Hypervitaminosis D and Metastatic Calcification in a Colony of Inbred Strain 13 Guinea Pigs, Cavia porcellus.

A commercial diet fed to a colony of inbred strain 13 guinea pigs for approximately 6 weeks was subsequently recalled for excessive levels of vitamin D. Twenty-one of 62 animals exhibited clinical signs, including anorexia, lethargy, and poor body condition. Nine affected and 4 clinically normal animals were euthanized for further evaluation, including serum chemistry, urinalysis, and gross and/or histopathology. Macroscopic findings included white discoloration in multiple organs in 8 animals, and microscopic evaluation confirmed multiorgan mineralization in tissues from 7 animals. Serum 25-hydroxyvitamin D levels were elevated in 10 animals. Serum inorganic phosphorus and alkaline phosphatase levels were increased in all exposed animals; however, total calcium and ionized calcium levels were not significantly higher in exposed animals than in control strain 13 guinea pigs from a different institution. The data support a diagnosis of hypervitaminosis D with metastatic calcification. Following the diet recall, the remaining guinea pigs increased their food intake and regained body condition. Diagnostic testing of 8 animals euthanized approximately 3 months after returning to a normal diet demonstrated that serum parathyroid hormone remained significantly lower, and ionized calcium and ionized magnesium were significantly higher, in recovered animals compared to controls and exposed animals. These results indicate that diagnostic tests other than serum calcium are necessary for a diagnosis of hypervitaminosis D in guinea pigs.

Pneumocystis carinii causes a distinctive interstitial pneumonia in immunocompetent laboratory rats that had been attributed to "rat respiratory virus".

A prevalent and distinctive infectious interstitial pneumonia (IIP) of immunocompetent laboratory rats was suspected to be caused by a putative virus, termed rat respiratory virus, but this was never substantiated. To study this disease, 2 isolators were independently populated with rats from colonies with endemic disease, which was perpetuated by the regular addition of naive rats. After Pneumocystis was demonstrated by histopathology and polymerase chain reaction (PCR) in the lungs of rats from both isolators and an earlier bedding transmission study, the relationship between Pneumocystis and IIP was explored further by analyzing specimens from 3 contact transmission experiments, diagnostic submissions, and barrier room breeding colonies, including 1 with and 49 without IIP. Quantitative (q) PCR and immunofluorescence assay only detected Pneumocystis infection and serum antibodies in rats from experiments or colonies in which IIP was diagnosed by histopathology. In immunocompetent hosts, the Pneumocystis concentration in lungs corresponded to the severity and prevalence of IIP; seroconversion occurred when IIP developed and was followed by the concurrent clearance of Pneumocystis from lungs and resolution of disease. Experimentally infected immunodeficient RNU rats, by contrast, did not seroconvert to Pneumocystis or recover from infection. qPCR found Pneumocystis at significantly higher concentrations and much more often in lungs than in bronchial and nasal washes and failed to detect Pneumocystis in oral swabs. The sequences of a mitochondrial ribosomal large-subunit gene region for Pneumocystis from 11 distinct IIP sources were all identical to that of P. carinii. These data provide substantial evidence that P. carinii causes IIP in immunocompetent rats.

Histopathology of naturally transmitted "rat respiratory virus": progression of lesions and proposed diagnostic criteria.

Rat respiratory virus (RRV) is the working name for a novel respiratory pathogen of laboratory rats in North America, Europe, and Asia. Although the agent has not been definitively identified, evidence supports a viral etiology. Because no serologic or molecular assays for RRV are available, diagnosis depends on histopathologic evaluation of the lung. We introduced 104 Wistar Han rats, free of known pathogens and of RRV-associated lesions, into a rat production colony positive for RRV-type lesions, but free of other histologic, serologic, or microbiologic evidence of infectious disease. Lungs of 8 of the naïve rats were examined grossly and microscopically each week, weeks 0-13. Irregular gray-white lesions suggestive of interstitial pneumonia were grossly evident from weeks 6 through 13. Primary histopathologic evaluation of all lungs by one pathologist found multifocal, lymphohistiocytic interstitial pneumonia or prominent perivascular lymphoid cuffing from weeks 5 through 13. Based on results of the initial evaluation, diagnostic criteria for RRV infection (i.e., changes seen only after exposure to the RRV-positive colony) were tentatively selected and used by 2 other pathologists to classify each lung as RRV positive, RRV equivocal, or RRV negative. The secondary evaluation found 95% concordance in RRV diagnosis between pathologists, and correlated well with the initial evaluation, thus confirming the consistency of the criteria. These data show that RRV-naïve rats introduced into an RRV-endemic colony develop equivocal microscopic lesions of RRV by 5 weeks of exposure, and positive diagnostic lesions by 7 weeks. Interstitial pneumonia becomes grossly evident after 6 weeks of exposure.

Cardiac tissue Doppler in steroid users.

Anabolic steroids cause a variety of side effects, among them a slight concentric left ventricular hypertrophy. The objective of the present study was to clarify if they also induce alterations in left ventricular function. 14 male body builders with substantial intake of anabolic steroids (users) were examined by standard echocardiography and cardiac tissue Doppler imaging. They were compared to 11 steroid-free strength athletes (non-users) and 15 sedentary control subjects. Users showed an increased left ventricular muscle mass index. The ratio of peak transmitral blood flow velocities during early diastolic filling and atrial contraction did not differ between groups (users: 1.4 +/- 0.3; non-users: 1.7 +/- 0.5; controls: 1.4 +/- 0.4). In contrast an analogous tissue Doppler parameter, the ratio of myocardial velocities during early and late ventricular filling in the basal septum, was significantly lower in users (1.2 +/- 0.4) when compared to non-users (1.6 +/- 0.5) or controls (1.6 +/- 0.6). The velocity gradient during myocardial E-wave in the posterior wall showed significantly lower values in users (3.8 +/- 1.3 1/s) as compared to controls (5.8 +/- 2.5 1/s). There were no differences in systolic function. Summarizing strength athletes abusing anabolic steroids show negative alterations in diastolic function.

Are the cardiac effects of anabolic steroid abuse in strength athletes reversible?

OBJECTIVE: To investigate the reversibility of adverse cardiovascular effects after chronic abuse of anabolic androgenic steroids (AAS) in athletes. METHODS: Doppler echocardiography and cycle ergometry including measurements of blood pressure at rest and during exercise were undertaken in 32 bodybuilders or powerlifters, including 15 athletes who had not been taking AAS for at least 12 months (ex-users) and 17 currently abusing AAS (users), as well as in 15 anabolic-free weightlifters. RESULTS: Systolic blood pressure was higher in users (mean (SD) 140 (10) mm Hg) than in ex-users (130 (5) mm Hg) (p < 0.05) or weightlifters (125 (10) mm Hg; p < 0.001). Left ventricular muscle mass related to fat-free body mass and the ratio of mean left ventricular wall thickness to internal diameter were not significantly higher in users (3.32 (0.48) g/kg and 42.1 (4.4)%) than in ex-users (3.16 (0.53) g/kg and 40.3 (3.8)%), but were lower in weightlifters (2.43 (0.26) g/kg and 36.5 (4.0)%; p < 0.001). Left ventricular wall thickness related to fat-free body mass was also lower in weightlifters, but did not differ between users and ex-users. Left ventricular wall thickness was correlated with a point score estimating AAS abuse in users (r = 0.49, p < 0.05). In all groups, systolic left ventricular function was within the normal range. The maximum late transmitral Doppler flow velocity (Amax) was higher in users (61 (12) cm/s) and ex-users (60 (12) cm/s) than in weightlifters (50 (9) cm/s; p < 0.05 and p = 0.054). CONCLUSIONS: Several years after discontinuation of anabolic steroid abuse, strength athletes still show a slight concentric left ventricular hypertrophy in comparison with AAS-free strength athletes.

Histochemical and ultrastructural characterization of primary cardiac chondrosarcoma.

A cardiac chondrosarcoma was found in the right atrium of a Golden Retriever dog. Macroscopically, the right atrial lumen was filled with a 6- x 12- x 8-mm white glossy mass, which was diffusely attached to the underlying myocardium. The mass was composed of spindle-shaped mesenchymal neoplastic cells loosely packed in light basophilic matrix, with focal areas of tightly packed cells in linear formation similar to the pattern of a growth plate. Tumor cells were positive when stained for vimentin and neuron-specific enolase, and weakly positive for S-100 protein. Ultrastructurally, neoplastic cells and abundant, dilate rough endoplasmic reticulum. Golgi apparatus, bundles of intermediate fibers, and primitive intercellular junctions between adjacent tumor cells. Based on morphologic, ultrastructural, histochemical, and immunohistochemical characteristics, this tumor was diagnosed as a chondrosarcoma.

Structural and functional roles of cytoskeletal proteins during repair of native guinea pig intestinal epithelium.

The cytoskeletal events that assist restitution of the native intestinal epithelium are poorly understood. To enhance our understanding of repair mechanisms in the native intestinal epithelium we assessed the functional role of actin and the temporal and spatial alterations in actin and villin that occur in native enterocytes migrating in response to injury. Using a well-characterized in vitro Ussing chamber model of native intestine epithelial restitution, the actin inhibitor cytochalasin D (CD) was applied to determine the functional importance of actin to restitution as assessed by sensitive electrophysiological means and structural techniques. Additionally we used phalloidin and indirect immunohistochemistry to localize and semiquantitate F-actin and villin in migrating cells during restitution. We report new data that shows that when cytoskeletal changes were impaired with CD, the epithelial monolayer was re-established in fewer than 20% of CD-treated villi, cells bordering the epithelial defect did not assume the characteristic phenotype associated with migrating cells, and transepithelial resistance did not return to pre-injury levels. F-actin and villin were present at the leading edge of the migrating cells, basolateral F-actin was decreased, and cytoplasmic villin was increased as determined by phalloidin and immunohistochemical methods. We conclude that in vitro repair of the native intestinal epithelium is functionally and structurally dependent on major changes in the cytoskeleton of cells involved in re-establishing the epithelial monolayer over a complex extracellular matrix.

Use of a lectin as an enterocyte-specific cell surface marker for flow cytometric analysis of isolated native small intestinal epithelial cells.

Little use has been made of flow cytometry in evaluating small intestinal epithelial cells. Obtaining pure epithelial cell populations devoid of peripheral blood contaminants and intraepithelial lymphocytes contributes to the difficulties encountered in flow cytometry studies. We have investigated the use of lectins as enterocyte specific cell markers using lectin histochemistry, and have identified one lectin, UEA-1, which binds exclusively and specifically to intestinal epithelial cell brush border. Additionally, we have exploited that specificity using flow cytometry and FITC-UEA-1 to identify and separate native intestinal epithelial cells from a mixed cell population isolated by mechanical vibration. This fluorescent-lectin technique is a unique and simple method to identify native small intestinal epithelial cells in a mixed cell population; it may be exploited by flow cytometric sorting of a pure population for biochemical study or as an enterocyte specific label for surface receptor flow cytometric studies in the research or clinical setting.

Fate of polarized membrane components and evidence for microvillus disassembly on migrating enterocytes during repair of native intestinal epithelium.

BACKGROUND: Maintenance of enterocyte polarity is crucial to normal intestinal function. Tight junctions and cell-matrix interactions play a role in maintaining polarized cell membrane domains. In many intestinal epithelial wounds, normal cell-cell associations mediated by tight junctions are lost. The goal of this study was to examine the fate of an apically restricted fucosylated glycoconjugate (FGC) and basolaterally restricted Na/K-ATPase in a model of intestinal epithelial repair. Comparisons are made to isolated enterocytes in which cell-matrix interactions are disrupted as well. EXPERIMENTAL DESIGN: We present a novel physiologically relevant model of intestinal epithelial injury and restitution that was used to examine the fate of two polarized membrane components by fluorescent and ultrastructural techniques. In addition, we used mechanical vibration to isolate enterocytes as individual and short sheets of cells from the intestinal basement membrane and evaluated the fate of these restricted membrane components using immuno- and lectin histochemistry. RESULTS: Na/K-ATPase maintained its basolateral membrane location in restituting epithelial but relocated to a nonbasolateral position in the majority of isolated enterocytes. The FGC maintained its apical restriction in isolated enterocytes and in epithelial cells migrating across denuded basement membrane. An additional and important observation noted in this study was a drastic alteration in shape of migrating epithelial cells characterized by diminution and loss of microvilli as the cells migrated across the injury. CONCLUSIONS: We conclude from our results that maintenance of Na/K-ATPase to a basolateral membrane position is influenced by cell-matrix interactions. In contrast, restriction of FGC to the apical membrane of enterocytes is dependent on the presence of microvilli and is not related to either cell-cell or cell-matrix interactions. Additionally, we suggest a new model of intestinal repair in which microvilli are disassembled. We speculate that membrane from disassembled microvilli, as well as lateral cell membrane, are used at the leading edge of the migrating cell.