Infusion of alglucerase at dose of 5 U/kg bodyweight normalised acid beta-glucosidase activity of splenic Gaucher's cells in vivo. When the enzyme was administered at a seven-fold higher dose (35 U/kg over 1 h), the receptor-mediated uptake in vivo was saturated, as shown by the increase in blood-clearance half-life of tracer enzyme from 4.5 min to 12 min. INTERPRETATION: Avid and saturable uptake of modified glucocerebrosidase was found, which indicates high-affinity targeting to the macrophage system in vivo. The rate of enzyme turnover suggests a rational basis for use of this therapy in treatment of Gaucher's disease.

Osteopontin is not a marker for proliferating human vascular smooth muscle cells.

Osteopontin (OP) is a secreted glycoprotein that contains the Arg-Gly-Asp (RGD) cell-binding sequence that binds calcium and is chemotactic and adhesive for rat vascular smooth muscle cells (VSMCs). OP gene expression is upregulated in cultured rat VSMCs in vitro and after balloon carotid injury in vivo, suggesting that OP may be a marker for proliferating VSMCs in vivo and in vitro. Our in situ hybridization studies of human atherosclerotic coronary vessels, however, have shown OP mRNA expression in plaque macrophages but not VSMCs. The current study investigated OP mRNA expression in cultured human VSMCs and macrophages and in an organ culture model of neointima formation in human saphenous vein. OP mRNA expression was not detected by Northern blot analysis of total RNA from subconfluent or confluent cultures of human VSMCs of any passage maintained in normal growth medium or after stimulation with TGF beta 1 (20 ng/mL), angiotensin II (1 mumol/L), or basic fibroblast growth factor (10 mg/mL) but was just detectable after stimulation with activation vitamin D3 (1 mumol/L). In contrast, cultured human macrophages expressed high levels of OP mRNA that were not dependent on lipid loading. OP mRNA was detected in isolated foci in all layers of saphenous veins maintained in organ culture for 14 days, including <2% of neointimal cells, a distribution that paralleled that of tissue macrophages. These results suggest that OP gene expression is not a marker for proliferation of human VSMCs in vitro and highlight a fundamental difference in the biology of human and rodent VSMCs.

Successful treatment of bone marrow failure in Gaucher's disease with low-dose modified glucocerebrosidase.

We report the beneficial effects of enzyme replacement therapy with mannose-terminated human glucocerebrosidase ('Ceredase') in a patient suffering from transfusion-dependent bone marrow failure due to Gaucher's disease. Treatment with low-dose enzyme infusions, given twice weekly, rapidly reversed the haematopoietic failure and incapacitating skeletal disease. It appears likely that prior splenectomy favourably influenced the response to this therapy.

Genetic diagnosis of Gaucher's disease.

The inherited disorder Gaucher's disease can be caused by various mutations in the glucocerebrosidase gene. Some mutations may be associated with greater severity, and there is a need for methods of gene analysis that would facilitate screening and diagnosis. We have studied the molecular basis of Gaucher's disease in twelve unrelated patients of diverse ethnic origin by means of the amplification refractory mutation system (ARMS). Primers for the polymerase chain reaction were designed to discriminate between mutant and wild-type alleles of glucocerebrosidase and to allow separation from products of the related pseudogene. The nucleotide 1226 mutation (asparagine 370----serine) and 84GG (an insertional frameshift mutation) were found exclusively in five patients of Ashkenazi Jewish descent (7 and 2 of the 10 disease alleles, respectively). Two point mutations, at nucleotides 1448 (leucine 444----proline) and 1504 (arginine 463----cysteine), were found in 4 and 3 alleles, respectively; they were associated with rapidly progressive disease and neurological involvement in non-Jewish patients. The ARMS procedure for direct detection of common mutations in glucocerebrosidase will facilitate genetic counselling and screening programmes for individuals at risk of Gaucher's disease.