Two-dimensional electrophoresis of liver proteins: characterization of a drug-induced hepatomegaly in rats.

Two-dimensional electrophoresis (2-DE) of liver proteins was applied to further characterize an unusual drug-induced increase in hepatocellular rough endoplasmic reticulum (RER) in Sprague-Dawley rats given a substituted pyrimidine derivative. Absolute liver weights of drug-treated rats (9.9 +/- 0.4 g) increased above vehicle-treated controls (7.2 +/- 0.2 g) by 37%. Light microscopy revealed diffuse granular basophilia of the hepatocellular cytoplasm, uncharacteristic of hepatocytes and suggested cells rich in ribosomes, which was confirmed by electron microscopy. Immunostaining for cell proliferation, viz., 5-bromo-2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA), indicated marked hepatocellular proliferative activity. 2-DE of solubilized liver using an ISO-DALT gel system indicated significant (p<0.001) quantitative changes in at least 17 liver proteins (12 increased, 5 decreased) compared to controls. The protein with the largest increase was homologous to acute-phase reactant, contrapsin-like protein inhibitor-6. Other markedly upregulated proteins were methionine adenosyltransferase, a catalyst in methionine/ATP metabolism and mitochondrial HMG-CoA synthase, involved in cholesterol synthesis. The complementary strategies of 2-DE coupled either with database spot mapping or protein isolation and amino acid sequencing successfully identified a subset of proteins from xenobiotic-damaged rodent livers, the expression of which differed from controls. However, the current bioinformatics platform for rodent hepatic proteins and limited knowledge of specific protein functionality restricted application of this proteomics profile to further define a mechanistic basis for this unusual hepatotoxicity.

Intralesional cytokines in chronic oxazolone-induced contact sensitivity suggest roles for tumor necrosis factor alpha and interleukin-4.

An analysis was conducted of the cytokine profile and inflammatory response in oxazolone sensitized mouse skin. Following exposure to oxazolone, the intralesional production of inflammatory cytokines was demonstrable at the levels of both mRNA and protein. An initial challenge led to a transient increase in tumor necrosis factor-alpha production followed predominately by the T helper (Th)1 cytokine, interferon-gamma. There was a minimal production of interleukin-4, a Th2 cytokine. Continued exposure to oxazolone led to a downregulation of interferon-gamma and an upregulation of interleukin-4 production. A strong relationship was found between interleukin-4 and the inflammatory response, as measured by ear thickness. Similar experiments conducted in mast cell-deficient mice revealed reduced neutrophil influx but only minor changes in cytokine profile. An irritant response induced by chronic exposure of mouse skin to phorbol ester did not reveal any significant interferon-gamma or interleukin-4 response but was characterized by a tumor necrosis factor-alpha response that correlated with the inflammatory response. These observations suggest that the major source of interferon-gamma and interleukin-4 in the oxazolone response may be the infiltrating lymphocytes; whereas the tumor necrosis factor-alpha may result from the local irritation seen with both oxazolone and phorbol ester. At the end of 4 wk of chronic exposure to oxazolone, it was found that serum IgE levels had significantly increased. Histologic analysis of the skin lesion revealed that a mixed infiltrate including eosinophils developed upon repeat exposure to oxazolone. These findings are consistent with an early predominate Th1 response that is reduced and largely replaced with a Th2 response upon chronic T cell activation.

Immunohistochemical evaluation of chemically induced rhabdomyosarcomas in rats: diagnostic utility of MyoD1.

Monoclonal antibodies (mAbs) to selected muscle proteins were assessed as potential immunohistochemical markers to assist in the definitive diagnosis of poorly differentiated soft tissue sarcomas in rats. A series of 7 rat rhabdomyosarcomas (RMS) induced with nickel subsulfide were studied by light microscopy and were evaluated for immunoreactivity to desmin, vimentin, fast (type II isoform) skeletal myosin, alpha-actin (smooth muscle isoform), or MyoD1 (myogenic regulatory protein) mAbs using an avidin-biotin-chromogen technique. Consecutive RMS slices were fixed in 10% neutral buffered formalin (the fixative routinely used in carcinogenicity bioassays) for periods of 3 days or 2 mo prior to paraffin embedding to determine the effect of fixation time on immunoreactivity. Desmin and vimentin mAbs bound to many cells of all tumors, but fixation for 2 mo resulted in irretrievable loss of desmin and vimentin binding. Fast myosin and alpha-actin mAbs bound to many cells in 1 RMS but to < 1% of the cells in the remainder. MyoD1 mAb bound to tumor cell nuclei in 5/7 RMS with no loss of staining in tissue fixed for 2 mo. Results indicate that MyoD1 immunostaining, in contrast to desmin, maintains its sensitivity following prolonged formalin fixation and may be of value to distinguish RMS from other soft tissue sarcomas in the rat.

Persistent airway eosinophilia after leukotriene (LT) D4 administration in the guinea pig: modulation by the LTD4 receptor antagonist, pranlukast, or an interleukin-5 monoclonal antibody.

Aerosolized cysteinyl leukotrienes (CysLTs) elicit migration of eosinophils into guinea pig lungs and the airways of patients with asthma. The present studies were designed to analyze the concentration-response relationship, time course, and pharmacologic and histologic characteristics of leukotriene D4 (LTD4)-induced eosinophil influx into the airways of conscious guinea pigs. Animals were exposed to aerosols of 0.3 to 30 microg/ml LTD4 for 1 min, during which specific airway conductance (sGaw) was monitored. Bronchoalveolar lavages (BALs) of guinea pig airways were conducted at selected times from 4 h to 4 wk after LTD4 challenge. LTD4 produced maximal decreases in sGaw (70 to 90% reduction) at all concentrations tested and concentration-related increases in eosinophil levels in BALs, assessed 24 h after challenge. Increased numbers of eosinophils in the bronchial epithelium and subepithelium were confirmed histologically. Significant eosinophilia was maintained for up to 4 wk postchallenge. Pretreatment with the LTD4 receptor antagonist, pranlukast (ONO-1078, SB 205312) (20 mg/kg, intragastrically), significantly inhibited both the bronchoconstriction and the eosinophilia at 24 h, whereas the cyclooxygenase inhibitor, meclofenamic acid (5 mg/kg, intragastrically), had no effect on either parameter. Histologic observations were consistent with BAL results. Pretreatment with the rat anti-mouse antibody to interleukin-5 (IL-5), TRFK-5 (10-300 microg, intraperitoneally), produced dose-related inhibition of LTD4-induced eosinophilia, measured in 24 h or 3 wk BAL, but did not affect the acute bronchoconstriction. These results indicate that LTD4 elicits airway eosinophil influx in guinea pigs which persists as long as 4 wk after a single exposure, and provide the first evidence that IL-5 may have a role in LTD4-induced airways inflammation. This and other previously reported proinflammatory effects of LTD4 may contribute significantly to its overall influential role in the pathophysiology of asthma, and may underlie the therapeutic benefit of CysLT receptor antagonists, such as pranlukast, in this disorder.

Morphology and incidence of hepatic foci of cellular alteration in Sprague-Dawley rats.

Morphology and incidence of altered hepatocellular foci (AHF) were evaluated in standard H&E-stained liver sections from 3 groups of control Sprague-Dawley (SD) rats (initially 70/sex/group) used in 2 2-yr carcinogenicity studies. All AHF observed could be classified as basophilic, eosinophilic, clear, vacuolated, or mixed types using criteria applied previously to the Fischer-344 (F-344) rat. Some eosinophilic foci were large and posed a diagnostic challenge in differentiation from hepatocellular adenoma. Rats were arbitrarily divided into 3 classes by age at death: 14-17, 18-23, and 24-26 mo. As reported for F-344 rats, the incidence of SD rats with AHF increased with age, and males with eosinophilic foci predominated over males with basophilic foci whereas the opposite held true for females. Mean incidence of rats with AHF at 18-23 mo was 28 and 38% for males and females, respectively, and at 24-26 months was 68 and 71%, respectively. These data indicate a strikingly lower incidence of spontaneous AHF in SD rats than that reported for F-344 rats.

Inhibition of antigen-induced bronchoconstriction and eosinophil infiltration in the guinea pig by the cyclic AMP-specific phosphodiesterase inhibitor, rolipram.

Selective inhibition of the low Km cyclic AMP-specific phosphodiesterase has been shown to inhibit inflammatory cell function and relax airway smooth muscle. These studies were conducted to characterize the bronchodilator and anti-inflammatory activity of rolipram, an archetypical cyclic AMP-specific phosphodiesterase inhibitor, in in vitro and in vivo guinea pig airway models. In isolated tracheal rings from ovalbumin (OA)-sensitive guinea pigs, both R- and S-enantiomers of rolipram (1 microM) significantly antagonized OA-induced contractions. In contrast, neither enantiomer at concentrations up to 1 microM significantly inhibited histamine- or LTD4-induced contractions. In superfusion and mediator release experiments, both enantiomers of rolipram significantly reduced antigen-induced prostaglandin D2 release, but had minimal effect on histamine release. In anesthetized, ventilated OA-sensitive guinea pigs, racemic rolipram or enantiomers reduced OA-induced bronchoconstriction with ID50 values of approximately 0.25 mg/kg i.v. Histamine- and leukotriene D4-induced bronchoconstriction were not affected by doses of rolipram which abolished the response to OA. Higher doses (3-10 mg/kg) reduced histamine-, but not the leukotriene D4-induced bronchoconstriction. In conscious OA-sensitive guinea pigs, intragastric pretreatment with rolipram dose-dependently reduced both the OA-induced decreases in specific conductance as well as the corresponding pulmonary eosinophil influx as assessed by both bronchoalveolar lavage and histological evaluation. Therefore, rolipram produces significant inhibition of antigen-induced bronchoconstrictor and inflammatory responses, thus providing strong evidence that this pharmacological approach may be of significant therapeutic value in allergic asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist-related differences in the relationship between cAMP content and protein kinase activity in canine trachealis.

We investigated the relationships between relaxation, cyclic AMP (cAMP) accumulation and cAMP-dependent protein kinase (cAMP-PK) activity in canine tracheal smooth muscle. In time course and concentration-response studies, forskolin and isoproterenol elicited relaxation of isolated trachealis strips that was accompanied by an increase in cAMP content and an activation of cAMP-PK. Although these results were consistent with the proposal that cAMP is a second messenger mediating relaxation of airway smooth muscle, close inspection of the data revealed a discrepancy in the relationship between cAMP accumulation and relaxation. To induce equivalent degrees of tracheal relaxation, forskolin generated greater increments in cAMP accumulation than did isoproterenol. On the other hand, the activation state of cAMP-PK correlated reasonably well with relaxation regardless of which agonist was used. Further analysis of the data revealed that the apparent disparity between cAMP accumulation and relaxation could largely be explained at the level of the relationship between cAMP content and cAMP-PK activity: compared to isoproterenol, forskolin induced greater increases in cAMP accumulation to achieve the same activation state of cAMP-PK. These observations lend support to the proposal that in canine trachealis, various components of the cAMP/cAMP-PK cascade exist in distinct subcellular compartments such that not all of the cAMP generated in response to forskolin has access to its target enzyme, cAMP-PK.

Distribution of fibre types and fibre sizes in the tibialis cranialis muscle of beagle dogs.

The percentages of Type I muscle fibres were measured systematically in ATPase-stained, transverse cryostat sections of whole tibialis cranialis muscles from 8 young, adult beagles. The distance of the section from the origin of the muscle does not significantly affect the mean percentage. There are no identifiable differences in mean percentages between right and left muscles. Differences in mean percentages between individuals are significant when sexes are combined (P less than 0.01) and within sexes (males: P less than 0.01; females: P less than 0.05). Within sections, the percentage tends to be lowest at the superficial (craniolateral) border and to vary less from site to site deeper within the muscle. Fibre cross sectional areas were measured systematically in the same sections of the right muscle from 3 males and 3 females. Mean areas for each section were greater for Type II than for Type I fibres. Mean areas for each fibre-type varied moderately and non-systematically between the sample sites within sections. A needle biopsy taken from deep within this muscle should provide a more consistent and reliable estimate of fibre-type proportion in the whole muscle than a superficial specimen. Proportions are not affected by the distance of the sample site from the muscle origin, and left or right muscles are suitable for sequential samples.

Myocardial pathology of domestic ruminants in southern Africa.

Myocardial pathology of ruminants in southern Africa, including lesions associated with toxic plants, other toxic agents, infectious agents and nutritional deficiency, is discussed with regard to recognition and to aetiological diagnosis. Findings are included which have not been published elsewhere. The importance and difficulties in recognition of myocardial lesions at an early stage are emphasized. Further research into the pathology of cardiac failure caused by toxic plants is clearly needed.

A mycobacteriosis in a sheep resembling paratuberculosis (Johne's disease).

In a sheep which was euthanased because of severe emaciation and weakness, slight thickening of the ileum was seen grossly. Microscopically there was a granulomatous ileitis with obliterative lymphangitis and lymphangiectasis. Granulomatous lesions were also present in the liver and some mesenteric lymph nodes. Large numbers of acid-fast bacilli were present within epithelioid macrophages in the lamina propria of the ileum. Although the identity of the Mycobacterium spp. involved was not established, the possibility of paratuberculosis is discussed. The apparent rarity of this disease in sheep in South Africa is considered. Particular attention is drawn to the absence of diarrhoea in this case, to the slightness of the gross changes and to the importance of submitting material for mycobacterial culture.

Cerebral mycosis in a dog caused by Cladosporium trichoides Emmons 1952.

The fungus, Cladosporium trichoides, was isolated and cultured from a lesion in the cerebellum and from smaller lesions in the liver, kidney and spleen of a dog which had a history of behavioural changes, ataxia and collapse. Histopathological examination showed the cerebellar lesion to be a purulant granuloma which contained brown, septate hypae and structures resembling conidia. The source of infection was not traced and no predisposing factors were apparent. As far as is known, this is the first record of the condition in animals in southern Africa and the first report anywhere of this condition in the dog.