Use of nonviral promoters in adenovirus-mediated gene therapy: reduction of lysosomal storage in the aspartylglucosaminuria mouse.

BACKGROUND: Aspartylglucosaminuria (AGU) is a lysosomal storage disease with severe neurodegenerative clinical features resulting from the deficiency of lysosomal aspartylglucosaminidase (AGA). The AGU knockout mouse is a good model to test different therapy strategies, as it mimics well the human pathogenesis of the disease exhibiting storage vacuoles in all tissues. In this study we investigated the efficiency of nonviral promoters in adenovirus-mediated gene therapy. METHODS: The deficient corrective enzyme, AGA, was expressed using two tissue-specific promoters, neuron-specific enolase (NSE), astrocyte-specific (GFAP) and the endogenous AGA promoter. An intrastriatal injection site was chosen due to its wide connections in the central nervous system (CNS). The expression of AGA was analyzed 1 week, 2 weeks, 4 weeks, 2 months and 4 months after the virus injection by lysosomal AGA-specific immunostaining. A correction of the lysosomal storage in the brain of treated mice was also studied using toluidine blue stained thin sections. RESULTS: The overexpressed AGA enzyme was detected in addition to the injection site, also in the ipsilateral parietal cortex indicating migration of AGA in the brain tissue. Duration of AGA expression was markedly longer with all the viruses used compared to the green fluorescent protein (GFP) expression driven by the viral cytomegalovirus (CMV) promoter. In most animals the storage was decreased by at least 50% as compared to untreated AGU mouse brains. Remarkably, >90% correction of storage at the ipsilateral cortex was found with the NSE promoter at 4 weeks and 2 months after injection. Additionally, partial clearance of storage was demonstrated also in the contralateral side of the brain. CONCLUSIONS: These data implicate that tissue-specific promoters are especially useful in virus-mediated gene therapy aiming at long-term gene expression.

Kidneys with heavy proteinuria show fibrosis, inflammation, and oxidative stress, but no tubular phenotypic change.

BACKGROUND: Sustained proteinuria is a major factor leading to kidney fibrosis and end-stage renal failure. Tubular epithelial cells are believed to play a crucial role in this process by producing mediators leading to fibrosis and inflammation. Congenital nephrotic syndrome of the Finnish type (NPHS1) is a genetic disease caused by mutations in a podocyte protein nephrin, which leads to constant heavy proteinuria from birth. In this work we studied the tubulointerstitial changes that occur in NPHS1 kidneys during infancy. METHODS: The pathologic lesions and expression of profibrotic and proinflammatory factors in nephrectomized NPHS1 kidneys were studied by immunohistochemistry, Western blotting, and cytokine antibody array. Oxidative stress in kidneys was assessed by measurement of gluthatione redox state. RESULTS: The results indicated that (1) severe tubulointerstitial lesions developed in NPHS1 kidneys during infancy; (2) tubular epithelial cells did not show transition into myofibroblasts as studied by the expression of vimentin, alpha-smooth muscle actin (alpha-SMA), collagen, and matrix metalloproteinases 2 and 9 (MMP-2 and -9); (3) the most abundant chemokines in NPHS1 tissue were neutrophil activating protein-2 (NAP-2), macrophage inhibiting factor (MIF), and monocyte chemoattractant protein-1 (MCP-1); (4) monocyte/macrophage cells expressing CD14 antigen were the major inflammatory cells invading the interstitium; (5) the arteries and arterioles showed intimal hypertrophy, but the microvasculature in NPHS1 kidneys remained quite normal; and (6) excessive oxidative stress was evident in NPHS1 kidneys. CONCLUSION: Heavy proteinuria in NPHS1 kidneys was associated with interstitial fibrosis, inflammation, and oxidative stress. The tubular epithelial cells, however, were resistant to proteinuria and did not show epithelial-mesenchymal transition.

Mice with Ppt1Deltaex4 mutation replicate the INCL phenotype and show an inflammation-associated loss of interneurons.

Infantile Neuronal Ceroid Lipofuscinosis (INCL) results from mutations in the palmitoyl protein thioesterase (PPT1, CLN1) gene and is characterized by dramatic death of cortical neurons. We generated Ppt1Deltaex4 mice by a targeted deletion of exon 4 of the mouse Ppt1 gene. Similar to the clinical phenotype, the homozygous mutants show loss of vision from the age of 8 weeks, seizures after 4 months and paralysis of hind limbs at the age of 5 months. Autopsy revealed a dramatic loss of brain mass and histopathology demonstrated accumulation of autofluorescent granular osmiophilic deposits (GRODS), both characteristic of INCL. At 6 months, the homozygous Ppt1Deltaex4 mice showed a prominent loss of GABAergic interneurons in several brain areas. The transcript profiles of wild-type and mutant mouse brains revealed that most prominent alterations involved parts of the immune response, implicating alterations similar to those of the aging brain and neurodegeneration. These findings make the Ppt1Deltaex4 mouse an interesting model for the inflammation-associated death of interneurons.

A mouse model for Finnish variant late infantile neuronal ceroid lipofuscinosis, CLN5, reveals neuropathology associated with early aging.

Neuronal ceroid lipofuscinoses (NCL) comprise the most common group of childhood encephalopathies caused by mutations in eight genetic loci, CLN1-CLN8. Here, we have developed a novel mouse model for the human vLINCL (CLN5) by targeted deletion of exon 3 of the mouse Cln5 gene. The Cln5-/- mice showed loss of vision and accumulation of autofluorescent storage material in the central nervous system (CNS) and peripheral tissues without prominent brain atrophy. The ultrastructure of the storage material accurately replicated the abnormalities in human patients revealing mixture of lamellar profiles including fingerprint profiles as well as curvilinear and rectilinear bodies in electronmicroscopic analysis. Prominent loss of a subset of GABAergic interneurons in several brain areas was seen in the Cln5-/- mice. Transcript profiling of the brains of the Cln5-/- mice revealed altered expression in several genes involved in neurodegeneration, as well as in defense and immune response, typical of age-associated changes in the CNS. Downregulation of structural components of myelin was detected and this agrees well with the hypomyelination seen in the human vLINCL patients. In general, the progressive pathology of the Cln5-/- brain mimics the symptoms of the corresponding neurodegenerative disorder in man. Since the Cln5-/- mice do not exhibit significant brain atrophy, these mice could serve as models for studies on molecular processes associated with advanced aging.

Mulibrey heart disease: clinical manifestations, long-term course, and results of pericardiectomy in a series of 49 patients born before 1985.

BACKGROUND: Mulibrey nanism is a rare inherited disease characterized by growth failure and multiorgan manifestations, including constrictive pericarditis. Its long-term course, the results of pericardiectomy, and the details of myocardial involvement have not been reported previously. METHODS AND RESULTS: We studied 49 patients (26 men) born before 1985 and followed for up to 25 years. By 1999, 25 patients (51%) had developed congestive heart failure (CHF), 19 (39%) had undergone pericardiectomy for constrictive pericarditis, 10 (22%) had died of cardiac causes, and 5 (10%) had died of noncardiac causes. Of the 19 pericardiectomized patients, 12 derived lasting clinical benefit, whereas 1 patient suffered an early noncardiac death and 6 died later of unrelieved or recurrent CHF. At echocardiography in 34 living patients, left ventricular mass adjusted for body height and weight averaged (+/-SEM) 149+/-5 g in 21 unoperated patients, 144+/-8 g in 13 pericardiectomized patients, and 104+/-7 g in 16 healthy persons matched for age and sex (P=0.000). Autopsies of 11 patients showed fibrotic thickening of the pericardial leaves with myocardial hypertrophy and variable but mostly mild myocardial fibrosis. Endocardial thickening was seen in 3 patients. CONCLUSIONS: Constrictive pericarditis, myocardial hypertrophy, and variable myocardial fibrosis constitute the main elements of Mulibrey heart disease. At least one half of patients ultimately develop CHF. Pericardiectomy generally provides clinical benefit, but in approximately one third of patients, CHF may recur because of coexisting myocardial involvement.

Hepatotoxicity in patients with juvenile idiopathic arthritis receiving longterm methotrexate therapy.

OBJECTIVE: To evaluate hepatotoxicity in patients with juvenile idiopathic arthritis (JIA) receiving methotrexate (MTX) therapy with doses of 20-30 mg/m2 of body surface area. METHODS: We graded the histology of percutaneous liver biopsies from 34 patients with JIA receiving longterm (> 2.4 years) MTX therapy at the Rheumatism Foundation Hospital, Heinola, Finland, using the Roenigk classification scale. Medical records of the patients with JIA were retrospectively analyzed. RESULTS: Of 10 patients with MTX doses >/= 20 mg/m2, 4 had grade II, 5 had grade I histology, and one specimen with extensive steatosis as the only pathologic finding could not be classified. All 24 patients treated with low dose MTX had grade I histology. No specimen showed fibrosis or cirrhosis. In 2 patients with grade II histology, extensive portal tract inflammation resolved when MTX was discontinued for 6 months. CONCLUSION: Aggressive medical treatment of JIA with MTX at 20-30 mg/m2 with concomitant disease modifying antirheumatic drugs and corticosteroids may contribute to minor liver abnormalities that seem to be reversible.

Proteinuria and prenatal diagnosis of congenital nephrosis in fetal carriers of nephrin gene mutations.

High concentrations of alpha-fetoprotein (AFP) are used for prenatal diagnosis of the Finnish type of congenital nephrotic syndrome (NPHS1). We investigated the validity of this test. We retrospectively established fetal NPHS1 genotype and assessed renal pathology in 21 pregnancies that had been terminated because of raised concentrations of AFP in amniotic fluid. 12 fetuses were homozygous and nine were heterozygous (carriers) for NPHS1 mutations. Raised concentrations of AFP and similar proteinuric features in fetal kidneys were seen in both groups, indicating that these signs are unreliable for prenatal diagnosis of congenital nephrosis. We strongly recommend the use of mutation analysis of the NPHS1 gene to confirm the AFP results in prenatal diagnosis of NPHS1.

Pathology of lethal fetal growth retardation syndrome with aminoaciduria, iron overload, and lactic acidosis (GRACILE).

Autopsy study of 17 newborn infants with lethal autosomal recessive disease presenting as growth retardation with lactic acidosis, Fanconi aminoaciduria, and hepatic hemosiderosis is reported. The patients succumbed between day 1 and 4 months of life; 9 patients died within the first month. All patients showed severe pathologic changes of liver with cholestasis in all livers. Extensive accumulation of stainable iron of the hepatocytes was present in 9/17 autopsy tissues and in two biopsy specimens. Moderate to abundant iron storage in the Kupffer cells was seen in all liver specimens. The amount of hepatocytic iron was high in livers up to 1 month of age and decreased thereafter. The general features and liver findings of this disorder suggest the name Growth Retardation Aminoaciduria Cholestasis Iron Overload, Lactacidosis and Early Death (GRACILE, OMIM 603358). Calcified concrements were seen in the medulla of 13/16 kidney specimens. Pancreas of 13/14 patients showed interstitial fibrosis and exocrine atrophy. Various pathologic findings such as renal tubular dysgenesis, paucity of hepatic bile ducts and iron storage in the macrophages of spleen and pulmonary alveoli were observed in some cases. Previous extensive clinical genetic and laboratory investigations have revealed that the patients had a previously unrecognized genetic disease. It is inherited as an autosomal recessive trait. The gene locus is 2q33-37. The basic defect of the disease remains unknown.