A transgenic mouse bearing an antisense construct of regulatory subunit type 1A of protein kinase A develops endocrine and other tumours: comparison with Carney complex and other PRKAR1A induced lesions

Background: Inactivation of the human type Iα regulatory subunit (RIα) of cyclic AMP dependent protein kinase (PKA) (PRKAR1A) leads to altered kinase activity, primary pigmented nodular adrenocortical disease (PPNAD), and sporadic adrenal and other tumours.

Methods and results: A transgenic mouse carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline responsive promoter (the Tg(Prkar1a*x2as)1Stra, Tg(tTAhCMV)3Uh or tTA/X2AS line) developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia and other features reminiscent of PPNAD, including late onset weight gain, visceral adiposity, and non-dexamethasone suppressible hypercorticosteronaemia, with histiocytic, epithelial hyperplasias, lymphomas, and other mesenchymal tumours. These lesions were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIß protein levels; the latter biochemical and protein changes were also documented in Carney complex tumours associated with PRKAR1A inactivating mutations and chromosome 17 PRKAR1A locus changes.

Conclusion: We conclude that the tTA/X2AS mouse line with a downregulated Prkar1a gene replicates several of the findings in Carney complex patients and their affected tissues, supporting the role of RIα as a candidate tumour suppressor gene.

     

Genetic resistance to experimental infection with Mycobacterium bovis in red deer (Cervus elaphus)

Tuberculosis (Tb) caused by Mycobacterium bovis is a worldwide threat to livestock and humans. One control strategy is to breed livestock that are more resistant to Mycobacterium bovis. In a 3-year heritability study 6 farmed red deer stags were selected from 39 on the basis of their differing responses to experimental challenge via the tonsillar sac with approximately 500 CFU of M. bovis. Two stags remained uninfected, two were moderately affected, and two developed serious spreading Tb. Seventy offspring, bred from these six stags by artificial insemination using stored semen, were similarly challenged with M. bovis. The offspring showed patterns of response to M. bovis challenge similar to those of their sires, providing evidence for a strong genetic basis to resistance to Tb, with an estimated heritability of 0.48 (standard error, 0.096; P < 0. 01). This is the first time the heritability of Tb resistance in domestic livestock has been measured. The breeding of selection lines of resistant and susceptible deer will provide an ideal model to study the mechanisms of Tb resistance in a ruminant and could provide an additional strategy for reducing the number and severity of outbreaks of Tb in farmed deer herds. Laboratory studies to identify genetic and immunological markers for resistance to Tb are under way. Preliminary studies showed no associations between NRAMP or DRB genes and resistance to Tb in deer. Patterns of immune responses seen in resistant animals suggest that both innate and acquired pathways of immunity are necessary to produce the resistant phenotype.     

Effect of the 21-aminosteroid,U-74389F,on hyperoxic lung injury in rats

Hyperoxia (>95% oxygen) in rats caused an increase in lung weight and an accumulation of fluid in the thorax. The mean lung wet weight of air-breathing controls at 60 h was 1.2±0.01 g, and that of vehicle-treated, oxygen-exposed animals was 2.45±0.05 g. Treatment with the 21-aminosteroid U-74389F, 3, 10, and 30 mg/kg twice daily throughout oxygen exposure, produced 8, 42, and 18% inhibition of the oxygen-induced increase in lung weight, respectively. However, U-74389F did not inhibit the hyperoxia-induced accumulation of neutrophils in bronchoalveolar lavage fluid.

No pleural fluid could be aspirated from the thorax of air-breathing controls. The volume of pleural fluid in oxygen-exposed, vehicle-treated animals and animals treated with 3, 10, and 30 mg/kg U-74389F b.i.d. was 6.5±0.9, 2.6±0.6, 0.8±0.3, and 1.3±0.5 ml, respectively.

U-74389F or its biologs are of potential value for the treatment of lung diseases in which oxidant damage has been implicated.

     

Peak Identification in Visual Evoked Potentials

Waveform patterns evoked by 4 intensities of flash in normal subjects were studied in relation to intersubject variability. Time-frequency distribution curves of all peaks occurring between 11 and 280 msec after flash onset and meeting minimal criteria were obtained from 46 males. These distributions closely corresponded to similar data reported by others for single intensity stimulation. An algorithm was developed which identified in 67 to 100% of instances a single “peak event’ within the time ranges of each of 6 peak distributions. Many peak events appeared and disappeared within the 4 intensity sets of individuals. Latencies were obtained for these peak events. Application of the algorithm to a replicate sample of 29 Ss, which included 8 females, indicated generalizability. Test-retest data on 15 Ss showed its reliability. The data suggest that methodology significantly contributes to the variability of peak identification among subjects. This may be reduced by employing multiple intensities of stimulation.     

Extensive immune-mediated hippocampal damage in mice surviving infection with neuroadapted Sindbis virus

Viral infections of the central nervous system and immune responses to these infections cause a variety of neurological diseases. Infection of weanling mice with Sindbis virus causes acute nonfatal encephalomyelitis followed by clearance of infectious virus, but persistence of viral RNA. Infection with a neuroadapted strain of Sindbis virus (NSV) causes fatal encephalomyelitis, but passive transfer of immune serum after infection protects from fatal disease and infectious virus is cleared. To determine whether persistent NSV RNA is associated with neurological damage, we examined the brains of recovered mice and found progressive loss of the hippocampal gyrus, adjacent white matter, and deep cerebral cortex associated with mononuclear cell infiltration. Mice deficient in CD4(+) T cells showed less tissue loss, while mice lacking CD8(+) T cells showed lesions comparable to those in immunocompetent mice. Mice deficient in both CD4(+) and CD8(+) T cells developed severe tissue loss similar to immunocompetent mice and this was associated with extensive infiltration of macrophages. The number of CD4(+) cells and macrophage/microglial cells, but not CD8(+) cells, infiltrating the hippocampal gyrus was correlated with the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling positive pyramidal neurons. These results suggest that CD4(+) T cells can promote progressive neuronal death and tissue injury, despite clearance of infectious virus.     

Isolation of brain parenchymal lymphocytes for flow cytometric analysis. Application to acute viral encephalitis.

A strategy for the isolation of mononuclear cells from the brain parenchyma of mice with ongoing central nervous system (CNS) inflammation has been developed in order to permit flow cytometric (FCM) analysis of these cell populations. Sindbis virus (SV) encephalitis in mice is characterized morphologically by an infiltration of mononuclear cells into both brain parenchyma and cerebrospinal fluid (CSF). Perfused brain tissue from infected animals is collected, homogenized, and subjected to a mild enzymatic digestion. A sedimentation at unit gravity is performed to remove any large particulate debris, and the remaining tissue is then centrifuged over a modified density gradient which separates intact cells from smaller tissue fragments. Cells collected directly from these gradients can be stained with monoclonal antibodies and analyzed by FCM without further manipulation. Data generated by this method correlates with previous studies of SV encephalitis using immunohistochemical analysis of brain tissue sections to quantify mononuclear cell types. This suggests that representative samples of the cellular infiltrate are obtained using this technique. The approach however, offers the possibility of more sophisticated and quantitative analyses of CNS inflammatory cells which is unobtainable by tissue section staining.     

Changes in neuronal size and neurotransmitter marker in hereditary canine spinal muscular atrophy

Hereditary canine spinal muscular atrophy (HCSMA) is a dominantly inherited motor neuron disease that produces muscle weakness and atrophy. Immunocytochemical and computer-imaging morphometric methods were used to compare early changes that occurred in dogs with HCSMA (n = 4) versus controls (n = 2). The size and number of neurons in the ventral horn and the number of motor neurons expressing choline acetyltransferase were quantitated. The density of all ventral horn neurons per micrometer squared in dogs with HCSMA was greater than controls, and there were more small neurons than in controls. Immunocytochemical methods revealed more small cholinergic neurons and fewer large cholinergic neurons in HCSMA than in controls, suggesting growth arrest in HCSMA or a shift in size class from large cholinergic neurons to small ones. The density of cholinergic neurons per micrometer squared was not significantly different between the two groups. Analysis of predicted distributions of cholinergic and noncholinergic neurons revealed that HCSMA cholinergic neurons were smaller and that, in some size classes, fewer neurons expressed choline acetyltransferase. These observations indicate that in HCSMA the motor neuron fails to achieve normal size and/or undergoes atrophy.