Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis in cystatin B-deficient mice.

Loss-of-function mutations in the gene (CSTB) encoding human cystatin B, a widely expressed cysteine protease inhibitor, are responsible for a severe neurological disorder known as Unverricht-Lundborg disease (EPM1). The primary cellular events and mechanisms underlying the disease are unknown. We found that mice lacking cystatin B develop myoclonic seizures and ataxia, similar to symptoms seen in the human disease. The principal cytopathology appears to be a loss of cerebellar granule cells, which frequently display condensed nuclei, fragmented DNA and other cellular changes characteristic of apoptosis. This mouse model of EPM1 provides evidence that cystatin B, a non-caspase cysteine protease inhibitor, has a role in preventing cerebellar apoptosis.

A biochemical function for attractin in agouti-induced pigmentation and obesity.

Agouti protein, a paracrine signaling molecule normally limited to skin, is ectopically expressed in lethal yellow (A(y)) mice, and causes obesity by mimicking agouti-related protein (Agrp), found primarily in the hypothalamus. Mouse attractin (Atrn) is a widely expressed transmembrane protein whose loss of function in mahogany (Atrn(mg-3J)/ Atrn(mg-3J)) mutant mice blocks the pleiotropic effects of A(y). Here we demonstrate in transgenic, biochemical and genetic-interaction experiments that attractin is a low-affinity receptor for agouti protein, but not Agrp, in vitro and in vivo. Additional histopathologic abnormalities in Atrn(mg-3J)/Atrn(mg-3J) mice and cross-species genomic comparisons indicate that Atrn has multiple functions distinct from both a physiologic and an evolutionary perspective.

Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype.

BACKGROUND: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs. METHODS: We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared. RESULTS: MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts. CONCLUSION: CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo.

Loss of polyubiquitin gene Ubb leads to metabolic and sleep abnormalities in mice.

AIMS: Ubiquitin performs essential roles in a myriad of signalling pathways required for cellular function and survival. Recently, we reported that disruption of the stress-inducible ubiquitin-encoding gene Ubb reduces ubiquitin content in the hypothalamus and leads to adult-onset obesity coupled with a loss of arcuate nucleus neurones and disrupted energy homeostasis in mice. Neuropeptides expressed in the hypothalamus control both metabolic and sleep behaviours. In order to demonstrate that the loss of Ubb results in broad hypothalamic abnormalities, we attempted to determine whether metabolic and sleep behaviours were altered in Ubb knockout mice. METHODS: Metabolic rate and energy expenditure were measured in a metabolic chamber, and sleep stage was monitored via electroencephalographic/electromyographic recording. The presence of neurodegeneration and increased reactive gliosis in the hypothalamus were also evaluated. RESULTS: We found that Ubb disruption leads to early-onset reduced activity and metabolic rate. Additionally, we have demonstrated that sleep behaviour is altered and sleep homeostasis is disrupted in Ubb knockout mice. These early metabolic and sleep abnormalities are accompanied by persistent reactive gliosis and the loss of arcuate nucleus neurones, but are independent of neurodegeneration in the lateral hypothalamus. CONCLUSIONS: Ubb knockout mice exhibit phenotypes consistent with hypothalamic dysfunction. Our data also indicate that Ubb is essential for the maintenance of the ubiquitin levels required for proper regulation of metabolic and sleep behaviours in mice.

Spontaneous murine neuroaxonal dystrophy: a model of infantile neuroaxonal dystrophy.

The neuroaxonal dystrophies (NADs) in human beings are fatal, inherited, neurodegenerative diseases with distinctive pathological features. This report describes a new mouse model of NAD that was identified as a spontaneous mutation in a BALB/c congenic mouse strain. The affected animals developed clinical signs of a sensory axonopathy consisting of hindlimb spasticity and ataxia as early as 3 weeks of age, with progression to paraparesis and severe morbidity by 6 months of age. Hallmark histological lesions consisted of spheroids (swollen axons), in the grey and white matter of the midbrain, brain stem, and all levels of the spinal cord. Ultrastructural analysis of the spheroids revealed accumulations of layered stacks of membranes and tubulovesicular elements, strongly resembling the ultrastructural changes seen in the axons of human patients with endogenous forms of NAD. Mouse NAD would therefore seem a potentially valuable model of human NADs.

Molecular and phenotypic analysis of Attractin mutant mice.

Mutations of the mouse Attractin (Atrn; formerly mahogany) gene were originally recognized because they suppress Agouti pigment type switching. More recently, effects independent of Agouti have been recognized: mice homozygous for the Atrn(mg-3J) allele are resistant to diet-induced obesity and also develop abnormal myelination and vacuolation in the central nervous system. To better understand the pathophysiology and relationship of these pleiotropic effects, we further characterized the molecular abnormalities responsible for two additional Atrn alleles, Atrn(mg) and Atrn(mg-L), and examined in parallel the phenotypes of homozygous and compound heterozygous animals. We find that the three alleles have similar effects on pigmentation and neurodegeneration, with a relative severity of Atrn(mg-3J) > Atrn(mg) > Atrn(mg-L), which also corresponds to the effects of the three alleles on levels of normal Atrn mRNA. Animals homozygous for Atrn(mg-3J) or Atrn(mg), but not Atrn(mg-L), show reduced body weight, reduced adiposity, and increased locomotor activity, all in the presence of normal food intake. These results confirm that the mechanism responsible for the neuropathological alteration is a loss--rather than gain--of function, indicate that abnormal body weight in Atrn mutant mice is caused by a central process leading to increased energy expenditure, and demonstrate that pigmentation is more sensitive to levels of Atrn mRNA than are nonpigmentary phenotypes.

Disease attributed to Mycobacterium chelonae in South African clawed frogs (Xenopus laevis).

The fast-growing nontuberculous mycobacterial species Mycobacterium chelonae was isolated from six captive South African clawed frogs (Xenopus laevis) with chronic weight loss and nonhealing ulcerative skin lesions. Three of the M. chelonae isolates were evaluated to confirm the species identification using polymerase chain reaction restriction analysis. Disease associated with M. chelonae is reported mainly in people and in fish. To our knowledge, this is the first report of disease associated with M. chelonae in a colony of captive Xenopus sp.

Canine motor neuron disease: clinicopathologic features and selected indicators of oxidative stress.

Hereditary canine spinal muscular atrophy (HCSMA) is an inherited motor neuron disease affecting a kindred of Brittanies. We have examined the clinicopathologic abnormalities in 57 animals with HCSMA, including 43 affected adult dogs and 14 homozygote pups. We also measured selected biochemical indices of oxidative stress: serum vitamin E (alpha-tocopherol) and Se concentrations; serum concentrations of Cu, Zn, Mg, and Fe; and total superoxide dismutase and glutathione peroxidase activities in red blood cells. Dogs with HCSMA had the following abnormalities: regenerative anemia, hypoglobulinemia, hypochloremia, and abnormally high creatine kinase and liver alkaline phosphatase activities. Serum Cu concentration was significantly (P = .01) increased in adult dogs with HCSMA compared to control dogs. Serum vitamin E concentrations tended to be lower in adult dogs with HCSMA compared to controls, and were significantly (P = .01) lower in homozygote pups compared to control pups.

Radiographic and scintigraphic evidence of focal pulmonary neoplasia in three cats with hyperthyroidism: diagnostic and therapeutic considerations.

Three cats were diagnosed as hyperthyroid based on clinical signs, historical findings, laboratory abnormalities, and basal serum thyroxine (T4) concentrations, and/or nuclear thyroid scans. Additionally, a presumptive diagnosis of thyroid carcinoma with pulmonary metastasis was made in each cat based on radiographic or scintigraphic evaluation. All three cats had solitary pulmonary nodules 1.5 to 2 cm in diameter on survey thoracic radiographs; one cat also had chylous pleural effusion and pulmonary lobar consolidation. Focal pulmonary accumulation of sodium pertechnetate (99mTcO4-) and/or radioiodine (131I) corresponding to radiographic lesions were seen in all cats. Two cats were treated with single ablative doses (1111 to 1480 MBq) of 131I; the remaining cat was euthanatized. One of the treated cats died 8 days later; the other cat was euthanatized 22 weeks following treatment. Histopathologic examination of tissue obtained at necropsy confirmed metastatic thyroid carcinoma in one cat and bronchogenic adenocarcinoma in two cats. Our findings indicate that increased radionuclide uptake in focal pulmonary lesions and cytologic evaluation of tissue obtained by fine-needle aspiration are not specific for thyroid tissue.

Identification and management of an outbreak of Flavobacterium meningosepticum infection in a colony of South African clawed frogs (Xenopus laevis).

During the summer of 1996, an outbreak of Flavobacterium meningosepticum infection developed in a colony of South African clawed frogs (Xenopus laevis). Clinical signs were consistent with septicemia: ascites, anasarca, dyspnea, extreme lethargy, congestion of web vessels, petechial hemorrhages, and sudden death. Mortality rate reached 35%, and all infections were fatal. The organism was resistant to most antibiotics but was susceptible to enrofloxacin, chloramphenicol, and trimethoprim-sulfadiazine. Treatment with trimethoprim-sulfadiazine was unsuccessful. Although the point source of the infection was not determined, several environmental reservoirs were identified, including a communal water barrel and various pieces of equipment. Molecular strain typing by pulsed-field gel electrophoresis and biochemical analyses revealed that frogs were infected with a single strain of F meningosepticum. Sanitation and management procedures were effective in controlling the outbreak.

Suspected hypovitaminosis A in a colony of captive green anoles (Anolis carolinensis).

In a colony of 18 green anoles (Anolis carolinensis), 3 animals experienced focally thickened lips, ulcerative cheilitis, lethargy, depression, and weight loss over a 5-month period. In addition to crickets fed fresh fruit and leafy green vegetables, the diet of the green anoles consisted of a supply of mealworms that had been dusted with a commercial liquid vitamin supplement. The history, clinical findings, and histopathologic lesions were suggestive of hypovitaminosis A, which is known to cause squamous metaplasia of the mucus secreting glands and epithelial surfaces in many species.

Effects of rainfall, host demography, and musth on strongyle fecal egg counts in African elephants (Loxodonta africana) in Namibia.

Wild African elephants (Loxodonta africana) are commonly infected with intestinal strongyle parasites. Our objective was to determine baseline fecal strongyle egg counts for elephants in the northeast region of Etosha National Park, Namibia and determine if these numbers were affected by annual rainfall, elephant demography (age of individuals and composition of groups), and hormonal state of males. We found that matriarchal family group members have significantly higher fecal egg counts than male elephants (bulls). Among family group members, strongyle egg counts increased with age, whereas among bulls, strongyle egg counts decreased with age. Years of higher rainfall were correlated with decreased numbers of strongyle eggs among bulls. Finally, bulls were not affected by their physiologic (hormonal) status (musth vs. nonmusth). These results suggest that infection by strongyle parasites in Namibian African elephants is a dynamic process affected by intrinsic and extrinsic factors including host demography and rainfall.

Experimental study of intracranial hematoma detection with flat panel detector C-arm CT.

BACKGROUND AND PURPOSE: Intracranial hemorrhage is a commonly acknowledged complication of interventional neuroradiology procedures, and the ability to image hemorrhage at the time of the procedure would be very beneficial. A new C-arm system with 3D functionality extends the capability of C-arm imaging to include soft-tissue applications by facilitating the detection of low-contrast objects. We evaluated its ability to detect small intracranial hematomas in a swine model. MATERIALS AND METHODS: Intracranial hematomas were created in 7 swine by autologous blood injection of various hematocrits (19%-37%) and volumes (1.5-5 mL). Four animals received intravascular contrast before obtaining autologous blood (group 1), and 3 did not (group 2). We scanned each animal by using the C-arm CT system, acquiring more than 500 images during a 20-second rotation through more than 200 degrees . Multiplanar reformatted images with isotropic resolution were reconstructed on the workstation by using product truncation, scatter, beam-hardening, and ring-artifact correction algorithms. The brains were harvested and sliced for hematoma measurement and compared with imaging findings. RESULTS: Five intracranial hematomas were created in group 1 animals, and all were visualized. Six were created in group 2, and 3 were visualized. One nonvisualized hematoma was not confirmed at necropsy. All the others in both groups were confirmed. In group 1 (with contrast), small hematomas were detectable even when the hematocrit was 19%-20%. In group 2 (without contrast) C-arm CT was able to detect small hematomas (<1.0 cm(2)) created with hematocrits of 29%-37%. The area of hematoma measured from the C-arm CT data was, on average, within 15% of the area measured from harvested brain. CONCLUSIONS: The image quality obtained with this implementation of C-arm CT was sufficient to detect experimentally created small intracranial hematomas. This capability should provide earlier detection of hemorrhagic complications that may occur during neurointerventional procedures.

The distribution, density and three-dimensional histomorphology of Pacinian corpuscles in the foot of the Asian elephant (Elephas maximus) and their potential role in seismic communication.

Both Asian (Elephas maximus) and African (Loxodonta africana) elephants produce low-frequency, high-amplitude rumbles that travel well through the ground as seismic waves, and field studies have shown that elephants may utilize these seismic signals as one form of communication. Unique elephant postures observed in field studies suggest that the elephants use their feet to 'listen' to these seismic signals, but the exact sensory mechanisms used by the elephant have never been characterized. The distribution, morphology and tissue density of Pacinian corpuscles, specialized mechanoreceptors, were studied in a forefoot and hindfoot of Asian elephants. Pacinian corpuscles were located in the dermis and distal digital cushion and were most densely localized to the anterior, posterior, medial and lateral region of each foot, with the highest numbers in the anterior region of the forefoot (52.19%) and the posterior region of the hindfoot (47.09%). Pacinian corpuscles were encapsulated, had a typical lamellar structure and were most often observed in large clusters. Three-dimensional reconstruction through serial sections of the dermis revealed that individual Pacinian corpuscles may be part of a cluster. By studying the distribution and density of these mechanoreceptors, we propose that Pacinian corpuscles are one possible anatomic mechanism used by elephants to detect seismic waves.

Correlation of contrast-enhanced MR images with the histopathology of minimally invasive thermal and cryoablation cancer treatments in normal dog prostates.

Magnetic Resonance Imaging (MRI) is a promising tool for visualizing the delivery of minimally invasive cancer treatments such as high intensity ultrasound (HUS) and cryoablation. We use an acute dog prostate model to correlate lesion histopathology with contrast-enhanced (CE) T1 weighted MR images, to aid the radiologists in real time interpretation of in vivo lesion boundaries and pre-existing lesions. Following thermal or cryo treatments, prostate glands are removed, sliced, stained with the vital dye triphenyl tetrazolium chloride, photographed, fixed and processed in oversized blocks for routine microscopy. Slides are scanned by Trestle Corporation at .32 microns/pixel resolution, the various lesions traced using annotation software, and digital images compared to CE MR images. Histologically, HUS results in discrete lesions characterized by a "heat-fixed" zone, in which glands subjected to the highest temperatures are minimally altered, surrounded by a rim or "transition zone" composed of severely fragmented, necrotic glands, interstitial edema and vascular congestion. The "heat-fixed" zone is non-enhancing on CE MRI while the "transition zone" appears as a bright, enhancing rim. Likewise, the CE MR images for cryo lesions appear similar to thermally induced lesions, yet the histopathology is significantly different. Glands subjected to prolonged freezing appear totally disrupted, coagulated and hemorrhagic, while less intensely frozen glands along the lesion edge are partially fragmented and contain apoptotic cells. In conclusion, thermal and cryo-induced lesions, as well as certain pre-existing lesions (cystic hyperplasia - non-enhancing, chronic prostatitis - enhancing) have particular MRI profiles, useful for treatment and diagnostic purposes.

The role of the innate immune system in the reconstituted SCID mouse model of herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) has an immunopathological basis, thought primarily to involve a CD4+ T cell-mediated immune response to viral antigen. Other cell types, however, particularly those involved in nonspecific immunity, such as natural killer (NK) cells or neutrophils, may also contribute to tissue destruction in the cornea. The reconstituted SCID mouse model of HSK provides a powerful system in which to study the interactions of the innate and adaptive immune responses to herpes simplex virus type 1 corneal infection. In the present study, reconstituted SCID mice depleted of NK cells had a reduced incidence and severity of clinical and histopathological HSK. The levels of T cell cytokine protein and message in restimulated splenocytes and cytokine message in corneas did not differ between experimental groups. However, significantly fewer neutrophils were seen within the inflamed corneas of NK-depleted SCID mice. Therefore, endogenous NK cells may indirectly influence the severity of HSK in reconstituted SCID mice by affecting neutrophil migration into the cornea.

Herpetic stromal keratitis in the reconstituted scid mouse model.

Infections of the cornea with herpes simplex virus type 1 cause inflammatory lesions which frequently lead to blindness. The disease is suspected to be immunopathological in nature. To establish this point and to study possible mechanisms involved, corneal infections in C.B-17 scid/scid and cell-reconstituted scid mice were investigated. Whereas unreconstituted scid mice failed to develop herpetic stromal keratitis (HSK) and died of encephalitis, mice reconstituted with T lymphocytes generated severe lesions. T cells of the CD4+ subset were found to be essential mediators of the HSK lesion, while T cells of the CD8+ subset protected mice from lethality. The results confirm that HSK is an immunopathological disease and that scid mice provide a convenient model that should prove valuable in establishing the biochemical mechanisms by which HSK is mediated.

Characterization of herpes simplex virus type-1 infection and herpetic stromal keratitis development in IFN-gamma knockout mice.

Herpetic stromal keratitis (HSK) has an immune-mediated pathogenesis that involves T cells that have a type 1 cytokine profile. IFN-gamma is suspected to be the type 1 cytokine involved in ocular pathology, and to test this notion more directly the pathogenesis of HSK was compared in mice deficient in the IFN-gamma gene (gamma knockout or gko) and control mice (wild-type littermates or BALB/c mice). The clinical course of HSK in gko mice closely paralleled that in control mice, yet virus persisted in the corneas of gko mice for an extended period of time, severe periocular skin lesions developed, and gko mice were far more susceptible to encephalitis. Delayed-type hypersensitivity to viral Ag was present, though diminished, in knockout mice, and serum herpes simplex virus-specific IgG isotypes indicated a Th2 shift. No differences existed in proliferative responses to in vitro Ag stimulation in gko vs control mice nor in T cell or proinflammatory cytokine mRNA levels in the corneas of infected mice. However, up-regulation of Th2 cytokine mRNA did occur in in vitro Ag-stimulated gko immune splenocytes. Histopathologic lesions were not statistically different between any of the groups of mice analyzed. These observations indicate that although IFN-gamma plays an important role in the clearance of virus from the eye, the pathogenesis of HSK lesions most likely involves additional cytokines, inflammatory mechanisms, or immune responses to nonviral Ags.

Murine cytomegalovirus CC chemokine homolog MCK-2 (m131-129) is a determinant of dissemination that increases inflammation at initial sites of infection.

The murine cytomegalovirus CC chemokine homolog MCK-2 (m131-129) is an important determinant of dissemination during primary infection. Reduced peak levels of viremia at day 5 were followed by reduced levels of virus in salivary glands starting at day 7 when mck insertion (RM461) and point (RM4511) mutants were compared to mck-expressing viruses. A dramatic MCK-2-enhanced inflammation occurred at the inoculation site over the first few days of infection, preceding viremia. The data further reinforce the role of MCK-2 as a proinflammatory signal that recruits leukocytes to increase the efficiency of viral dissemination in the host.