Adeno-associated virus serotypes 9 and rh10 mediate strong neuronal transduction of the dog brain.

Canine models have many advantages for evaluating therapy of human central nervous system (CNS) diseases. In contrast to nonhuman primate models, naturally occurring canine CNS diseases are common. In contrast to murine models, the dog's lifespan is long, its brain is large and the diseases affecting it commonly have the same molecular, pathological and clinical phenotype as the human diseases. We compared the ability of four intracerebrally injected adeno-associated virus vector (AAV) serotypes to transduce the dog brain with green fluorescent protein as the first step in using these vectors to evaluate both delivery and efficacy in naturally occurring canine homologs of human diseases. Quantitative measures of transduction, maximum diameter and area, identified both AAV2/9 and AAV2/rh10 as significantly more efficient than either AAV2/1 or AAV2/5 at transducing cerebral cortex, caudate nucleus, thalamus and internal capsule. Fluorescence co-labeling with cell-type-specific antibodies demonstrated that AAV2/9 and AAV2/rh10 were capable of primarily transducing neurons, although glial transduction was also identified and found to be more efficient with the AAV2/9 vector. These data are a prerequisite to evaluating the efficacy of recombinant AAV vectors carrying disease-modifying transgenes to treat naturally occurring canine models in preclinical studies of human CNS disease therapy.

Mucopolysaccharidosis in a cat with arylsulfatase B deficiency: a model of Maroteaux-Lamy syndrome.

A Siamese cat that presented clinical signs similar to those seen in humans with mucopolysaccharidoses was studied. The animal excreted increased amounts of polymeric glycosaminoglycans in the urine, consisting almost entirely of dermatan sulfate. Electron microscopy of circulating polymorphonuclear leukocytes revealed the presence of many membrane-bound lamellar inclusion bodies. Sulfate incorporation studies with cultured skin fibroblasts indicated defective glycosaminoglycan degradation. These cells showed a deficiency in arylsulfatase B activity. The disorder appears similar or identical to the Maroteaux-Lamy syndrome described in humans.

Genetic basis of XX male syndrome and XX true hermaphroditism: evidence in the dog.

Serological analysis of white blood cells from the members of a family of American cocker spaniels indicates that a form of abnormal sexual development, in which individuals with a female karyotype have testes or ovotestes, is caused by anomalous transmission of male-determining H-Y genes.

Clinical characterization of cardiovascular abnormalities associated with feline mucopolysaccharidosis I and VI.

OBJECTIVE: The purpose of this study was to define the cardiovascular abnormalities present in young and adult cats affected with the lysosomal storage diseases mucopolysaccharidosis (MPS) I and MPS VI. METHOD: Eighteen cats affected with MPS I and 10 cats affected with MPS VI were evaluated by physical examination, electrocardiography and echocardiography. Electrocardiography (ECG) was performed on all MPS I and 9 of the MPS VI cats. Twelve unaffected cats underwent complete examinations for comparison purposes. RESULTS: No cardiovascular abnormalities were noted on physical examination. Measured ECG intervals were normal in affected cats; however, sinus arrhythmia was noted more frequently than in the unaffected cats. Significant echocardiographic abnormalities included aortic valve thickening, regurgitation and aortic root dilation. Significant mitral valve thickening was also noted. The severity of changes increased in older affected cats. CONCLUSION: As affected animals increased in age, more cardiac abnormalities were found with increasing severity. Significant lesions included the mitral and aortic valves and ascending aorta, but myocardial changes were not recognized. MPS I and MPS VI cats have similar cardiovascular findings to those seen in children and constitute important models for testing new MPS therapies.

Enhancement of residual arylsulfatase B activity in feline mucopolysaccharidosis VI by thiol-induced subunit association.

The molecular pathology of the deficient arylsulfatase B activity in feline mucopolysaccharidosis (MPS) VI was investigated. Compared with the highly purified normal feline hepatic enzyme, the purified MPS VI residual activity had a 100-fold higher Michaelis constant (K(m)), an altered electrophoretic mobility, half the apparent native molecular weight, and markedly decreased thermo-, cryo-, and pH stabilities. Molecular weight and alkylation studies were consistent with the normal enzyme being a homodimer and the residual MPS VI enzyme a monomer. When incubated with various sulfhydryl reagents, the residual specific activity was enhanced several-fold, whereas the activity of the purified normal enzyme was un-affected or slightly inhibited. In the presence of dithiothreitol (DTT) and cysteamine, a lysosomotropic aminothiol, the residual activity had an electrophoretic mobility and native molecular weight similar to those of the normal feline enzyme. These findings suggested that the monomeric residual enzyme was dimerized in the presence of thiol-reducing agents. To determine if thiol-induced subunit association could therapeutically increase the residual activity and degrade the accumulated dermatan sulfate, in vitro and in vivo experiments were undertaken. When 2 mM DTT or cysteamine was incubated with heparinized whole blood from an MPS VI cat, the leukocyte residual arylsulfatase B activity increased 11- and 20-fold, respectively, and the accumulated dermatan sulfate was degraded in the presence of both thiol reagents. Intravenous administration of DTT (50 mg/kg) effected an immediate, but transient, increase in leukocyte residual activity; however, the substrate levels were not significantly decreased. In contrast, intravenous administration of cysteamine (15 mg/kg) increased leukocyte residual activity more than sixfold 30 min postinfusion; concomitantly, the leukocyte substrate was decreased to 35% of the initial level immediately after infusion and to about 45% of preinfusion values during the 120-min period studied. These results suggest that the defective residual activity in feline MPS VI can be therapeutically manipulated by thiol-induced subunit association. Furthermore, this animal analog provides a prototype for the investigation of human inborn errors of metabolism resulting from enzymatic defects that might be amenable to enzyme manipulation therapy.

Arylsulfatase B activities and glycosaminoglycan levels in retrovirally transduced mucopolysaccharidosis type VI cells. Prospects for gene therapy.

Mucopolysacchariodosis type VI (MPS VI) is the lysosomal storage disorder caused by the deficient activity of arylsulfatase B (ASB; N-acetylgalactosamine 4-sulfatase) and the subsequent accumulation of the glycosaminoglycan (GAG), dermatan sulfate. In this study, a retroviral vector containing the full-length human ASB cDNA was constructed and used to transduce skin fibroblasts, chondrocytes, and bone marrow cells from human patients, cats, or rats with MPS VI. The ASB vector expressed high levels of enzymatic activity in each of the cell types tested and, in the case of cat and rat cells, enzymatic expression led to complete normalization of 35SO4 incorporation. In contrast, overexpression of ASB in human MPS VI skin fibroblasts did not lead to metabolic correction. High-level ASB expression was detected for up to eight weeks in transduced MPS VI cat and rat bone marrow cultures, and PCR analysis demonstrated retroviral-mediated gene transfer to approximately 30-50% of the CFU GM-derived colonies. Notably, overexpression of ASB in bone marrow cells led to release of the enzyme into the media and uptake by MPS VI cat and rat skin fibroblasts and/or chondrocytes via the mannose-6-phosphate receptor system, leading to metabolic correction. Thus, these studies provide important rationale for the development of gene therapy for this disorder and lay the frame-work for future in vivo studies in the animal model systems.

Optimized transduction of canine paediatric CD34(+) cells using an MSCV-based bicistronic vector.

We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (gammac) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34(+) bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting approximately 75% of Cf2Th cells. An average of 11% of highly enriched canine CD34(+) cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine gammac cDNA produced a gammac protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34(+) BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.

Gene therapy ameliorates cardiovascular disease in dogs with mucopolysaccharidosis VII.

BACKGROUND: Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease caused by deficient beta-glucuronidase (GUSB) activity resulting in defective catabolism of glycosaminoglycans (GAGs). Cardiac disease is a major cause of death in MPS VII because of accumulation of GAGs in cardiovascular cells. Manifestations include cardiomyopathy, mitral and aortic valve thickening, and aortic root dilation and may cause death in the early months of life or may be compatible with a fairly normal lifespan. We previously reported that neonatal administration of a retroviral vector (RV) resulted in transduction of hepatocytes, which secreted GUSB into the blood and could be taken up by cells throughout the body. The goal of this study was to evaluate the effect on cardiac disease. METHODS AND RESULTS: Six MPS VII dogs were treated intravenously with an RV-expressing canine GUSB. Echocardiographic parameters, cardiovascular lesions, and biochemical parameters of these dogs were compared with those of normal and untreated MPS VII dogs. CONCLUSIONS: RV-treated dogs were markedly improved compared with untreated MPS VII dogs. Most RV-treated MPS VII dogs had mild or moderate mitral regurgitation at 4 to 5 months after birth, which improved or disappeared when evaluated at 9 to 11 and at 24 months. Similarly, mitral valve thickening present early in some animals disappeared over time, whereas aortic dilation and aortic valve thickening were absent at all times. Both myocardium and aorta had significant levels of GUSB and reduction in GAGs.

Mucopolysaccharidosis VII in a Cat Caused by 2 Adjacent Missense Mutations in the GUSB Gene.

BACKGROUND: Mucopolysaccharidoses (MPS) are common lysosomal storage disorders causing typically progressive skeletal and ocular abnormalities. OBJECTIVES: To describe the clinic features, metabolic profile and a unique mutation in a domestic shorthair (DSH) kitten with MPS VII. ANIMALS: Affected kitten and 80 healthy cats. METHODS: Serum lysosomal enzyme activities and urinary glycosaminoglycan (GAG) accumulation were assessed. Exons of the ß-glucuronidase gene (GUSB) were sequenced from genomic DNA and genotyping was conducted. RESULTS: A 3-month-old DSH cat was presented for stunted growth, paresis, facial dysmorphia, multiple skeletal deformities, and corneal opacities. Evaluation of blood smears disclosed metachromatic granules in leukocytes and a urinary mucopolysaccharide spot test was positive. The proband had no GUSB activity but normal or increased activities for other lysosomal enzymes. Sequencing of the GUSB gene from the proband and comparison to the sequence of 2 healthy cats and the published feline genome sequence demonstrated 2 unique single base transitions (c.1421T>G and c.1424C>T) in exon 9, altering 2 adjacent codons (p.Ser475Ala and p.Arg476Trp). These amino acid changes are in a highly conserved domain of the GUSB protein and nontolerable to maintain function. Moreover, the p.Arg476Trp mutation previously has been identified in human patients. None of the other clinically healthy cats had these mutations. CONCLUSIONS AND CLINIC IMPORTANCE: The diagnostic approach to MPS disorders is delineated. This is only the second mutation known to cause MPS VII in cats. Similarly, 2 different mutations have been described in MPS VII dogs, thereby showing the molecular heterogeneity of MPS VII in companion animals.

Retrovirus vector-mediated correction and cross-correction of lysosomal alpha-mannosidase deficiency in human and feline fibroblasts.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease alpha-mannosidosis. Retrovirus vector transfer of a new human alpha-mannosidase cDNA resulted in high-level expression of alpha-mannosidase enzymatic activity in deficient human and feline fibroblasts. The expressed alpha-mannosidase had the same biochemical properties (thermal stability, pH profile, inhibitor/activator sensitivity) as the native enzyme expressed in normal cells. The transferred enzyme colocalized with a control lysosomal hydrolase in cell fractionation experiments. The vector-encoded enzyme also was released at high levels from the corrected cells, and was taken up by untreated mutant cells via the mannose 6-phosphate receptor-mediated endocytic pathway (cross-correction). It is envisioned that genetic correction of a subset of cells (e.g., hematopoietic stem cells) in patients will provide a source of corrective enzyme for other affected tissues in this multisystem disease. Development of a vector expressing high levels of alpha-mannosidase that cross-corrects mutant cells will enable somatic gene transfer experiments in the cat model of human alpha-mannosidosis.

Neonatal intramuscular injection with recombinant adeno-associated virus results in prolonged beta-glucuronidase expression in situ and correction of liver pathology in mucopolysaccharidosis type VII mice.

For many metabolic diseases, early correction of the inherited deficiency is required to prevent long-term sequelae. We examined the ability of adeno-associated virus (AAV) to mediate efficient gene transfer during the neonatal period in mice with the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII). Quadriceps of newborn MPS VII mice were injected with an AAV vector containing human beta-glucuronidase (GUSB) cDNA. High-level intramuscular GUSB expression was seen as early as 2 weeks of age, and persisted for at least 16 weeks with no reduction in activity. In addition, GUSB activity was detected in both liver and spleen at later time points. The level of GUSB activity resulted in a significant reduction in lysosomal storage in the liver and a minimal reduction in the spleen at 16 weeks. However, the temporal and spatial pattern of hepatic GUSB activity, coupled with the presence of GUSB cDNA in liver sections, suggests that hematogenous dissemination of virus at the time of injection led to gene transfer to hepatic cells. These results demonstrate that AAV vectors can successfully infect neonatal muscle and persist through the rapid growth phase following birth. However, GUSB secretion from an intramuscular source is inefficient, limiting the therapeutic efficacy of this approach.

Meningiomas in young cats with mucopolysaccharidosis I.

Meningiomas are the most common central nervous system tumor of the cat, with an age at diagnosis of greater than nine years in 73% of the affected animals. Four of seven cats with alpha-L-iduronidase deficient mucopolysaccharidosis I had meningiomas at postmortem examination. All four cats were less than three years of age. The relationship between the meningiomas and the metabolic defect of MPS I is not clear.

Histopathology, electrodiagnostic testing, and magnetic resonance imaging show significant peripheral and central nervous system myelin abnormalities in the cat model of alpha-mannosidosis.

Alpha-mannosidosis is a disease caused by the deficient activity of alpha-mannosidase, a lysosomal hydrolase involved in the degradation of glycoproteins. The disease is characterized by the accumulation of mannose-rich oligosaccharides within lysosomes. The purpose of this study was to characterize the peripheral nervous system (PNS) and central nervous system (CNS) myelin abnormalities in cats from a breeding colony with a uniform mutation in the gene encoding alpha-mannosidase. Three affected cats and 3 normal cats from 2 litters were examined weekly from 4 to 18 wk of age. Progressively worsening neurological signs developed in affected cats that included tremors, loss of balance, and nystagmus. In the PNS, affected cats showed slow motor nerve conduction velocity and increased F-wave latency. Single nerve fiber teasing revealed significant demyelination/remyelination in affected cats. Mean G-ratios of nerves showed a significant increase in affected cats compared to normal cats. Magnetic resonance imaging of the CNS revealed diffuse white matter signal abnormalities throughout the brain of affected cats. Quantitative magnetization transfer imaging showed a 8%-16% decrease in the magnetization transfer ratio in brain white matter of affected cats compared to normal cats, consistent with myelin abnormalities. Histology confirmed myelin loss throughout the cerebrum and cerebellum. Thus, histology, electrodiagnostic testing, and magnetic resonance imaging identified significant myelination abnormalities in both the PNS and CNS that have not been described previously in alpha-mannosidosis.

Mixed lymphocyte reactivity in cats.

A method was developed to perform primary one-way mixed lymphocyte cultures (MLC) with cat cells. A polymorphic system of MLC reactivities was found within a cat population. In family studies, alloreactivity segregated as a single genetic locus with codominantly expressed products, designated feline lymphocyte defined (Fld)--although closely linked multigene control remains possible. The relationship of Fld to a putative feline major histocompatibility complex is discussed.

Biochemical, pathological, and clinical response to transplantation of normal bone marrow cells into acid sphingomyelinase-deficient mice.

BACKGROUND: Acid sphingomyelinase knock-out (ASMKO) mice are a model of types A and B Niemann-Pick disease. In the present study, we evaluated whether bone marrow transplantation (BMT) carried out on newborn ASMKO mice could prevent or alter the Niemann-Pick disease phenotype. METHODS: Previous work from our laboratory had shown that ASMKO mice were highly susceptible to irradiation-induced death. Therefore, we preconditioned 1-day-old ASMKO (n=35) mice with a "sublethal" dose of 200 cGy of total body irradiation before BMT. The transplantation effects were then analyzed by biochemical, pathological, and clinical approaches. RESULTS: Engraftment ranging from 7% to 100% was achieved in 97% of the transplanted animals. Growth of the engrafted animals was improved, and their survival was increased (from a mean of 5 months to 9 months). The onset of ataxia also was delayed in most of the engrafted animals. In accordance with these observations, biochemical and pathological analysis revealed significant changes in the transplanted group as compared with nontransplanted animals. Lipid storage was reduced in several organs, and there was evidence of histologic improvement seen throughout the reticuloendothelial system, even in animals that were engrafted as low as 14%. In the central nervous system, lipid storage also was reduced, and the Purkinje cells, which are almost absent in ASMKO mice, were present in certain areas of the transplanted animals cerebella. CONCLUSIONS: These results demonstrated that BMT could alter the pathologic phenotype in ASMKO mice, but that this procedure alone was not sufficient to elicit a complete therapeutic effect.

Bone marrow transplantation in newborn rats with mucopolysaccharidosis type VI: biochemical, pathological, and clinical findings.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is the lysosomal storage disorder caused by the deficient activity of arylsulfatase B (ASB). In this study, we evaluated bone marrow transplantation (BMT) for the treatment of MPS VI and the effects of irradiation on the survival and engraftment of bone marrow-transplanted neonatal rats. METHODS: One- to 2-day-old MPS VI rats were injected with normal bone marrow after irradiation with 200, 400, or 800 cGy. Ninety percent of the animals receiving a single dose of 200 cGy (n=30) survived the procedure, whereas irradiation with 400 cGy (n=23) or 800 cGy (n=12) resulted in significant mortality (78% and 100%, respectively). Engraftment was monitored by determining ASB activities in peripheral white blood cells and by Y chromosome in situ hybridization analysis. Fifty-two percent of the animals from the 200-cGy group engrafted for up to 8 months after BMT; among the five animals that survived the 400-cGy dose, all engrafted. In comparison, only 20% of nonirradiated animals engrafted at low levels. Of the 24 engrafted animals that were monitored for 8 months after BMT, clinical and/or radiographic improvements were noted in only one (BMT animal 3). Enzymatic analysis revealed that the ASB activities in the reticuloendothelial organs of this animal, as well as two other engrafted but clinically unimproved animals (BMT animals 1 and 2), were normal or near normal; correspondingly, the glycosaminoglycan levels in these organs were significantly reduced. Consistent with the clinical and biochemical observations, light and electron microscopic findings were more improved in BMT animal 3 as compared with BMT animals 1 and 2, although a reduction of storage was evident in each of these transplant recipients, particularly in the trachea and aorta, two tissues that are characteristic sites of pathology in human patients. CONCLUSIONS: These results indicate that BMT in newborn MPS VI patients may prevent many of the pathological and clinical findings in this disorder, but is likely to have very limited and unpredictable effects on the skeletal abnormalities.

Retinal pigment epithelial glycosaminoglycan metabolism: intracellular versus extracellular pathways. In vitro studies in normal and diseased cells.

The synthesis and turnover of glycosaminoglycans (GAGs) in different fractions of cultured feline retinal pigment epithelium (RPE) were characterized. In one method of fractionation, trypsin was used to separate the extracellular components (referred to as trypsin-soluble glycocalyx) from the intracellular components. As a second method, the basal extracellular matrix (basal ECM) was separated from the rest of the GAGs (cell-associated GAGs) by extracting the cell layer with NH4OH. The incorporation of 35SO4 into cetylpyridinium chloride-precipitable GAGs in the cell-associated and the intracellular fractions increased throughout the labeling period, while in the trypsin-soluble glycocalyx and the basal ECM incorporation approached a maximum. While heparan sulfate was the predominant GAG in all compartments, most was located extracellularly. The majority of dermatan sulfate was localized in the intracellular fraction. GAGs in the trypsin-soluble glycocalyx exhibited a rapid rate of turnover, while GAGs in the intracellular compartment and basal ECM turned over much more slowly. Ascorbic acid increased the incorporation of 35SO4 into ECM chondroitin sulfate/dermatan sulfate, but not heparan sulfate, on a per cell basis. Cycloheximide reduced incorporation of 35SO4-GAGs into both the cell-associated compartment and the basal ECM. In contrast, monensin caused a reduction in basal ECM GAGs while increasing the GAGs in the cell-associated compartment. The intracellular accumulation of GAGs and resultant pathology in alpha-L-iduronidase (alpha-L-id)-deficient RPE indicated that this pathway for the intracellular degradation of GAGs is important in normal RPE function. However, the turnover of GAGs in the trypsin-soluble glycocalyx was not affected by deficient alpha-L-id activity or by the subsequent intracellular accumulation of GAGs. Therefore, normal lysosomal activity in the RPE is not a prerequisite for maintaining the rate of extracellular GAG turnover within normal limits.

Retroviral cDNA transfer to the RPE: stable expression and modification of metabolism.

PURPOSE: To determine the versatility of retroviral vector-mediated rat beta-glucuronidase cDNA expression in the normal retinal pigment epithelium (RPE) of eyes of various species and in RPE of eyes with three types of mucopolysaccharidosis (MPS types I, VI, and VII) and to evaluate the effect of multiple transductions and long-term stable expression in the RPE. METHODS: A retroviral construct containing a rat beta-glucuronidase cDNA (NTK-BGEO) was used to infect RPE cells at subconfluence. The transduced cells were selected in G418, an antibiotic toxic to normal mammalian cells. Beta-glucuronidase activity was measured in transduced cells and media, using a fluorogenic substrate. Glycosaminoglycan profiles were examined by metabolically labeling RPE with Na2(35)SO4. RESULTS: Transduced RPE cells, regardless of species or disease status, expressed rat beta-glucuronidase. The expressed enzyme restored normal levels of glycosaminoglycans in the RPE cells of homozygous MPS VII-affected dogs by metabolizing stored glycosaminoglycans. The expressed enzyme failed to metabolize stored glycosaminoglycans of MPS I and MPS VI, indicating that overexpression could not bypass the exoglycosidase restriction. Multiple transductions increased beta-glucuronidase activity several times in the cell layer and in the media. The expression was stable in vitro for at least 12 weeks. CONCLUSIONS: A retroviral vector can mediate transfer of beta-glucuronidase in various species of normal and MPS-affected RPE. The expression is stable in vitro. The metabolism of stored glycosaminoglycans in MPS needs replacement of only the deficient enzyme to reverse the storage.

Arylsulfatase B activity in cultured retinal pigment epithelium: regional studies in feline mucopolysaccharidosis VI.

Feline mucopolysaccharidosis VI (MPS VI) is a recessively inherited lysosomal storage disease resulting from a deficiency of arylsulfatase B (ASB). Previous histopathologic findings have indicated that the disease is expressed morphologically in non-pigmented retinal pigment epithelial cells (RPE) in the posterior pole and superior equatorial regions by the accumulation of vacuolated inclusions and eventual cellular hypertrophy, while pigmented regions in the periphery are minimally affected. To determine if the regional and age-dependent variations in disease severity result from differences in residual enzyme activity, primary cultures of feline MPS VI-affected RPE were initiated from defined regions of the eye and maintained in vitro for 14 days. Cultures initiated from nonpigmented areas of affected adult eyes (posterior pole, superior equatorial) were more diseased than those from pigmented (inferior-equatorial, peripheral) areas. In the nonpigmented cultures, the disease was expressed by the accumulation of single membrane-bound inclusions and cellular hypertrophy. These inclusions were indistinguishable in their morphologic appearance and distribution from those found in situ. In contrast, the cultures initiated from pigmented areas remained normal or minimally affected. The same spatial disease distribution was present in young affected eyes, but the expression of the disease was much less severe. It is apparent that temporal, spatial, and pigmentation factors were correlated with disease expression in vitro as well as in situ. Arylsulfatase B activity was measured biochemically, and found to be deficient in all regions of young and adult eyes. It was notable that there was no correlation between the level of residual enzyme activity, and the pigmentation or spatial position from which the cells were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Inherited platelet delta-storage pool disease in dogs causing severe bleeding: an animal model for a specific ADP deficiency.

The nature of a disorder producing moderate to severe bleeding after minor trauma, venipuncture, and surgery was studied in 3 families of American cocker spaniel dogs. In the 5 affected dogs tested, platelet counts and measurements of plasma coagulant function and von Willebrand factor were normal. However, bleeding times were prolonged in 4 of the 5 affected dogs tested, and platelet aggregation in response to ADP and collagen was consistently abnormal in 3, suggesting that the bleeding disorder was due to abnormal platelet function. Measurements of 14C-serotonin uptake and retention by the affected platelets were normal. However, their ADP content was decreased, while their ATP content was normal, resulting in a mean ATP/ADP ratio of 8.32, compared to a mean ratio of 1.9 in normal canine platelets. Electron microscopy revealed that the number and appearance of the dense granules in the affected platelets were indistinguishable from those of normal controls. These studies suggest that this bleeding disorder results from a deficient delta-granule storage pool of ADP; given the normal serotonin uptake and retention by affected platelets and the apparently normal number of dense granules, the ADP deficiency may be the consequence of a selective defect in delta-granule ADP transport. Additional studies of this unique platelet disorder will provide an opportunity to understand the mechanism of adenine nucleotide storage in platelet delta-granules.