A multicentre randomised double masked clinical trial of a new formulation of topical cysteamine for the treatment of corneal cystine crystals in cystinosis.

AIM: To evaluate the safety and efficacy of a new topical cysteamine formulation, stable at room temperature, for the treatment of corneal cystine crystals in cystinosis. METHODS: 20 study subjects were enrolled in the safety study and 16 in the efficacy study. Both studies were randomised and double blind. The primary outcome for the safety study was the occurrence of predefined serious adverse reactions over 6 months and for the efficacy study the reduction of corneal cystine crystal score (CCCS) by 1.00 or more units on photographs graded by a reading centre using a standardised protocol. RESULTS: No study subject developed any serious adverse reactions. In the efficacy study, 47% of eyes receiving the standard formulation experienced a reduction in the CCCS of >/=1.00 after 1 year, while 7% of eyes on the new formulation experienced such a decrease (p=0.04). CONCLUSION: Although no serious adverse reactions were observed with either formulation, the new formulation was not as effective as the standard formulation.

Expression of CTNS alleles: subcellular localization and aminoglycoside correction in vitro.

Mutations in CTNS result in one of three forms of cystinosis: benign, intermediate, or nephropathic. Homozygosity for a nonsense mutation in CTNS (753G -->A), encoding a premature termination codon (PTC) at amino acid 138 (W138X), results in nephropathic cystinosis. Gentamicin is known to induce PTC readthrough and hence full-length protein production. We demonstrate that addition of gentamicin (300 microg/ml) to cystinotic fibroblasts leads to depletion of intracellular cystine in cell lines with a premature termination codon, but not in those with a large deletion or a deletion leading to a frameshift mutation. Plasmids were constructed with GFP as a C-terminal or N-terminal fusion to CTNS. The normal CTNS protein fused with either N- or C-terminal GFP colocalized with Lysotracker red, a fluorescent stain which selectively accumulates in lysosomes. PTC-GFP, a construct with GFP fused to the C-terminus of CTNS containing a PTC, allowed GFP to serve as a reporter of PTC readthrough. No significant fluorescence was observed in PTC-GFP-transfected cells in the absence of gentamicin but was seen and localized to lysosomes in its presence. A patient with a splice site mutation (IVS11 + 2T -->C) that eliminates the GYDQL lysosomal targeting sequence of cystinosin on one allele, and a PTC mutation (753G -->A) on the other, displays the intermediate phenotype. Transfection of the splice site mutant allele into CTNS null fibroblasts produced cystine depletion. Plasmids with GFP fused to the N-terminus of CTNS containing the splice site mutation (GFP-SS) were constructed. While the normal CTNS-GFP fusion protein was found to colocalize with Lysotracker red almost exclusively, the GFP-SS fusion product was found in the plasma membrane and cytoplasm, as well as lysosomes. A second lysosomal targeting motif in CTNS is present in this sequence, just proximal to the mutation, accounting for the partial lysosomal localization.

CTNS mutations in African American patients with cystinosis.

Cystinosis, an autosomal recessive lysosomal storage disorder, is rarely diagnosed in African Americans. The disease results from mutations in the gene CTNS; at least 55 such mutations have been reported. By far the most common is a 57,257-bp deletion of Northern European origin encompassing most of the CTNS gene. We performed mutation analysis on DNA from four African American patients with cystinosis. In one individual with classical, nephropathic cystinosis, we identified a new molecular defect, i.e., a homozygous GT-->CC substitution at the +5 position of IVS 5 of CTNS (IVS 5+5 GT-->CC). The out-of-frame splicing of exon 5 creates a null allele consistent with the patient's severe phenotype. Two patients were heterozygous and one homozygous for the common 57-kb deletion allele, reflecting the admixture of African and Northern European gene pools in North America. The two African Americans heterozygous for the 57-kb deletion were also hemizygous for a 928G-->A change, associated with ocular or nonnephropathic cystinosis. These two individuals are the only known African Americans with ocular cystinosis. We conclude that the diagnosis of cystinosis should be entertained in African Americans with symptoms of the disease, and that mutation analysis for the 57-kb deletion should be considered in this group of patients.

Ocular nonnephropathic cystinosis: clinical, biochemical, and molecular correlations.

Ocular nonnephropathic cystinosis, a variant of the classic nephropathic type of cystinosis, is an autosomal recessive lysosomal storage disorder characterized by photophobia due to corneal cystine crystals but absence of renal disease. We determined the molecular basis for ocular cystinosis in four individuals. All had mutations in the cystinosis gene CTNS, indicating that ocular cystinosis is allelic with classic nephropathic cystinosis. The ocular cystinosis patients each had one severe mutation and one mild mutation, the latter consisting of either a 928 G-->A (G197R) mutation or an IVS10-3 C-->G splicing mutation resulting in the insertion of 182 bp of IVS10 into the CTNS mRNA. The mild mutations appear to allow for residual CTNS mRNA production, significant amounts of lysosomal cystine transport, and lower levels of cellular cystine compared with those in nephropathic cystinosis. The lack of kidney involvement in ocular cystinosis may be explained by two different mechanisms. On the one hand (e.g. the G197R mutation), significant residual cystinosin activity may be present in every tissue. On the other hand (e.g. the IVS 10-3 C-->G mutation), substantial cystinosin activity may exist in the kidney because of that tissue's specific expression of factors that promote splicing of a normal CTNS transcript. Each of these mechanisms could result in minimally reduced lysosomal cystine transport in the kidneys.

Mutations of CTNS causing intermediate cystinosis.

Six patients with the intermediate form of cystinosis are described. Two have new mutations not previously described. The disease occurs due either to the combination of one mild mutation and one which is known to cause nephropathic cystinosis or to homozygosity for a predicted mild mutation. Partial phenotypic correction of cystinotic fibroblasts by transfection with normal cDNA or a cDNA derived from a mutation causing intermediate cystinosis is demonstrated.

CTNS mutations in an American-based population of cystinosis patients.

Nephropathic cystinosis is an autosomal recessive lysosomal storage disease characterized by renal failure at 10 years of age and other systemic complications. The gene for cystinosis, CTNS, has 12 exons. Its 2.6-kb mRNA codes for a 367-amino-acid putative cystine transporter with seven transmembrane domains. Previously reported mutations include a 65-kb "European" deletion involving marker D17S829 and 11 small mutations. Mutation analysis of 108 American-based nephropathic cystinosis patients revealed that 48 patients (44%) were homozygous for the 65-kb deletion, 2 had a smaller major deletion, 11 were homozygous and 3 were heterozygous for 753G-->A (W138X), and 24 had 21 other mutations. In 20 patients (19%), no mutations were found. Of 82 alleles bearing the 65-kb deletion, 38 derived from Germany, 28 from the British Isles, and 4 from Iceland. Eighteen new mutations were identified, including the first reported missense mutations, two in-frame deletions, and mutations in patients of African American, Mexican, and Indian ancestry. CTNS mutations are spread throughout the leader sequence, transmembrane, and nontransmembrane regions. According to a cystinosis clinical severity score, homozygotes for the 65-kb deletion and for W138X have average disease, whereas mutations involving the first amino acids prior to transmembrane domains are associated with mild disease. By northern blot analysis, CTNS was not expressed in patients homozygous for the 65-kb deletion but was expressed in all 15 other patients tested. These data demonstrate the origins of CTNS mutations in America and provide a basis for possible molecular diagnosis in this population.

Characterization of cysteamine as a potential contraceptive anti-HIV agent.

Cysteamine (beta-mercaptoethylamine, or MEA) is a thiol-reducing agent and has anti-HIV activity. Because of these properties, cysteamine was evaluated as a vaginal contraceptive and tested for its effects on sperm function and on other sexually transmitted microbes. Cysteamine was contraceptive in the rabbit. Conception was inhibited completely when sperm were pretreated with 500 microg/ml cysteamine and was inhibited by more than 60% when 7.5 mg cysteamine was applied vaginally as a suspension in 50% K-Y Jelly. Cysteamine had multiple effects on spermatozoa. Both acrosin (EC 3.4.21.10) and hyaluronidase (EC 3.2.1.35) were reversibly inhibited by cysteamine. Calculated IC50 values were 370 microg/ml and 150 microg/ml for acrosin and hyaluronidase, respectively. Cysteamine behaved as a poor spermicide when activity was measured by the 30-second Sander-Cramer test. However, sperm motility was inhibited completely when cysteamine was preincubated for 10 minutes prior to motility evaluation, at concentrations as low as 50 microg/ml. The calcium ionophore A23187-induced human acrosome reaction was inhibited by cysteamine (IC50 = 0.5 microg/ml). Neither herpes simplex virus nor Neisseria gonorrhoeae was affected by cysteamine at concentrations as high as 500 microg/ml and 100 microg/ml, respectively. Cysteamine appears to have no effect on normal vaginal flora (i.e., lactobacillus). These results, together with published data, strongly support the further development of cysteamine as a topical contraceptive anti-HIV agent.

Safety and feasibility of liver-directed ex vivo gene therapy for homozygous familial hypercholesterolemia.

OBJECTIVE: The purpose of this report was to provide detailed information on the safety and feasibility of surgical procedures associated with the first ex vivo liver-directed gene therapy trial for the treatment of vivo gene therapy for homozygous familial hypercholesterolemia (FH). SUMMARY BACKGROUND DATA: Familial hypercholesterolemia is an autosomal dominant disease in which the gene encoding the low density lipoprotein receptor is defective. Patients homozygous for this mutation have extraordinarily high levels of cholesterol and accelerated atherosclerosis and die prematurely of myocardial infarction. The concept of liver-directed gene therapy was based on the report of normalization of cholesterol levels by orthotopic cardiac/liver transplant in a child with homozygous FH. METHODS: Five patients with homozygous FH were selected for inclusion in this trial. The patients underwent hepatic resection and placement of a portal venous catheter. Primary hepatocytes cultures were prepared from the resected liver and transduced with a recombinant retrovirus encoding the gene for the human low density lipoprotein receptor. The genetically modified cells were then transplanted into the liver through the portal venous catheter. RESULTS: Numerous clinical, laboratory, and radiologic parameters were analyzed. Elevations of the hepatic transaminases and leukocyte counts and a decline in hematocrit count were noted. Transient elevations of the portal pressure were observed during cell infusion. No major perioperative morbidity--specifically, myocardial infarct, perioperative hemorrhage, or portal vein thrombosis--or death occurred as a result of this protocol. CONCLUSION: Liver-directed ex vivo gene therapy can be accomplished safely in humans and is appropriate for selected patients.

Detection and characterization of a transport system mediating cysteamine entry into human fibroblast lysosomes. Specificity for aminoethylthiol and aminoethylsulfide derivatives.

The uptake of [3H]cysteamine by Percoll-purified human fibroblast lysosomes was investigated to determine whether lysosomes contain a transport system recognizing cysteamine. Lysosomal cysteamine uptake is a Na(+)-independent process which rapidly attains a steady state within 1 min at pH 7.0 and 37 degrees C. A biphasic Arrhenius plot is observed for cysteamine uptake, giving a Q10 of 2.2 from 17 to 26 degrees C and a Q10 of 1.2 from 27 to 35 degrees C. The rate of lysosomal cysteamine uptake is maximal at pH 8.2, half-maximal at pH 6.8, and declines approximately 50-fold from the maximum to show very little transport at pH 5.0. Cysteamine uptake into fibroblast lysosomes displays complete saturability with a Km of 0.88 mM and Vmax of 1410 pmol of beta-N-acetylhexosaminidase/min at pH 7.0 and 37 degrees C. Analog inhibition studies demonstrated that all analogs recognized thus far by the cysteamine carrier are either aminothiols or aminosulfides and contain an amino group and sulfur atom separated by a carbon chain, 2 carbon atoms in length. The Ki constants for these analogs as competitive inhibitors of lysosomal cysteamine uptake are 2-(ethylthio)ethylamine (0.64 mM), 1-amino-2-methyl-2-propanethiol (0.74 mM), 2-dimethylaminoethanethiol (0.87 mM), thiocholine (1.6 mM), and bis(2-aminoethyl)sulfide (4.9 mM). L-Cysteine, D-penicillamine, and analogs lacking either a sulfur atom or amino group are not recognized by the cysteamine carrier including ethanolamine, choline, taurine, beta-mercaptoethanol, ethylenediamine, cadaverine, spermine, spermidine, histamine, dopamine, and 3-hydroxytyramine. In a cystine-depletion assay, a 2-h exposure of cystinotic fibroblasts to 1 mM 1-amino-2-methyl-2-propanethiol lowers cell cystine levels to the same low level obtained with cysteamine. Thus, all four aminothiols, known to deplete cystinotic fibroblasts of their accumulated cystine, are recognized as substrates by the lysosomal cysteamine carrier, suggesting the importance of this transporter in the delivery of aminothiols to the lysosomal compartment.

Recent advances in the treatment of cystinosis.

Cysteamine bitartrate capsules (Cystagon) have been approved by the US Food and Drug Administration for use in patients with nephropathic cystinosis. Plasma cysteamine concentrations were virtually identical at various times following ingestion of either cysteamine hydrochloride or Cystagon capsules in 24 normal control subjects. A transfer study was done with eight cystinosis patients who had been receiving either cysteamine hydrochloride or phosphocysteamine for many years. The plasma cysteamine concentration was significantly higher 2h after Cystagon and the leukocyte cystine content was significantly lower at all times after Cystagon compared to older forms of the drug. These differences are probably the result of greater patient compliance in taking the capsules compared to the older, liquid forms of the drug. A new method for following the course of renal glomerular deterioration in diseases such as cystinosis has been published recently. This method was used to re-analyse data on the efficacy of cysteamine treatment and to re-analyse new data on treating cystinosis patients with either of two doses of cysteamine (1.30 g/m2 per day and 1.95 g/m2 per day). This new method agrees well with other methods and shows that both doses of drug are equally effective in maintaining glomerular function.

Cystinosis.

Nephropathic cystinosis is an autosomal recessive inborn error of metabolism characterized by the lysosomal storage of the disulphide amino acid cystine. It produces a variety of clinical manifestations including failure to thrive, the renal Fanconi syndrome, eye findings, and end-stage renal disease. A variety of phenotypes are known; however, the molecular defect underlying any of the forms has not yet been identified. Therapy of cystinosis with cysteamine averts the otherwise inevitable renal failure, but systemic therapy does not improve the corneal keratopathy. A number of presentations in this review detail approaches to gene identification, systemic therapy with cysteamine, measurement of cystine, and pathophysiological effects at the cellular and clinical level.

Current status of orphan disease drug development.

The Orphan Drug Act has successfully stimulated the production of many orphan products for a number of orphan diseases. The success of its exclusive marketing provision in bringing otherwise unprofitable products to market has attracted the attention of manufacturers who use this provision to gain a monopoly for products with much larger annual sales than were contemplated by the original legislation. Corrective legislation to close this loophole is being prepared for introduction to Congress.

Metabolic studies in a mouse model of hepatorenal tyrosinemia: absence of perinatal abnormalities.

Radiation induced chromosomal deletions at the albino locus in the mouse, lethal when homozygous, cause abnormalities of expression of several unlinked liver specific genes. Recently, the gene encoding FAH was shown to be included in the deletions. Since in humans FAH mutations cause tyrosinemia type I, deletion homozygous mice were suspected of having tyrosinemia. Studies of plasma amino acids did not confirm this suspicion. Also, succinylacetone levels were normal in fetal and newborn livers of deletion homozygotes. The present evidence, therefore, does not support the assumption that the earlier described ultrastructural and enzyme abnormalities in deletion homozygotes are secondary effects of tyrosinemia caused by the deletion of FAH.

Ex vivo gene therapy of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by a deficiency in the receptor that clears low density lipoprotein (LDL) from the serum (reviewed in Ref. 1 and 2). Patients with one abnormal LDL receptor allele have moderate elevations in plasma LDL and suffer premature coronary artery disease (CAD). Approximately 5% of all patients under 45 who have had a myocardial infarction carry this trait. Patients with two abnormal LDL receptor genes (homozygous deficient patients) have severe hypercholesterolemia and life-threatening coronary artery disease in childhood. Strategies for treating patients with FH are directed at lowering the plasma level of LDL. In heterozygotes, this is accomplished through the administration of drugs that stimulate the expression of LDL receptor from the normal allele (2). This therapeutic approach is not effective in the treatment of homozygous deficient patients, especially those that retain less than 2% of residual LDL receptor activity. Partial amelioration of hyperlipidemia has been achieved in some homozygous deficient patients by diverting the portal circulation through a portacaval anastomosis (3) and by chronic plasmapheresis therapy (4). A more direct approach has been to correct the deficiency of hepatic LDL receptor by transplanting a liver that expresses normal levels of LDL receptor. Three patients that survived this procedure normalized their serum LDL-cholesterol (5-9). We have used an authentic animal model for FH, the Watanabe Heritable Hyperlipidemic rabbit (WHHL), to develop gene therapies for the homozygous form of FH (10-13). The WHHL rabbit has a mutation in its LDL receptor gene which renders the receptor completely dysfunctional (12) leading to severe hypercholesterolemia, diffuse atherosclerosis, and premature death. The potential efficacy of gene therapy for FH is supported by a series of studies we have performed in the WHHL rabbit in which we have achieved metabolic improvement (14-18). Liver tissue was removed from WHHL rabbits and used to isolate hepatocytes and establish primary cultures. A functional rabbit LDL receptor gene was transduced into a high proportion of hepatocytes using recombinant retroviruses, and the genetically corrected cells were transplanted into the animal from which they were derived. Transplantation of the genetically corrected, autologous hepatocytes was associated with a 30-40% decrease in serum cholesterol that persisted for the duration of the experiment (4 months, Ref. 18). Recombinant derived LDL receptor RNA was detected in liver for at least 6 months. There was no apparent immunological response to the recombinant derived LDL receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Leucine and tissue distribution of bulky and small neutral amino acids in rats: dissociation between transport and insulin-mediated effects.

The mechanism of the observed decrease in the plasma concentration of several amino acids in the presence of high levels of Leu has remained unexplained. In the present study a decrease in the plasma concentration of Ile, Val, Phe, Tyr, Met, Ala, Pro and Gly was observed after the intraperitoneal injection of Leu to weanling rats. Decreases in net intracellular concentrations in muscle accompanied the decrease in plasma of all of these amino acids except Pro and Gly. An increase in the distribution ratio muscle/plasma was observed exclusively for Gly after administration of Leu or of a non-insulinogenic transport system L analogue. Diazoxide suppressed the Leu-induced decreases in plasma and muscle intracellular concentrations of Ile and Val as well as of Pro in plasma. An increase in the distribution ratio liver/plasma was observed for Pro and Gly in the absence but not in the presence of diazoxide. All the above changes were statistically significant. Hence insulin probably mediates Leu effects, promoting an increased utilization of Ile and Val in muscle and of Pro in liver. A more direct effect of Leu appears to be involved in the apparent increased utilization of Phe, Tyr and Ala in the same tissue. Gly depletion in plasma can be explained by its trapping by inhibitory action of Leu on the exodus of Gly through transport system L.

Description of a selection method highly cytotoxic for cystinotic fibroblasts but not normal human fibroblasts.

Nephropathic cystinosis is an inherited disorder characterized by a high intralysosomal accumulation of cystine due to a defect in lysosomal cystine transport. Cystine can be specifically loaded into the lysosomal compartment of intact cells by incubating cells with cystine dimethyl ester (CDME). We have applied this methyl ester loading technique to develop a selection method that is highly cytotoxic for cystinotic fibroblasts but not normal human fibroblasts and that is based on the inherent differences in lysosomal cystine transport activity of normal and cystinotic fibroblasts. Thus, only 0-0.03% of fetal cystinotic fibroblasts survive exposure to 2 mM CDME for 20 min whereas 70-80% of normal fetal fibroblasts survive these same conditions. Following transfection of cystinotic fibroblasts with normal human genomic DNA or cDNA, this CDME selection method can be used to select for those cells that have been transformed to the normal phenotype and thus aid in the identification of the gene coding for the lysosomal cystine transport protein.

Mediated calcium transport by isolated human fibroblast lysosomes.

Lysosomes purified by Percoll gradient from normal human fibroblasts (GM0010A) show uptake of Ca2+ in a mediated manner. The uptake is linear over the first 1.5 min and approaches a steady state by 10 min. Uptake is saturable, displaying a Vmax of about 10 pmol/min/unit hexosaminidase at 20 mM Ca2+ (7 nmol/min/mg protein), and a Km of 5.7 mM. Ca2+ uptake increases with increasing extralysosomal pH from 5.0 to 8.5. The Q10 is 1.6, and Ea 8.7 kcal/mol. Uptake of 0.1 mM Ca2+ was inhibited to the extent indicated by 1.0 mM of the following: Cd2+, 100%; Hg2+, 100%; Zn2+, 89%; Mg2+, 77%; Ba2+, 60%; Sr2+, 37%; Fe2+, 20%; Cu2+, 0%. Mono- and trivalent cations had no effect. ATP (1.0 mM) inhibited uptake by 80%, and chloroquine (0.1 mM) inhibited by 60%, as did 1.0 mM L-cystine. Cysteamine, N-ethylmaleimide, and the anions Cl-, SO(2-)4, and acetate had no effect. The calcium ionophore A23187 augmented uptake by 10-fold at 10 microM. Surprisingly, Pb2+ greatly augmented lysosomal Ca2+ uptake in a concentration-dependent manner. Pb2+, however, adversely affected lysosomal latency. Lysosomal calcium uptake was not affected by inositol 1,4,5-triphosphate, and calcium-induced calcium release from lysosomes was not observed. A role for lysosomes in cellular calcium homeostasis has not been previously suggested. This work shows that Ca2+ can be transported into and out of lysosomes and could assist in lysosomal proteolysis. The extent of further lysosomal participation in cellular calcium regulation is unclear.

Seropositivity to Helicobacter pylori: lack of association with length of hospitalization.

To examine the possibility of nosocomial spread of Helicobacter pylori, a serosurvey (n = 238) was conducted at Perry Point Department of Veterans Affairs Medical Center, an institution providing both acute and chronic care. We hypothesized that if significant nosocomial transmission was occurring, seropositivity (as measured by enzyme-linked immunosorbent assay [ELISA]) would correlate with length of stay in the facility. Whether treated as a continuous or dichotomous variable, the ELISA results did not correlate significantly with length of stay even after adjustments were made for age, race, antibiotic use, gastrointestinal instrumentation, and diagnoses by using multiple-regression models. Age was found to be a significant risk factor for a higher ELISA optical density value. A history of chronic obstructive pulmonary disease was significantly protective among internal medicine ward patients in adjusted analysis; black race was a significant risk factor among psychiatry ward patients. Our results confirm the association of H. pylori infection with age but provide no indication that extended hospitalization is a risk factor for infection.