AAV-based gene therapy prevents neuropathology and results in normal cognitive development in the hyperargininemic mouse.

Complete arginase I deficiency is the least severe urea cycle disorder, characterized by hyperargininemia and infrequent episodes of hyperammonemia. Patients suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation and seizures, and is associated with intellectual disability. In mice, onset is heralded by weight loss beginning around day 15; gait instability follows progressing to inability to stand and development of tail tremor with seizure-like activity and death. Here we report that hyperargininemic mice treated neonatally with an adeno-associated virus (AAV)-expressing arginase and followed long-term lack any presentation consistent with brain dysfunction. Behavioral and histopathological evaluation demonstrated that treated mice are indistinguishable from littermates, and that putative compounds associated with neurotoxicity are diminished. In addition, treatment results in near complete resolution of metabolic abnormalities early in life; however, there is the development of some derangement later with decline in transgene expression. Ammonium challenging revealed that treated mice are affected by exogenous loading much greater than littermates. These results demonstrate that AAV-based therapy for hyperargininemia is effective and prevents development of neurological abnormalities and cognitive dysfunction in a mouse model of hyperargininemia; however, nitrogen challenging reveals that these mice remain impaired in the handling of waste nitrogen.

Kir4.1 potassium channel subunit is crucial for oligodendrocyte development and in vivo myelination.

To understand the cellular and in vivo functions of specific K(+) channels in glia, we have studied mice with a null mutation in the weakly inwardly rectifying K(+) channel subunit Kir4.1. Kir4.1-/- mice display marked motor impairment, and the cellular basis is hypomyelination in the spinal cord, accompanied by severe spongiform vacuolation, axonal swellings, and degeneration. Immunostaining in the spinal cord of wild-type mice up to postnatal day 18 reveals that Kir4.1 is expressed in myelin-synthesizing oligodendrocytes, but probably not in neurons or glial fibrillary acidic protein-positive (GFAP-positive) astrocytes. Cultured oligodendrocytes from developing spinal cord of Kir4.1-/- mice lack most of the wild-type K(+) conductance, have depolarized membrane potentials, and display immature morphology. By contrast, cultured neurons from spinal cord of Kir4.1-/- mice have normal physiological characteristics. We conclude that Kir4.1 forms the major K(+) conductance of oligodendrocytes and is therefore crucial for myelination. The Kir4.1 knock-out mouse is one of the few CNS dysmyelinating or demyelinating phenotypes that does not involve a gene directly involved in the structure, synthesis, degradation, or immune response to myelin. Therefore, this mouse shows how an ion channel mutation could contribute to the polygenic demyelinating diseases.

c-Abl is required for development and optimal cell proliferation in the context of p53 deficiency.

The c-Abl tyrosine kinase and the p53 tumor suppressor protein interact functionally and biochemically in cellular genotoxic stress response pathways and are implicated as downstream mediators of ATM (ataxia-telangiectasia mutated). This fact led us to study genetic interactions in vivo between c-Abl and p53 by examining the phenotype of mice and cells deficient in both proteins. c-Abl-null mice show high neonatal mortality and decreased B lymphocytes, whereas p53-null mice are prone to tumor development. Surprisingly, mice doubly deficient in both c-Abl and p53 are not viable, suggesting that c-Abl and p53 together contribute to an essential function required for normal development. Fibroblasts lacking both c-Abl and p53 were similar to fibroblasts deficient in p53 alone, showing loss of the G(1)/S cell-cycle checkpoint and similar clonogenic survival after ionizing radiation. Fibroblasts deficient in both c-Abl and p53 show reduced growth in culture, as manifested by reduction in the rate of proliferation, saturation density, and colony formation, compared with fibroblasts lacking p53 alone. This defect could be restored by reconstitution of c-Abl expression. Taken together, these results indicate that the ATM phenotype cannot be explained solely by loss of c-Abl and p53 and that c-Abl contributes to enhanced proliferation of p53-deficient cells. Inhibition of c-Abl function may be a therapeutic strategy to target p53-deficient cells selectively.

Short-term enzyme replacement in the murine model of Sanfilippo syndrome type B.

The Sanfilippo syndrome type B (MPS III B) is an autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase (EC 3. 2.1.50), one of the lysosomal enzymes required for the degradation of heparan sulfate. The disease is characterized by profound neurodegeneration but relatively mild somatic manifestations, and is usually fatal in the second decade. A mouse model had been generated by disruption of the Naglu gene in order to facilitate the study of pathogenesis and the development of therapy for this currently untreatable disease. Recombinant human alpha-N-acetylglucosaminidase (rhNAGLU) was prepared from secretions of Lec1 mutant Chinese hamster ovary cells. The enzyme, which has only unphosphorylated high-mannose carbohydrate chains, was endocytosed by mouse peritoneal macrophages via mannose receptors, with half-maximal uptake at ca. 10(-7) M. When administered intravenously to 3 month-old mice, rhNAGLU was taken up avidly by liver and spleen but marginally if at all by thymus, lung, kidney, heart, and brain (in order of diminishing uptake). The half-life of the enzyme was 2.5 days in liver and spleen. Immunohistochemistry and electron microscopy showed that only macrophages were involved in enzyme uptake and correction in these two organs, yet the storage of glycosaminoglycan was reduced to almost normal levels. The results show that the macrophage-targeted rhNAGLU can substantially reduce the body burden of glycosaminoglycan storage in the mouse model of Sanfilippo syndrome III B.

Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding alpha-N-acetylglucosaminidase.

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding alpha-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu-/- mice were healthy and fertile while young and could survive for 8-12 mo. They were totally deficient in alpha-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides G(M2) and G(M3) in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu-/- mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the alpha-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

Duodenal adenomas in Balb/-c mice monoinfected with Clostridium perfringens.

An outbreak of disease in multiparous females occurred in one isolator in a colony of Balb/-c germ-free mice. The affected isolator had been accidentally contaminated with Copyright Clostridium perfringens. Gross and histological examination of the diseased mice revealed lesions in the lungs, heart and intestinal tract. Lesions in the valvular endocardium and vascular walls were closely associated with bacterial colonies and septic thrombi containing Gram-positive rods. C perfringens type B was recovered in pure culture from the faeces, intestinal contents and atrial thrombi of the sick mice. Intestinal lesions varied, depending on the region of the intestine. The ileum showed shortened villi and ulceration of the mucosa. The duodenum of all the affected mice showed microscopic foci of polypoid adenomatous growth of the crypt epithelium. The significance of these unusual neoplastic lesions is discussed in the context of the growing evidence of an association between cell growth and bacterial cell products.

A cluster of cases of feline dysautonomia (Key-Gaskell syndrome) in a closed colony of cats.

Twenty-five cases of feline dysautonomia (Key-Gaskell syndrome) occurred in a closed cat colony over a period of three weeks. The clinical and pathological signs were sufficiently similar to those reported during the 1982-1986 outbreak to establish a positive diagnosis. The special epidemiological and environmental circumstances of the outbreak provide a new insight into the cause(s) of the syndrome.

Comparison of a PCR-based diagnostic assay for Mycoplasma pulmonis with traditional detection techniques.

Current diagnosis of infection by Mycoplasma pulmonis, an important pathogen of laboratory rodent colonies worldwide, is based on serological, histopathological and culture techniques which can be slow and unreliable. A polymerase chain reaction (PCR) assay for M. pulmonis diagnosis was compared to current diagnostic methods. This PCR based technique allows a more specific, sensitive and rapid diagnosis of M. pulmonis from various tissues by comparison with culture and histopathology.

Endocardial fibroelastosis in common domestic cats in the UK.

Endocardial fibroelastosis (EFE) is a pathological disorder characterized by a diffuse and elastic thickening of the endocardium. It is often thought to be congenital as most victims are infants who die of congestive heart failure before reaching one year of age. A naturally occurring animal model of EFE was proposed more than a decade ago on the basis of one pair of adult pure-bred Burmese cats which produced offspring consistently affected with EFE. In the present study a case of EFE is described which occurred in a closed colony of experimental SPF cats. A 5-week-old kitten which was found dead showed hydrothorax, hydropericardium and oedematous lungs at necropsy. Histological examination of the heart revealed left atrio-ventricular dilation with severe, diffuse endocardial thickening which did not extend into the myocardium. A retrospective study was carried out on stored tissues of two further kittens and one adult male cat from the colony which had died during a period of 2 years. The hearts of all the animals showed endocardial thickening in the left atria and ventricle but not in the right chambers. Over a period of 12 years of existence of this colony considerable inbreeding had occurred and it is concluded that an inherited factor was responsible for the disorder.

A spontaneous outbreak of Theiler's encephalomyelitis in a colony of severe combined immunodeficient mice in the UK.

Severe Combined Immunodeficient (SCID) mice lack both T and B lymphocytes which makes them highly susceptible to infectious agents. In the present communication, we describe an outbreak of Theiler's encephalomyelitis in a colony of SCID mice which was characterized by an unusually high prevalence of clinically overt cases. Diagnosis was based on the clinical signs, histological lesions and presence of antibodies in contemporaneous immunocompetent mice. This is the first report of this disease in SCID mice and, to our knowledge, also the first reported outbreak of Theiler's encephalomyelitis in the UK.

Vacuolar neuronal degeneration in the ventral horns of SCID mice in naturally occurring Theiler's encephalomyelitis.

During a spontaneous outbreak of Theiler's encephalomyelitis severe combined immunodeficient mice developed high morbidity and high mortality. Histological lesions were localized in the ventral horns of the spinal cord and brain stem. The salient features were the severe vacuolar degeneration of neurones and glial cells and the absence of inflammatory cellular infiltrates. The clinical and pathological features of this outbreak indicate that the SCID mouse would be a much improved model for studying the mechanism of poliovirus infection and of virus-induced demyelinating diseases.

Localization of endothelin-like immunoreactivity in airway epithelium of rats and mice.

We have examined lungs from adult Wistar rats (n = 6) and four different strains of juvenile and adult mice (n = 40) to localize endothelin-like immunoreactivity. Paraffin sections of lung tissue fixed by distension in Bouin's fluid were stained by the peroxidase-antiperoxidase (PAP) method using 10 different rabbit antisera to endothelin. Immunoreactivity was detected in the majority of epithelial cells of conducting airways from the hilum to the periphery and was similar in rats and all four strains of mice studied. Intense immunostaining was detected in mucous, serous and Clara cells and in occasional alveolar pneumocytes type II. Basal cells and most ciliated cells did not immunostain. From these results it is concluded that endothelin-like immunoreactivity is present in bronchiolar epithelial cells in vivo in rats and mice.

Urinary cytology of a canine bladder carcinoma.

This is a study of a case of transitional cell carcinoma of the urinary bladder in a dog. Clinical and radiological signs were inconclusive. The morphology of cells exfoliated from the tumour was very similar to that of cells exfoliated from transitional cell carcinomas in human patients. On the basis of this information a diagnosis was made which was confirmed at post-mortem examination. The findings in this case report demonstrate the usefulness of this technique in the diagnosis of poorly differentiated transitional cell carcinoma.