Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits.

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

Design of a simplified histopathologic model for gastrointestinal inflammation in dogs.

Significant interobserver variability in the diagnostic interpretation of endoscopic gastrointestinal (GI) specimens exists even with the use of World Small Animal Veterinary Association (WSAVA) standardization criteria. Chi-square analyses compared the extent of pathologists' agreement for microarchitectural features of inflammation in endoscopic specimens obtained from 253 animals of the original WSAVA study. Patterns of agreement between pathologists were classified as broad (3/4 pathologists agreed), dichotomous (2/4 pathologists agreed), or divergent (no agreement between pathologists). The simplified model for GI inflammation was based on those parameters for which the pathologists had either broad or minimally divergent opinions of histopathologic significance. In this model, the parameters chosen were as follows: gastric parameters (intraepithelial lymphocytes [IELs], lamina propria [LP] infiltrates, and mucosal fibrosis), duodenal parameters (villus atrophy, epithelial injury, IELs, crypt changes, and LP infiltrates), and colonic parameters (epithelial injury, crypt dilation, fibrosis, LP infiltrates, and goblet cell depletion). Preliminary data using this simplified model showed excellent correlation between pathologists in defining the presence and extent of GI inflammation in dogs.

Assessment of the histological quality of endoscopic biopsies obtained from the canine gastro-esophageal junction.

OBJECTIVE: In the dog biopsy samples from the gastro-esophageal junction (GEJ) are rarely obtained during routine gastroscopy. The aim of this pilot study was to assess the histological quality of endoscopic biopsies sampled from the canine esophagus and cardia. It was hypothesised that it is possible to sample adequate specimens from these sites. MATERIALS AND METHODS: For this purpose 10 dogs with an indication for gastroscopy were enrolled in a prospective study. Biopsy samples were obtained with standard biopsy forceps for single use exactly from the GEJ thus containing preferably columnar epithelium from the cardia and squamous epithelium from the esophagus, respectively. In every dog the specimens were examined for size, layers and site, respectively. Study endpoint was reached when specimens originated from cardia and esophagus, showing at least epithelium and lamina propria mucosae, and a diameter >2mm on the slide, respectively. RESULTS: 72 biopsy specimens (median 7, range 5-10) obtained from the GEJ were examined in 10 dogs. Specimens from the esophagus containing squamous epithelium with lamina propria mucosae were found in 5 of 10 (50.0%) dogs. Specimens from the cardia containing columnar epithelium with lamina propria mucosae were found in 10 of 10 (100.0%) dogs. Four of 10 (40.0%), and 10 of 10 (100.0%) dogs showed at least one specimen >2mm on the slide originating from the esophagus, and from the cardia, respectively. Histological quality was found to be adequate in 4 of 10 (40.0%) dogs, showing specimens of adequate size, originating from both esophagus and cardia, and containing at least epithelium and lamina propria mucosae. CONCLUSION AND CLINICAL RELEVANCE: The pilot study provides evidence that during routine gastroscopy it is possible to sample endoscopic biopsies from the cardia and with limitations from the esophagus showing a quality adequate for histological examination of the epithelium and the lamina propria mucosae.

Hereditary polyneuropathy in the Alaskan Malamute.

OBJECTIVE: To prove the hypothesis that a polyneuropathy in Alaskan Malamutes has a genetic background. MATERIAL AND METHODS: Pedigrees of 131 related Alaskan Malamutes were included in the current study. Neurological examination, electrodiagnosis as well as muscle and nerve biopsies could be performed in 10 dogs. Information about the disease status of the other 121 Alaskan Malamutes were supplied by referring veterinarians, breeders and owners. Segregation analysis using four different models (monogenic, polygenic, mixed monogenic-polygenic and the phenotypic model) was performed on 71 dogs to test the different mechanisms of genetic transmission. RESULTS: In seven clinically affected dogs abnormal electromyographic findings and reduced nerve conduction velocity were detected. Suspected diagnosis of polyneuropathy was confirmed by nerve biopsy results, characterized by axonal degeneration and hypomyelination. Muscle specimens revealed signs of neurogenic myopathy. Three related clinically normal Alaskan Malamutes also displayed moderate neuromuscular changes in histopathology. In the segregation analysis the polygenic model proved as best suitable to explain the observed segregation pattern among all other models tested. CONCLUSION: The current study could demonstrate that polyneuropathy in Alaskan Malamutes is a hereditary disease with variable phenotypic expression ranging from severely affected to subclinical forms, which has to be considered in future gene analysis studies.

Effect of tissue processing on assessment of endoscopic intestinal biopsies in dogs and cats.

BACKGROUND: Prior studies failed to detect significant association between hypoalbuminemia and small intestinal lesions. HYPOTHESIS: Use of pictorial templates will enhance consistency of interpathologist interpretation and identification of intestinal lesions associated with hypoalbuminemia. ANIMALS: Tissues from 62 dogs and 25 cats examined as clinical cases at 7 referral veterinary practices in 4 countries. METHODS: Retrospective, observational study. Histopathology slides from sequential cases undergoing endoscopic biopsy were examined by 4 pathologists by pictorial templates. Changes for 9 microscopic features were recorded as normal, mild, moderate or severe, and 2- and 4-point scales were tested for consistency of interpretation. Logistic regression models determined odds ratios (OR) of histologic lesions being associated with hypoalbuminemia while kappa statistics determined agreement between pathologists on histologic lesions. RESULTS: There was poor agreement (kappa = -0.013 to 0.3) between pathologists, and institution of origin of slides had effect (kappa = 1.0 for 3 of 4 lesions on slides from Institution 5) on agreement between pathologists on selected histologic features. Using 2 point as opposed to 4-point grading scale increased agreement between pathologists (maximum kappa = 0.69 using 4-point scale versus maximum kappa = 1.0 using 2-point scale). Significant association (P = .019- .04; 95% OR = 3.14-10.84) between lacteal dilation and hypoalbuminemia was found by 3 pathologists. CONCLUSIONS AND CLINICAL IMPORTANCE: Substantial inconsistency between pathologists remains despite use of pictorial template because of differences in slide processing. Distinguishing between mild and moderate lesions might be important source of the disagreement among pathologists.

Pulmonary dysfunction and skeletal muscle changes in horses with RAO.

BACKGROUND: Chronic pulmonary diseases (recurrent airway obstruction [RAO]) have been reported to alter skeletal muscle cells in humans. The purpose of this study was to evaluate a potential relationship between pulmonary and muscle variables in horses with a clinical diagnosis of RAO. Muscle biopsies from healthy horses and from horses with RAO were investigated and the relationship between the severity of lung disease and the degree of muscular changes was determined. HYPOTHESIS: We hypothesized that chronic pulmonary disease can lead to changes of the skeletal muscle in horses. ANIMALS: Fifteen healthy horses (control) and 50 horses with RAO were examined. METHODS: In a prospective clinical trial, a complete lung examination was performed in all horses. In all horses, muscle enzyme activity at rest and after exercise and muscle biopsies from the M. gluteus medius were examined. RESULTS: None of the horses had clinical or histologic signs of primary or neurogenic myopathies. According to the clinical, endoscopic, and radiographic findings and with a scoring system, the horses with RAO were grouped according to the severity of pulmonary findings (15 horses mild, 24 horses moderate, 11 horses severe RAO). Pathologic changes of the skeletal muscle (fiber atrophy or fiber hypertrophy, myofibrillar degeneration, hyperplasia of mitochondria, and ragged-red-like fibers) were identified in most horses with RAO but in only a few individual control horses. In addition, a marked depletion of muscle glycogen storage was evident in the RAO horses but not in the control group. Other pathologic changes of skeletal muscle such as centralized nuclei and regenerating fibers were rare, but were more frequent in horses with lung diseases than in the control group. The degree of muscle cell changes was also graded with a scoring system and correlated with the severity of pulmonary disease (r= 0.55). CONCLUSION: Chronic pulmonary disease in horses is associated with structural changes in skeletal muscle. CLINICAL IMPORTANCE: Because chronic pulmonary disease may affect muscles, early and effective therapy may prevent these changes. This finding could be of clinical importance but requires further studies.

Histopathological standards for the diagnosis of gastrointestinal inflammation in endoscopic biopsy samples from the dog and cat: a report from the World Small Animal Veterinary Association Gastrointestinal Standardization Group.

The characterization of inflammatory change in endoscopic biopsy samples of the gastrointestinal mucosa is an increasingly important component in the diagnosis and management of canine and feline gastrointestinal disease. Interpretation has hitherto been limited by the lack of standard criteria that define morphological and inflammatory features, and the absence of such standardization has made it difficult, if not impossible, to compare results of retrospective or prospective studies. The World Small Animal Veterinary Association (WSAVA) Gastrointestinal Standardization Group was established, in part, to develop endoscopic and microscopical standards in small animal gastroenterology. This monograph presents a standardized pictorial and textual template of the major histopathological changes that occur in inflammatory disease of the canine and feline gastric body, gastric antrum, duodenum and colon. Additionally, a series of standard histopathological reporting forms is proposed, to encourage evaluation of biopsy samples in a systematic fashion. The Standardization Group believes that the international acceptance of these standard templates will advance the study of gastrointestinal disease in individual small companion animals as well as investigations that compare populations of animals.

Myotonic dystrophy in two European grey wolves (Canis lupus).

Two related European Grey wolves (Canis lupus) with the history of muscle stiffness beginning at 2 weeks of age were examined in this study. Muscle tone and muscle mass were increased in both animals. Muscle stiffness was worsened by stress so that the animals fell into lateral recumbency. Blood chemistry revealed mildly increased serum creatine kinase activity. Abnormal potentials typical of myotonic discharges were recorded by electromyography. Cataract, first-degree atrioventricular (AV) block and inhomogeneous myocardial texture by ultrasound suggested extramuscular involvement. Myopathology demonstrated dystrophic signs in the muscle biopsy specimen. The presumptive diagnosis based on the in vivo findings was myotonic dystrophy. Immunochemistry of the striated muscles revealed focal absence of dystrophin 1 and beta-dystroglycan in both cases. Cardiac and ophthalmologic involvement suggested a disorder very similar to a human form of myotonic dystrophy. This is the first description of myotonic dystrophy in wolves.

Evidence for MHC I-restricted CD8+ T-cell-mediated immunopathology in canine masticatory muscle myositis and polymyositis.

Masticatory muscle myositis (MMM) is the most common inflammatory myopathy (IM) in dogs, associated with antibodies against myosin. To further elucidate the immunopathogenesis, we investigated muscles of 53 dogs with MMM, 32 dogs with polymyositis (PM), and 4 dogs suffering from both, with regard to the presence and location of CD4(+) and CD8(+)T cells, B cells, macrophages, major histocompatibility complex (MHC) class I and class II antigens, and autoantibodies. CD8(+)T cells were found in MMM (91%) and PM (75%), mostly paralleled (68% and 61%) by enhanced expression of MHC class I antigen on muscle fibers. CD8(+)T cells invading intact and neighboring necrotic muscle fibers were present in MMM (39%) and PM (42%). Dogs with MMM lacking intramuscular (26%) and circulating (36%) autoantibodies also had CD8(+) T-cell infiltrations and muscle-fiber lesions. Since MHC class I antigen and CD8(+) T cells were detected in the presence of CD4(+) T cells, regardless of antimuscular antibodies, we consider MMM and PM in the dog as a CD8(+) T-cell-mediated immunopathological disease that initiates muscle-fiber destruction and leads to production of myosin autoantibodies.

Cortical cholinergic decline parallels the progression of Borna virus encephalitis.

Borna disease virus (BDV)-induced meningoencephalitis is associated with the dysfunction of the cholinergic system. Temporal development of this cholinergic decline during pre-encephalitic and encephalitic stages of BDV infection remains however elusive. Changes in choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were therefore determined in the cerebral cortex, hippocampus, striatum, amygdala and cholinergic basal forebrain nuclei (ChBFN) of rats infected with BDV. Immunocytochemistry for ChAT and vesicular acetylcholine transporter (VAChT) was employed to identify morphological consequences of BDV infection on cholinergic neurons. Whereas both ChAT and AChE activities changed only slightly under pre-encephalitic conditions, the encephalitic stage was characterized by a significant decrease of ChAT activity in the cerebral cortex, horizontal diagonal band of Broca (hDBB), hippocampus and amygdala concomitant with a marked reduction of AChE activity in the cerebral cortex, hDBB and hippocampus. The striatum and medial septum remained unaffected. ChAT and VAChT immunocytochemistry revealed prominent axonal degeneration in affected cortical and limbic projection areas of ChBFN. In summary, our data indicate progressive deterioration of forebrain cholinergic systems that parallels the progression of BDV encephalitis.

High-dose Borna disease virus infection induces a nucleoprotein-specific cytotoxic T-lymphocyte response and prevention of immunopathology.

Experimental Borna disease virus (BDV) infection of rats and natural infection of horses and sheep leads to severe central nervous system disease based on immunopathological pathways. The virus replicates slowly, and the cellular immune response results in immunopathology. CD8(+) T cells exert effector cell functions, and their activity results in the destruction of virus-infected cells. Previously, Oldach and colleagues (D. Oldach, M. C. Zink, J. M. Pyper, S. Herzog, R. Rott, O. Narayan, and J. E. Clements, Virology 206:426-434, 1995) have reported protection against Borna disease after inoculation of high-dose cell-adapted BDV. Here we show that the outcome of the infection, i.e., immunopathology versus protection, is simply dependent on the amount of virus used for infection. High-dose BDV (10(6) FFU) triggers an early virus-specific reaction of the immune system, as demonstrated by strong cellular and humoral responses. In particular, the early presence and function of nucleoprotein-specific CD8(+) T cells could be demonstrated in the brain. We present evidence that in a noncytolytic and usually persistent virus infection, high-dose input virus mediates early control of the pathogen due to an efficient induction of an antiviral immune mechanism. From these data, we conclude that immune reactivity, in particular the cytotoxic T-cell response, determines whether the virus is controlled with prevention of the ensuing immunopathological disease or whether a persistent infection is established.

[Muscle biopsy using the muscle punch--removal technique, transport and evaluation ability]. / Muskelbiopsien mit der Biopsiestanze--Entnahmetechnik, Versand und Beurteilungsfähigkeit.

Histopathological striated muscle examination is one of the most important and sensitive tests in diagnosis of muscle and/or nerve diseases. Although muscle biopsy is a relatively easy procedure, it is not frequently performed in small animal practice conditions. Different biopsy techniques have been described in veterinary literature. Punch biopsy of striated muscle appears to be a less invasive and quicker method in comparison with traditional surgical excision technique. Additionally, punch biopsy provide good quality and adequate amount of muscle tissue for diagnostic histopathological evaluation. The aim of this study is to describe striated muscle punch biopsy technique and to encourage use of it under the conditions of small animal practice. The described biopsy method can be specially advantageous in screening of inherited muscle diseases in affected litters or even wider animal population.

Neutralizing antibodies in persistent borna disease virus infection: prophylactic effect of gp94-specific monoclonal antibodies in preventing encephalitis.

Borna disease virus (BDV) infection triggers an immune-mediated encephalomyelitis and results in a persistent infection. The immune response in the acute phase of the disease is characterized by a cellular response in which CD8(+) T cells are responsible for the destruction of virus-infected brain cells. CD4(+) T cells function as helper cells and support the production of antiviral antibodies. Antibodies generated in the acute phase of the disease against the nucleoprotein and the phosphoprotein are nonneutralizing. In the chronic phase of the disease, neutralizing antibodies directed against the matrix protein and glycoprotein are synthesized. In the present work, the biological role of the neutralizing-antibody response to BDV was further investigated. By analyzing the blood of rats infected intracerebrally with BDV, a highly neurotropic virus, nucleic acid could be detected between 30 and 50 days after infection. Neutralizing antibodies were found between 60 and 100 days after infection. Furthermore, we produced hybridomas secreting BDV-specific neutralizing monoclonal antibodies. These antibodies, directed against the major glycoprotein (gp94) of BDV, were able to prevent Borna disease if given prophylactically. These data suggest that the late appearance of BDV-specific neutralizing antibodies is due to the presence of BDV in the blood of chronically infected rats. Furthermore, these antibodies have the potential to neutralize the infectious virus when given early, which is an important finding with respect to the development of a vaccine.

Borna disease virus in human brains with a rare form of hippocampal degeneration but not in brains of patients with common neuropsychiatric disorders.

To estimate the frequency of persistent Borna disease virus (BDV) infections of the human central nervous system and to determine which neuropsychiatric disorders might be associated with this viral infection, reverse transcription-nested polymerase chain reaction was used to screen a large collection of autopsy brain samples for the presence of BDV-specific nucleic acids. The presence of BDV RNA was found in 3 brains of persons with psychiatric symptoms and prominent hippocampal degeneration previously reported to be positive by others. However, no BDV RNA was detected in 86 randomly collected brains from persons with various psychiatric disorders, including schizophrenia, affective disorders, and Alzheimer's disease, or from suicide victims or in 52 brains from healthy controls. Furthermore, no BDV-RNA was detected in 16 surgical brain samples from persons with epilepsy-associated hippocampal sclerosis. These results indicate that life-long persistent BDV infections are rare in humans and that such infections may be associated with certain forms of hippocampal degeneration.

A functional role for neutralizing antibodies in Borna disease: influence on virus tropism outside the central nervous system.

Borna disease virus (BDV) is a negative-strand RNA virus that infects the central nervous systems (CNS) of warm-blooded animals and causes disturbances of movement and behavior. The basis for neurotropism remains poorly understood; however, the observation that the distribution of infectious virus in immunocompetent rats is different from that in immunoincompetent rats indicates a role for the immune system in BDV tropism: whereas in immunocompetent rats virus is restricted to the central, peripheral, and autonomic nervous systems, immunoincompetent rats also have virus in nonneural tissues. In an effort to examine the influence of the humoral immune response on BDV pathogenesis, we examined the effects of passive immunization with neutralizing antiserum in immunoincompetent rats. Serum transfer into immunoincompetent rats did not prevent persistent CNS infection but did result in restriction of virus to neural tissues. These results indicate that neutralizing antibodies may play a role in preventing generalized infection with BDV.

Lack of antiviral effect of amantadine in Borna disease virus infection.

The antiviral effect of amantadine (1-aminoadamantane) was tested in vitro as well as in vivo. Treatment of persistently Borna disease virus (BDV)-infected cell lines of different origin and for various length of time did not result in a general reduction of virus titer or clearance of virus from infected cells. In vivo, rats were treated with amantadine by daily oral application or by use of osmotic pumps, and in both cases treatment was started before infection. Neither route of application of the drug had any influence on the time of onset of disease, on antiviral antibody titers, on virus titer in the brain, on the severity of the inflammatory reaction in the brain, or on the severity of neurological symptoms. These experiments, although revealing negative results and obtained using a virus from a natural case of Borna disease grown after isolation in vitro for a long period of time, should caution from the general use of amantadine as a curative agent against BDV infection as has been implicated recently [Bode et al. (1997) Lancet 349:178-179].

Virus-specific CD4+ T cells eliminate borna disease virus from the brain via induction of cytotoxic CD8+ T cells.

Persistent Borna disease virus infection of the brain can be prevented by treatment of naive rats with a virus-specific CD4+ T-cell line prior to infection. In rats receiving this treatment, only a transient low-level encephalitis was seen compared to an increasingly inflammatory reaction in untreated infected control rats. Virus replication was found in the brain for several days after infection before the virus was cleared from the central nervous system. The loss of infectivity from the brain was confirmed by negative results by reverse transcription-PCR with primers for mRNA, by in situ hybridization for both genomic and mRNA, and by immunohistology. Most importantly, in vitro assays revealed that the T-cell line used for transfusion had no cytotoxic capacity. The kinetics of virus clearance were paralleled by the appearance of CD8+ T cells and the expression of perforin in the brain. Testing of lymphocytes isolated from the brains of CD4+ T-cell-treated rats after challenge revealed high cytotoxic activity due to the presence of CD8+ cytotoxic T cells at time points when brain lymphocytes from infected control rats induced low-level cytolysis of target cells. Neutralizing antiviral antibodies and gamma interferon were shown not to be involved in the elimination of virus from the brain.