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.     

Method for the determination of cerebral blood flow in conscious rabbits

A procedure for the determination of cerebral blood flow by the local clearance method after intracarotid injection of 133Xe in the conscious rabbit is described. The inert radioactive indicator is injected into a permanent nylon catheter equipped with a two-way Gordth's needle inserted into the common carotid artery and filled with heparin, emerging behind the shoulders of the animals. All branches of the homolateral common carotid artery except the internal carotid artery were ligated. Studies of the distribution of colored tracers (dark blue ink) and radioactive tracers (99mTc albumin microspheres) show that the main localization of the injected indicator is within the homolateral hemisphere. Brain to blood partition coefficients of 133Xe are worked out for rabbit's gray matter (0.576 +/- 0.048) and white matter (0.808 +/- 0.023). The slope method for first and second component of the wash-out Xenon curve is used for CBF calculations. CBF determinations in 9 normal rabbits result in 84.27 +/- 5.59 and 16.69 +/- 2.44 ml/min x 100 g tissue, respectively, for the fast and slow component. Significant changes do not occur in serial determinations within 2 hr.     

Differential responsiveness of murine T lymphomas to local growth and invasion factors may determine metastasis formation in the ovaries

Iodine-123-metaiodobenzylguanidine [123I-MIBG] has been used to evaluate the cardiac sympathetic nervous system. We evaluated the effect of pulmonary hypertension on the sympathetic neuronal function of the left ventricle in patients with pulmonary hypertension. We studied 20 patients with either chronic lung disease or pulmonary vascular disease. The patients were divided into a pulmonary hypertensive group and a control group. Single photon emission tomography was performed in the resting state 15 min and 4 h after administration of 123I-MIBG. Regions of interest (ROI) were set in the left ventricular (LV) free wall, the interventricular septum (IVS) and outside the LV free wall on short-axis images. The washout rate and the ROI/LV uptake ratio were calculated in each ROI. The IVS:LV uptake ratio was significantly lower in the pulmonary hypertensive group than in the control group. Our results suggest that left heart sympathetic neuronal dysfunction initially occurs in the IVS before it involves the LV free wall subsequently.     

Gaucher's disease: the best laid schemes of mice and men

The creation of animal models of Gaucher's disease, the inherited deficiency of the enzyme glucocerebrosidase, has led to new clinical insights and to a new appreciation of the complexity of the glucocerebrosidase gene locus. Murine embryonic stem cells with targeted modifications in the glucocerebrosidase gene were used to generate mouse models of Gaucher's disease, the first having a null glucocerebrosidase allele. The resulting knockout mice have no glucocerebrosidase activity and die within 12 hours of birth. Ultrastructural studies of liver, spleen, brain and bone marrow demonstrate the characteristic storage material seen in Gaucher patients. In the nervous system, storage of lipid increased in a rostral-caudal distribution. Analysis of skin from the knockout mice revealed histological, ultrastructural and biochemical abnormalities. The null allele Gaucher mice are analogous to neonates with Type 2 Gaucher's disease who present with hydrops foetalis and/or congenital ichthyosis. Moreover, the epidermal changes seen in Type 2 mice are also found in Type 2 patients and may provide a means to presymptomatically discriminate Type 2 from Type 1 and 3 Gaucher's disease. Another targeted modification in the murine glucocerebrosidase gene locus led to the discovery of a contiguous gene, metaxin. Closer analysis of the glucocerebrosidase gene locus, including sequencing of 75 kb of genomic DNA, reveals that this is a gene-rich region coding for seven genes and two pseudogenes. Further study of these closely arrayed genes may contribute to our understanding of the clinical variation encountered among patients with Gaucher's disease.     

Acid phosphatase isoenzymes in Gaucher's disease

Acid phosphatase (EC 3.1.3.2) isoenzyme profiles of extracts of splenic tissue and serum from patients with Gaucher's disease were measured by a mini-column ion-exchange chromatographic method [Clin. Chem., 23, 000 (1977)]. Diagnosis of Gaucher's disease in the five patients studied was confirmed by demonstrating decreased (2.3 to 4.1% of normal) glucocerebrosidase activity in the spleen. With p-nitrophenyl phosphate as substrate, increased acid phosphatase activity (three-to eight-fold normal) was demonstrated in spleen tissue from Gaucher;s disease patients; isoenzyme profiles by the ion-exchange column technique showed acid phosphatase isoenzyme 5 to be the predominant isoenzyme. Comparison of acid phosphatase isoenzyme profiles from patients with Gaucher's disease and prostatic carcinoma revealed distinct differences in the activities of isoenzymes 2 and 5. The isoenzyme-5 measurement thus appears to provide a diagnostic test for Gaucher's disease that can be done reapidly and easily in the routine clinical chemistry laboratory.     

Technetium 99m sulfur colloid scintigraphy to evaluate reticuloendothelial system function in dogs with portasystemic shunts

In a retrospective study, technetium 99m sulfur colloid scintigraphy was used to evaluate reticuloendothelial system function in 61 dogs with single congenital and 40 dogs with multiple acquired portasystemic shunts. Whole body reticuloendothelial function was measured by calculating the plasma clearance rate constant from a dynamic study of liver uptake of 99mTc sulfur colloid. Relative liver, spleen, and lung uptake, and a ratio of hepatic:extrahepatic uptake were measured on static equilibrium images. Results were compared with those of a group of 26 normal dogs. Compared with values for the group of normal dogs, the plasma clearance rate constant, relative liver uptake, and hepatic:extrahepatic uptake ratio were significantly decreased, and relative spleen and lung uptake were significantly increased in dogs with portasystemic shunts (P < .0001). The only significant difference between dogs with single congenital versus multiple acquired shunts was that the relative splenic uptake was higher in the former group (P < .0002). Based on these results, we concluded that dogs with portasystemic shunts have significantly impaired reticuloendothelial function. The primary cause of this dysfunction is likely a reduction in effective liver blood flow. Increases in spleen and lung reticuloendothelial activity did occur, but only partially compensated for the reduction of liver reticuloendothelial activity. Possible mechanisms for the increased spleen and lung uptake are discussed.     

Gaucher's disease: clinical features and natural history

Gaucher's disease is an inherited disorder characterized by pathological storage of glycolipid in mononuclear phagocytes: it is a multi-system disease associated with striking variation in its clinical manifestations, severity and course. Although molecular analysis of the glucocerebrosidase gene in patients with Gaucher's disease has permitted broad correlations between genotype and phenotype to be made, with few exceptions genetic variation at this locus does not allow confident prediction of clinical phenotype or prognosis. Partial deficiency of glucocerebrosidase is associated principally with parenchymal disease of the liver, spleen, bone marrow and, in severe cases, the lung, in non-neuronopathic, Type 1, Gaucher's disease: here storage material in macrophages originates from turnover of exogenous glycolipids. Severe deficiency of glucocerebrosidase caused by disabling mutations is additionally associated with neurological manifestations that in part reflect a failure to degrade endogenous neuronal glycosphingolipids, the so-called neuronopathic, Type 2 and Type 3 disease categories. Here we describe the clinical features, complications and natural history principally of Type 1 Gaucher's disease: emphasis is placed on emerging pulmonary, osseous and other manifestations of obscure pathogenesis that respond poorly to enzyme-replacement therapy.     

Studies on 133Xe wash-out from human skin: quantitative measurements of blood flow in normal and corticosteroid-treated skin

Blood flow was measured by the 133Xe technique in normal and corticosteroid-treated skin. Epicutaneous and intracutaneous methods of tracer application were compared in normal skin. The two labeling methods were equally suitable for measuring cutaneous blood flow provided calculations in both cases were based on a biexponential resolution of the wash-out curve in its cutaneous and subcutaneous components and provided the traumatic hyperemia phase was considered, when intracutaneous application of the tracer was used. Results were invalidated if calculations were based on initial slope of the wash-out curves.Topical application of beta-methasone valerate in a reduction in cutaneous blood flow as measured by the intracutaneous technique with curve resolution, whereas no effect could be demonstrated when calculations were based on the initial slopes of the curves. The 133Xe technique is a simple and reliable method for measuring cutaneous blood flow, which might prove useful in estimations of penetration ability and potency of topical corticosteroids.     

Renal disease. II. Urinary tract infection in women

The paper is devoted to the investigation of the lipid peroxidation level according to measurements of the accumulation level of malon dialdehyde in fragments of the liver tissue of newborn and adult pigs, depending on the cooling rate and cryoprotectants used. The conclusion has been made on the basis of the data obtained. That the lipid peroxidation level in the liver tissue fragments is decreased with the increase of cooling rates. That is activated the least of all with the use of average cooling rate (100 degrees C/min) with dimethylsulphoxide (DMSO) as cryoprotectant in concentrations of 10, 20% and rapid cooling rate (80,000 degrees C/min) with polyethylenglycol-1500 (PEG-1500) and DMSO in analogous concentrations. These cryopreservation data have shown the lowest of the lipid peroxidation level in the liver tissue fragments when comparing one with native tissue (before freezing).     

Comparison of technetium-99m-ECD to Xenon-133 SPECT in normal controls and in patients with mild to moderate regional cerebral blood flow abnormalities

Technetium-99m-1,1-ethyl cysteinate dimer (ECD) has been proposed as a "chemical microsphere" for SPECT measurement of regional cerebral blood flow (rCBF). However, its distribution has not yet been compared in humans to an established rCBF measure. Therefore, we compared the uptake and distribution of ECD with rCBF measured by 133Xe SPECT in subjects with mild to moderate flow abnormalities and in normal volunteers. Blood and urine chemistries and vital signs were unchanged from pre-ECD values up to seven days postinjection. Profile plots demonstrated pattern agreement between rCBF ratios (133Xe) and ECD count density ratios. A significant correlation of rCBF ratios to ECD count density ratios was observed (r = 0.77), with a slope of 0.64 and intercept of 0.36. To explore whether or not the relationship between rCBF and ECD was dependent on absolute flow, ECD region of interest data were expressed in units of ml/min/100 g by equating global CBF (133Xe) and ECD global count density. A closer correlation (r = 0.88) was found for these data than for the count ratio data. The slope was closer to one (m = 0.83) and the intercept was closer to zero (b = 8.2). Also, a significant correlation was observed between ECD-derived rCBF and 133Xe rCBF in the lesion area (r = 0.92) for patients with well-demarcated rCBF lesions. The slope (0.80) suggested a slight underestimation of lesion flow by ECD. Finally, ECD clearance from cortical gray matter ROIs derived from high-resolution scans from 1 to 4 hr postinjection was slow (2.4%/hr). In summary, ECD is a safe and effective marker of regional cerebral perfusion. The distribution of ECD is linearly related to rCBF measured by 133Xe SPECT, although our data suggest a mild underestimation of flow at the high end of the normal range.     

Decreased uptake of 14C-labeled dicarboxylic amino acids in rapidly growing hepatomas

In contrast to the increased uptake of amino acids which has been found in many neoplastic cells, we have observed a decrease in the net uptake of [14C]aspartate and [14C]glutamate in rapidly growing hepatomas relative to rat host liver. When measured 10 min after s.c. injection, the radioactivity from 14C-labeled dicarboxylic amino acids was greater in liver than in all other tissues examined (blood, skeletal, muscle, heart, spleen, lung, and brain) except kidney, where there was an approximately 2-fold greater uptake of aspartate and 10-fold greater uptake of glutamate. Mean uptakes in the rapidly growing Morris hepatomas 7288CTC and 7777 were 19 to 26% of corresponding values for the host livers. Comparison with uptake of 3H2O indicated that these low values were not solely due to differences in circulation. Decreased uptake was not accompanied by equivalent decreases in the concentration of aspartate and glutamate in the tumors. There were small changes in the net uptake of these amino acids in the slowly growing hepatoma 7787 and no significant differences in regenerating liver and hepatoma 5123C, a tumor of intermediate growth rate. The net uptake of [14C]arginine and [14C]lysine in the hepatomas was similar to that in host livers, except for a 250% increase in uptake of [14C]lysine in hepatoma 5123C. A decreased uptake of the magnitude seen with dicarboxylic amino acids in rapidly growing hepatomas has not been observed with other amino acids.     

Color functional images of the cerebral blood flow

Functional gamma imaging, in color, was established for regional cerebral blood flow (rCBF) using 133Xe. During 10 min after intracarotid injection of 133Xe in saline, 60 picture frames of the 133Xe clearance curve for the entire hemisphere were obtained. After nine-point smoothing, the rCBF for each of the 4,096 picture elements was calculated by two methods: the half-time method and the height-over-area method. Both the 133Xe clearance half-times and the calculated CBF values were displayed, using 13 steps of color, as functional CBF images of the brain. Images of peak count and total count were also displayed on the same frame of the color television. Forty-six studies, performed on 37 patients with various cerebral disorders, were divided into two types: diffuse and focal. In the diffuse type, a decrease in CBF was noted in cases of normal-pressure hydrocephalus; successful ventriculoperitoneal shunt operations were followed by recovery of CBF. Occlusion of the middle cerebral artery showed up as a wedge-shaped area of decreased CBF, even when the conventional brain scan looked normal. Increased perfusion to a tumor was frequently associated with decreased CBF in the rest of the lateral hemisphere; such a decrease could be improved by surgical removal of the tumor.     

What is the blood flow to resting human muscle?

1. An investigation was carried out in five healthy lean adults to assess whether forearm and calf plethysmography largely reflect muscle blood flow as measured by 133Xe and whether there is substantial variability in the blood flow to muscles located at different sites in the body. 2. Blood flow to forearm and calf flexors and extensors, biceps, triceps and quadriceps was assessed using the 133Xe clearance technique. Blood flow to forearm skin and subcutaneous adipose tissue was also measured using the 133Xe clearance technique, whereas blood flow to the forearm and calf was measured using strain gauge plethysmography. 3. The mean blood flow to different muscles ranged from 1.4 +/- 0.6 (gastrocnemius) to 1.8 +/- 0.7 (forearm extensor) ml min-1 100 g-1 muscle (1.4 +/- 0.6 and 1.9 +/- 0.8 ml min-1 100 ml-1 muscle, respectively) but there were no significant differences between them. Forearm and calf blood flows (2.7 +/- 0.3 and 3.0 +/- 0.7 ml min-1 100 ml-1 limb tissue, respectively) were about 50% to more than 100% greater (P < 0.025) than blood flow to the muscles within them (1.7 +/- 0.5 and 1.4 +/- 0.5 ml min-1 100 g-1 muscle, respectively, or 1.8 +/- 0.6 and 1.5 +/- 0.5 ml min-1 100 ml-1 muscle, respectively). In contrast, the blood flows to 100 g of forearm skin (9.1 +/- 2.6 ml min-1 100 g-1) and adipose tissue (3.8 +/- 1.1 ml min-1 100 g-1) were higher than the blood flow to 100 g of forearm (P < 0.01 and not significant, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calculation of coronary blood flow from myocardial clearance of systemically administered 133Xe

Clearance curves for arterial and coronary-venous blood were determined after systemic left-ventricle or pulmonary-artery infections of 133Xe, paired with selective left-coronary-artery injections of 133Xe in 20 dogs with closed chest. Coronary blood flows calculated from systemic and coronary-artery injections were comparable only when a correction was made for arteriol recirculation of 133Xe following the systemic injection (r = 0.962) for left ventricle and 0.932 for pulmonary artery, paired with coronary artery). Experiments in four other dogs, verified that clearance of 133Xe from the pulmonary circulation was only about 60%. The myocardium/blood 133Xe partition coefficient, determined in vivo in ten dogs, agreed within 10% with that previously determined in vitro.     

Modifications of glucose and lipid metabolism in cold-acclimated lean and genetically obese rats

Glucose turnover rate, 2-deoxy-D-[3H]glucose (2-DG) uptake, lipid synthesis in liver, white adipose tissue, and brown adipose tissue (BAT) were measured in lean FA/FA and genetically obese fa/fa rats either kept at 21 degrees C or acclimated to a cold environment (4 degrees C). After 10 days at 4 degrees C, lean rats increased their glucose turnover rate; 2-DG uptake as well as lipid synthesis in BAT were markedly stimulated. After cold acclimation, obese rats also increased glucose turnover; however, BAT glucose utilization was only slightly stimulated. Basal hyperinsulinemia and muscle insulin resistance of the obese group (as assessed by reduced 2-DG uptake in the soleus muscle) were present at room temperature and persisted at 4 degrees C. Total BAT lipid synthesis was increased to the same extent as in lean rats. Obese rat liver lipid synthesis, already much higher than normal at 21 degrees C, was further increased by cold exposure. We conclude that obese cold-acclimated fa/fa rats do not improve their muscle insulin resistance and barely improve BAT glucose utilization. We further suggest that an additional activation of hepatic lipid synthesis and oxidation thereof could participate in the heat production needed by the cold-acclimated obese rats.     

Impairment of bacterial clearance induced by norepinephrine infusion in rabbits

OBJECTIVE: Purpose of the study was to investigate the potential influence of norepinephrine (NE) on immune functions in terms of systemic and organ-specific bacterial clearance in rabbits. DESIGN: To enable quantification of the clearance process, defined numbers of exogenous Escherichia coli (1.3 x 10(8) CFU) were injected intravenously 60 min after starting the NE infusion at a low dose (1 microgram/kg per min, n = 6), causing an increase (30 mmHg) in mean arterial pressure without affecting the oxygen uptake, and at a higher dose (7.5 micrograms/kg per min, n = 6), resulting in a marked decrease (20%) in oxygen uptake, after infusion of NaCl solution (control, n = 6). In additional experiments (n = 6) NE (1 microgram/kg per min) was tested in endotoxemia induced by simultaneous infusion of endotoxin (40 micrograms/kg per h). Parameters monitored were arterial pressure, oxygen uptake, and rates of bacterial elimination from the blood. At 180 min after E. coli injection, the animals were sacrificed, and tissue samples of liver, kidney, spleen, and lung were collected for bacterial counts. RESULTS: NE infusion resulted in a dose-dependent prolonged elimination of the injected E. coli from the blood and in significantly higher (p < 0.05) numbers of CFU in liver and lung compared to the controls. Significant impairment of bacterial clearance was found after shock-producing endotoxemia, whereas simultaneous infusion of NE and endotoxin caused only a slightly delayed blood clearance of the injected bacteria. CONCLUSION: NE dose dependently affected bacterial clearance, which might be due to ischemia-derived hypoxic impairment of the phagocytosis and lysis function of the reticuloendothelial system, whereas NE improved elimination of bacteria in a state of endotoxic shock.     

Relationship between gender and holistic representations of health and illness

Steady-state arterial spin tagging MRI approaches were used to quantitate regional cerebral blood flow increases during finger tapping tasks in seven normal subjects. Statistically significant increases in cerebral blood flow were observed in the contralateral primary sensorimotor cortex in all seven subjects and in the supplementary motor area in five subjects. The intrinsic spatial resolution of the cerebral blood flow images was approximately 4 mm. If no spatial filtering was applied, the average increase in cerebral blood flow in the activated primary sensorimotor cortex was 60 +/- 10 cc/100 g/min (91 +/- 32%). If the images were filtered to a spatial resolution of 15 mm, the average increase in cerebral blood flow in the activated primary sensorimotor cortex was 23 +/- 7 cc/100 g/min (42 +/- 15%), in agreement with previously reported 133Xe and PET results.     

Risk factors and outcome of human immunodeficiency virus-infected patients with sporadic multidrug-resistant tuberculosis in New York City

The discovery of neuroendocrine differentiation in hormone-refractory prostatic adenocarcinoma has opened a potentially new therapeutic approach in this group of patients with a poor prognosis and few effective therapy modalities. Based on previous findings of increased uptake of 11C-5-hydroxytryptophan (11C-5-HTP) in neuroendocrine tumours using the PET technique, this tracer was applied in the study of 10 patients with metastatic hormone-refractory prostatic adenocarcinoma. In three patients, the study was repeated after treatment. An increased uptake of 11C-5-HTP was observed in all investigated skeletal lesions, although the magnitude of the uptake was moderate. The difference between the standard uptake values (SUV) in normal bone and metastatic lesions was significant (p < 0.001). A kinetic analysis of the uptake of 11C-5-HTP demonstrates an increase during the first minutes followed by a wash-out and a stabilization of the tissue/blood ratio at about 2. The Patlak plots demonstrated a gradual increase in the transport rate during the first 20 to 30 min, after which a constant level was observed. The SUV varied between patients and between lesions over time and treatment. The uptake of 11C-5-HTP discriminates metastatic lesions from normal bone and may thus aid in the diagnosis and, potentially, in treatment monitoring of metastatic hormone-refractory prostatic adenocarcinoma. Uptake kinetics are characterized by a wash-out and cannot alone be used as proof of neuroendocrine differentiation in hormone-refractory prostatic adenocarcinoma.     

Isoenzymes of membrane-bound beta-glucosidase of human spleen

Normal human spleen contains two forms of membrane-bound beta-glucosidase, distinguishable by their thermostability and kinetic properties. The spleen from a patient with adult Gaucher's disease was deficient in the major, thermolabile, form of the enzyme.     

Enzyme replacement therapy for Gaucher's disease: the early Canadian experience

BACKGROUND: The management of severe Gaucher's disease was dramatically improved by the development of enzyme replacement therapy. However, this treatment is very costly (currently about $21,000 per infusion for adults at the starting dose recommended by the manufacturer). The goal of this study was to determine how enzyme replacement therapy was being prescribed and financially supported in various parts of Canada. In addition, demographic and outcome information was elicited. METHODS: Prescribing physicians were identified through professional associations and with the help of the manufacturer of the enzyme preparations used for the treatment of Gaucher's disease. The physicians were surveyed by questionnaire in July 1995. The study included all patients in Canada who had received enzyme replacement therapy for Gaucher's disease before July 1, 1995. RESULTS: A total of 25 patients (15 children and 10 adults) with type 1 Gaucher's disease, the common nonneuronopathic variant of the disease, were receiving enzyme replacement therapy by the end of 1995. The indications for treatment included massive splenomegaly, growth failure, and severe bony, hematologic and pulmonary complications of the disease; no patients with mild disease were receiving treatment. Treatment regimens varied markedly (from 12 to 160 units of enzyme/kg per month). All the patients were reported to have responded well to therapy, based on serial measurements of hematologic indices, liver and spleen volumes, and numbers of bony crises as well as patients' subjective impressions. Financial support for therapy varied markedly from one province to another. None of the reporting physicians was aware of any patients with severe Gaucher's disease who were denied therapy as a result of inability to pay for the medication. Various agencies provided financial support for therapy, including both federal and provincial governments, private insurance carriers and the commercial supplier of the enzyme. In Ontario provincial health care officials accepted the development, by a multidisciplinary panel of medical experts, of formal guidelines for determining eligibility, on the basis of objective medical criteria, for reimbursement for enzyme replacement treatment. INTERPRETATION: Although some differences were found across the country with respect to the details of treatment, the indications for enzyme replacement therapy and the selection of severely affected patients were similar in the various provinces. However, financial support was inconsistent and varied among provinces and patients. This will prove to be a challenge in future, not only with respect to this disease but also for other diseases for which effective, expensive therapy has been developed.