Upregulation of cyclooxygenase-2 expression in porcine macula densa with chronic nitric oxide synthase inhibition.

The objective of this study was to investigate the effects of chronic inhibition of nitric oxide synthase (NOS) on cyclooxygenase-2 (COX-2) expression in the macula densa (MD) of swine, as well as the effects on expression of related proteins. Adult female Yucatan swine were given either tap water (control, n = 6) or water with N (G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/liter, n = 5) for a minimum of 30 days. Duplicate samples of kidney were fixed or snap frozen. There was a significant (P = .0082) upregulation of COX-2 mRNA expression in the MD of L-NAME, as well as an apparent increase in COX-2 protein. Plasma renin activity also increased with L-NAME treatment (control, 0.34 ± 0.08 ng/ml; L-NAME, 1.26 ± 0.03 ng/ml; P = .00000003). There were no differences between groups in expression of either inducible NOS or renin protein or in serum electrolyte concentrations. In conclusion, with chronic inhibition of NOS, COX-2 in MD is upregulated, perhaps to compensate for loss of nitric oxide. Increases in COX-2 products may counteract renal arteriolar constriction and sustain renin release.

Reduced incidence and severity of experimental autoimmune arthritis in mice expressing catalytically inactive A disintegrin and metalloproteinase 8 (ADAM8).

A disintegrin and metalloproteinase 8 (ADAM8), a catalytically active member of the ADAMs family of enzymes, is expressed primarily on immune cells and thus probably involved in inflammatory responses. ADAM8 is also produced by chondrocytes, and recombinant ADAM8 can induce cartilage catabolism. We therefore decided to test the role of ADAM8 in autoimmune inflammatory arthritis using transgenic mice expressing catalytically inactive ADAM8. Transgenic DBA/1J mice expressing an inactivating point mutation in the ADAM8 gene to change Glu330 to Gln330 (ADAM8(EQ)) were generated to evaluate the proteolytic function of ADAM8 in an lipopolysaccharide-synchronized collagen-induced arthritis (LPS-CIA) model of autoimmune arthritis. The systemic inflammatory reaction to LPS was also evaluated in these mice. Expression profiling of paw joints from wild-type mice revealed that ADAM8 mRNA levels increased at the onset of clinical arthritis and correlated well with cellular macrophage markers. When subjected to LPS-CIA, ADAM8(EQ) mice demonstrated decreased incidence and severity of clinical arthritis compared to wild-type mice. Histological examination of paw joints from ADAM8(EQ) mice confirmed marked attenuation of synovial inflammation, cartilage degradation and bone resorption when compared to wild-type mice. However, transgenic mice and wild-type mice responded similarly to LPS-induced systemic inflammation with regard to mortality, organ weights, neutrophil sequestration and serum cytokine/chemokine production. We conclude that ADAM8 proteolytic activity plays a key role in the development of experimental arthritis and may thus be an attractive target for the treatment of arthritic disorders while minimizing risk of immunocompromise.

Xenotransplantation of cryopreserved equine squamous cell carcinoma to athymic nude and SCID mice.

Cryopreserved equine ocular squamous cell carcinoma (SCC) was inoculated subcutaneously into 15 athymic nude and 15 SCID mice. Xenotransplantation resulted in tumor growth in two athymic nude mice and 1 SCID mouse. Histological appearance and immunohistochemical characterization using cytokeratin 5/6 markers and p53 markers of the tumor grown in mice was in full accord with the original equine tumors. No evidence of metastasis was noted in any mouse. This model may serve as a relevant in vivo model for studying the biology of equine ocular SCC and for the testing of new therapeutic modalities.

Thyroid status and nitric oxide in rat arterial vessels.

Thyroid disease has profound effects on cardiovascular function. Hypo- and hyperthyroidism, for example, are associated with reduced and increased maximal endothelium-dependent vasodilation respectively. We therefore hypothesized that the capacity for vascular nitric oxide (NO) formation is decreased in hypothyroidism and increased in hyperthyroidism. To test this hypothesis, rats were made hypothyroid (HYPO) with propylthiouracil or hyperthyroid (HYPER) with triiodothyronine over 3-4 months. Compared with euthyroid control rats (EUT), HYPO exhibited blunted growth and lower citrate synthase activity in the soleus muscle; HYPER exhibited left ventricular hypertrophy and higher citrate synthase activity in the soleus muscle (P<0.05 for all effects). The capacity for NO formation was determined in aortic extracts by formation of [3H]L-citrulline from [3H]L-arginine, i.e. NO synthase (NOS) activity. Thyroid status modulated NOS activity (EUT, 36.8 +/- 5.5 fmol/h per mg protein; HYPO, 26.0 +/- 7.9; HYPER, 64.6 +/- 12.7; P<0.05, HYPER vs HYPO). Expression of endothelial and neural isoforms of NOS was modulated by thyroid status in a parallel fashion. Capacity for responding to NO was also determined via measuring cGMP concentration in aortae incubated with sodium nitroprusside. Stimulated cGMP formation was also modulated by thyroid status (EUT, 73.0 +/- 20.2 pmol/mg protein; HYPO, 152.4 +/- 48.7; HYPER, 10.4 +/- 2.6; P<0.05, HYPER vs HYPO). These data indicate that thyroid status alters capacities for both formation of and responding to NO. The former finding may contribute to previous findings concerning vascular function in thyroid disease states.

Isoform-specific modulation of coronary artery PKC by glucocorticoids.

Glucocorticoids (GC) exert diverse cellular effects in response to both acute and chronic stress, the functional consequences of which have been implicated in the development of cardiovascular pathology such as hypertension and atherosclerosis. However, the mechanisms by which GCs activate divergent signaling pathways are poorly understood. The present study examined the direct effects of natural (cortisol) and synthetic (dexamethasone) GCs on protein kinase C (PKC) isoform expression in coronary arteries. Porcine right coronary arteries were treated in vitro for 18 h in the presence and absence of either dexamethasone (10, 100, or 500 nM) or cortisol (50, 125, 250, or 500 nM). PKC isoform levels and subcellular distribution were determined by immmunoblotting of whole cell homogenates and immunocytofluorescence using PKC-alpha, -betaII, -epsilon, -delta, and -zeta specific antibodies. Dexamethasone caused a approximately 4-fold increase in PKC-alpha, a approximately 2.5-fold increase in PKC-betaII, and a 2-fold increase in PKC-epsilon (p<0.05). In contrast, dexamethasone had no effect on PKC-delta or PKC- zeta levels. Dexamethasone also caused an increase in the activity of PKC-alpha (285%), -betaII (170%), and -epsilon (210%). Cortisol produced similar effects on PKC isoform expression. Confocal microscopy revealed that while dexamethasone altered localization patterns for PKC-alpha, -betaII and -epsilon, no such effect was observed for PKC-delta or PKC-zeta. The stimulatory effects of dexamethasone and cortisol on coronary PKC levels and translocation were prevented by the GC receptor (GR) blocker, RU486. These results demonstrate, for the first time, that GCs modulate coronary PKC expression and subcellular distribution in an isoform-specific manner through a GR-dependent mechanism.

Hypercholesterolemia inhibits L-type calcium current in coronary macro-, not microcirculation.

Hypercholesterolemia (HC) is a mary risk factor for the development of coronary heart disease. Coronary ion regulation, especially calcium, is thought to be important in coronary heart disease development; however, the influence of high dietary fat and cholesterol on coronary arterial smooth muscle (CASM) ion channels is unknown. The purpose of this study was to determine the effect of diet-induced HC on CASM voltage-gated calcium current (I(Ca)). Male miniature swine were fed a high-fat, high-cholesterol diet (40% kcal fat, 2% wt cholesterol) for 20-24 wk, resulting in elevated serum total and low-density lipoprotein cholesterol. Histochemistry indicated early atherosclerosis in large coronary arteries. CASM were isolated from the right coronary artery (>1.0 mm ID), small arteries ( approximately 200 microm), and large arterioles ( approximately 100 microm). I(Ca) was determined by whole cell voltage clamp. L-type I(Ca) was reduced approximately 30% by HC compared with controls in the right coronary artery (-5.29 +/- 0.42 vs. -7.59 +/- 0.41 pA/pF) but not the microcirculation (small artery, -8.39 +/- 0.80 vs. -10.13 +/- 0.60; arterioles, -10.78 +/- 0.93 vs. -11.31 +/- 0.95 pA/pF). Voltage-dependent activation was unaffected by HC in both the macro- and microcirculation. L-type voltage-gated calcium channel (Ca(v)1.2) mRNA and membrane protein levels were unaffected by HC. Inhibition of I(Ca) by HC was reversed in vitro by the cholesterol scavenger methyl-beta-cyclodextrin and mimicked in control CASM by incubation with the cholesterol donor cholesterol:methyl-beta-cyclodextrin. These data indicate that CASM L-type I(Ca) is decreased in large coronary arteries in early stages of atherosclerosis, whereas I(Ca) in the microcirculation is unaffected. The inhibition of calcium channel activity in CASM of large coronary arteries is likely due to increases in membrane free cholesterol.

Short-term exercise training increases ACh-induced relaxation and eNOS protein in porcine pulmonary arteries.

We tested the hypothesis that short-term exercise (STEx) training and the associated increase in pulmonary blood flow during bouts of exercise cause enhanced endothelium-dependent vasorelaxation in porcine pulmonary arteries and increased expression of endothelial cell nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) protein. Mature, female Yucatan miniature swine exercised 1 h twice daily on a motorized treadmill for 1 wk (STEx group, n = 7); control pigs (Sed, n = 6) were kept in pens. Pulmonary arteries were isolated from the left caudal lung lobe, and vasomotor responses were determined in vitro. Arterial tissue from the distal portion of this pulmonary artery was processed for immunoblot analysis. Maximal endothelium-dependent (ACh-stimulated) relaxation was greater in STEx (71 +/- 5%) than in Sed (44 +/- 6%) arteries (P < 0.05), and endothelium-independent (sodium nitroprusside-mediated) responses did not differ. Sensitivity to ACh was not altered by STEx training. Immunoblot analysis indicated a 3.9-fold increase in eNOS protein in pulmonary artery tissue from STEx pigs (P < 0.05) with no change in SOD-1 or glyceraldehyde-3-phosphate dehydrogenase protein levels. We conclude that STEx training enhances ACh-stimulated vasorelaxation in pulmonary arterial tissue and that this adaptation is associated with increased expression of eNOS protein.

Toxicity of moniliformin from Fusarium fujikuroi culture material to growing barrows.

In two studies, the effects of moniliformin (M)-contaminated diets from Fusarium fujikuroi culture material on growing barrows were evaluated. In the first study, six barrows (three replicates of two each, mean body weight = 17.8 kg) per group (four groups; 24 barrows total) were fed diets calculated to contain 0 mg M/kg feed (control); 25 mg M/kg feed; 50 mg M/kg feed; or 100 mg M/kg feed for 28 days. In the second study, the same experimental design and numbers of barrows (mean body weight = 15.3 kg) were used, and diets were formulated to contain 0 mg M/kg feed (control); 50 mg M/kg feed; 100 mg M/kg feed; or 200 mg M/kg feed. Diets of 100 mg or 200 mg M/kg feed reduced body weight, body weight gain, and feed consumption. Serum biochemical analytes were affected by 100 to 200 mg M/kg feed. Hematologic values were affected by 50, 100, and 200 mg M/kg feed. In the first study, one barrow in the 100 mg M-treated group died, and in the second study. one barrow died in the 100 mg M-treated group, and five barrows died in the 200 mg M-treated group. Relative heart weight was increased in the 200 mg M-treated barrows, yet tissues from organs collected from treatment groups were generally histologically unimpressive. The most consistent sign of M toxicity in barrows appeared to be death induced within 2 to 5 days by 100 to 200 mg M/kg feed.

Chronic toxicity of fumonisin in weanling pigs.

Three gilts fed a diet containing 100 mg fumonisin B1/kg for 7 days followed by a diet containing 190 mg/kg for 83 days developed nodular hyperplasia of the liver. These nodules of various diameters were composed of solid sheets or nests of hepatocytes. There were no discernible central veins or portal triads, and the perilobular connective tissue and adjacent parenchyma were compressed. Three other gilts maintained on the same diet for 27-80 days developed severe hepatopathies, but not nodular hyperplasia, necessitating euthanasia prior to conclusion of the feeding trial. At necropsy, 1 of the 6 gilts had grossly apparent hyperplastic plaques within the distal esophageal mucosa. On histopathologic examination, 6 of 6 gilts had mild to severe hyperkeratosis, parakeratosis, and formation of papillary downgrowths of the stratum basale of the distal esophageal mucosa. The hyperplastic nodules in the liver and the changes in the distal esophageal mucosa illustrate the unique chronic toxicity of this mycotoxin in pigs.

Foxtail-induced ulcerative stomatitis outbreak in a Missouri stable.

Twenty of 25 horses in a well-managed Missouri boarding stable were diagnosed with gingivitis/stomatitis. Gross examination of the affected horses revealed varying degrees of gingivitis ranging from mild periodontal swelling to marked swelling and erythema with ulceration and hemorrhage. Fine hair-like material was embedded within the intensely affected areas. Gingival biopsies from 4 affected horses contained pyogranulomatous inflammation with, in some cases, numerous eosinophils and several grass awns in cross and longitudinal section. Numerous foxtail seed heads were identified in hay samples. Examination of the records revealed that all of the affected horses had been fed the suspect hay, with the exception of 1 horse. Although not deliberately fed the suspect hay, this horse did have access to the hay when turned out into the exercise paddock. The lesions resolved following a change in hay source.

Chlamydial infection in aborted and stillborn lambs.

Chlamydia psittaci is a major cause of ovine abortion in the fourth to fifth months of gestation. During the lambing seasons of 1986, 1987, and 1988, fetuses from 52 cases of ovine abortion, stillbirth, or perinatal death were submitted to the laboratory for necropsy examination. Placenta or fetal tissues from 34 cases were cultured on mouse L cells for C. psittaci. Chlamydia psittaci was identified by immunofluorescence on cultures in 20 of these cases. The major gross lesion consistently associated with chlamydial abortion was placentitis with multifocal cotyledonary necrosis and accumulation of red-brown exudate in the intercotyledonary placenta. Chlamydiae appeared as spherical organisms, less than 1 micron in diameter, in the cytoplasm of trophoblasts in impression smears of cotyledons. Histologically, placentitis was sometimes accompanied by pneumonia or encephalitis in the fetus. Chlamydia psittaci was considered the cause for fetal death when chlamydial isolation was associated with placentitis or inflammation of other fetal tissues and when other abortifacient agents were not detected.

Antimicrobial susceptibility and serotypes of Actinobacillus (Haemophilus) pleuropneumoniae recovered from Missouri swine.

The antimicrobial susceptibility of 73 Actinobacillus (Haemophilus) pleuropneumoniae isolates from swine in Missouri was determined with a microdilution minimal inhibitory concentration test system. Serotyping was accomplished by means of co-agglutination. Serotype 1 (39/73) and serotype 5 (30/73) were commonly found, whereas serotype 7 (4/73) was infrequently encountered. Most isolates (MIC90) were found susceptible to ampicillin (amoxicillin), cephalothin, penicillin, erythromycin, gentamicin, and kanamycin. Marked resistance was found with oxytetracycline, tylosin, and sulfadimethoxine. The data indicate that use of ampicillin (amoxicillin) or penicillin may correlate well with the favorable outcome of treatment.

Immunoreactivity of A103, an antibody to Melan A, in canine steroid-producing tissues and their tumors.

The monoclonal antibody A103 to the melanocytic differentiation antigen Melan A stains human steroid-producing cells and their tumors. A total of 200 formalin-fixed, paraffin-embedded canine normal tissues and hyperplastic and neoplastic lesions of the adrenal gland, testis, and ovary were immunohistochemically tested for Melan A with antibody A103. Leydig cell tumors (23/23, 100%), Sertoli cell tumors (14/15, 93%), and adrenocortical adenomas (12/13, 92%) were consistently positive. Adrenocortical carcinomas (23/35, 65%) and granulosa cell tumors (10/17, 59%) were less frequently positive. All pheochromocytomas, seminomas, and dysgerminomas were negative. The pattern of staining was cytoplasmic, but nuclear staining was also frequently seen in normal Leydig cells and their tumors. As in human tumors, immunohistochemistry for Melan A stains many canine steroid-producing tumors and can be used to distinguish these tumors from those of nonstereidogenic cells.

Intestinal adenocarcinoma in a chicken.

An obstructing intestinal stricture 65 cm distal to the pylorus in an aged hen was seen on histologic examination to be caused by a primary intestinal adenocarcinoma.

The chronic effects of Fusarium moniliforme culture material, containing known levels of fumonisin B1, in turkeys.

Fourteen 1-day-old male turkeys were randomly assigned to two adjacent floor pens and fed balanced rations containing 0 and 75 mg fumonisin B1 (FB1)/kg for 18 weeks. Inclusion of FB1 in the ration caused decreased body weight gain on weeks 4, 10, and 12 during the trial. Turkeys fed 75 mg FB1/kg had significantly heavier livers after treatment for 18 weeks. Chronic FB1 exposure resulted in an increased total white blood cell count, absolute heterophil count, absolute lymphocyte count, and heterophil-to-lymphocyte ratio. No mortality was noted in turkeys in either treatment group. Turkeys are relatively resistant to chronic FB1 exposure.

Effects on turkey poults of feeding Fusarium moniliforme M-1325 culture material grown under different environmental conditions.

The effects of feeding Fusarium moniliforme M-1325 culture material (CM), grown under different environmental conditions, were studied in turkey poults. Poults were fed a control diet or diets containing four levels of FB1 (75, 150, 225, or 300 mg/kg) prepared from F. moniliforme M-1325 cultures that produced 7800 (CM1) or 4000 mg FB1/kg (CM2). F. moniliforme M-1325 CM that produced a low concentration of FB1 (350 mg FB1/kg) was also used to prepare an additional diet containing 75 mg FB1/kg (CM3). Dose-dependent decreases in feed intake and body-weight gains and dose-dependent increases in liver weights and serum sphinganine (SA) to sphingosine (SO) ratios were observed in poults fed CM1 or CM2. Poults fed CM3 consumed more feed and had lower body-weight gains than controls or poults fed CM1 or CM2 (at 75 mg FB1/kg). Poults fed CM3 also had increased liver weights and SA:SO ratios compared with control poults. Generalized hepatocellular hyperplasia was observed in all FB1 treatment groups. Biliary hyperplasia was evident in turkeys fed 150 to 300 mg FB1/kg. Results indicate that at equivalent dietary FB1 levels, F. moniliforme cultures producing different concentrations of FB1 differ in their effects on turkey poults.

Moniliformin from Fusarium fujikuroi culture material and deoxynivalenol from naturally contaminated wheat incorporated into diets of broiler chicks.

The effects of feeding diets containing 100 mg moniliformin (M)/kg of feed from culture material and 16 mg deoxynivalenol (DON)/kg of feed from naturally contaminated wheat were evaluated in growing broiler chicks from 1 day to 21 days of age. Body weight (BW), body-weight gain, and feed consumption were decreased by feeding M and M plus DON diets. Relative heart weight was increased by the M diet, whereas relative weights of proventriculus, gizzard, and heart were increased by the M plus DON diet. The M diet increased alanine transferase and aspartate transaminase activities and creatinine concentration and decreased mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration (MCHC). The M and DON diet decreased glucose, hemoglobin, and MCHC. Histopathological lesions from the M diet were limited to the kidney and consisted of extensive renal tubular epithelial degeneration plus luminal mineralization. A moderation of the severity of lesions was seen in the tissues of the M plus DON-fed chicks, consisting of generally mild tubular epithelial degeneration. None of the parameters measured were affected by the DON diet. Results indicate additive or less-than-additive toxicity for most parameters when chicks were fed diets containing 100 mg M plus 16 mg DON/kg of feed. Although the concentration of M in this study was high compared with that reported for feedstuffs, additional information on the occurrence and toxicity of M will need to be collected in order to assess the importance of M to the poultry industry.

Growth and functional assessment of pulmonary valves in pigs after replacement of sinuses of Valsalva.

Neopulmonary artery stenosis may occur after the arterial switching procedure to correct transposition of the great arteries. One technique to reduce this complication is to use a single rectangular piece of autogenous pericardium to reconstruct two adjacent sinuses of Valsalva and maintain pulmonary artery size. The long-term effect of this technique on pulmonary artery and valve growth and function is unknown. To assess this technique, Yorkshire-cross pigs (n = 5) weighing 29 +/- 1.7 kg (mean +/- SEM) were anesthetized, and during cardiopulmonary bypass, the pulmonary artery was transected distal to the pulmonary valve. Pulmonary artery diameter and commissure distances were measured. Two adjacent pulmonary artery sinuses of Valsalva were completely excised from the anulus to 4 mm distal to the commissures, leaving 2 mm of pulmonary artery tissue attached to the skeletonized commissure and on each side of the one remaining intact sinus of Valsalva. A single rectangular patch of fresh autologous pericardium was sutured to the anulus and remnant of the pulmonary artery along the commissure and edges of the one intact sinus of Valsalva. Pericardium composed two thirds of the circumference of the proximal pulmonary artery; this was anastomosed to the distal pulmonary artery. Weight gain occurred at a rate of 0.6 kg/day (median). The animals underwent right heart catheterization and cineangiography. They were killed 157.2 +/- 12.9 days post-operatively. The reconstructed pulmonary artery grew from 17.6 +/- 0.8 mm to 30.8 +/- 1.5 mm (p < 0.01), and the commissure distances grew from 17.0 +/- 1 mm to 27.2 +/- 1.6 mm (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Naturally occurring scrapie in Southdown sheep.

The purpose of this study was to characterize naturally occurring scrapie in the Southdown breed of sheep. Experimental subjects included 4 Southdown ewes admitted to the University of Missouri, College of Veterinary Medicine Large Animal Clinic. All 4 sheep had signs compatible with clinical scrapie. Cerebrospinal fluid (CSF) cell counts ranged from a low of 1 nucleated cell/microL to high of 4 cells/microL with a median of 3 cells/microL. Cerebrospinal protein concentrations ranged from 26 to 78 mg/dL with a median of 53 mg/dL. Immunoassay of the CSF for the 14-3-3 protein yielded positive results in 3 of the 4 sheep. Sequencing of the prion protein (PrP) gene revealed that all 4 sheep were homozygous for glutamine at codon 171 and, hence, were of the QQ genotype. Histopathologic examination of brain stem tissue sections revealed intracytoplasmic neuronal vacuolation and mild spongiform changes in the gray matter neuropil in all 4 ewes. The diagnosis of scrapie was confirmed by immunohistochemical staining for the abnormal PrP Our results suggest that the genetics of scrapie susceptibility are probably similar in Suffolk and Southdown sheep. Positive immunoassay results for the 14-3-3 protein were observed in 3 of the 4 sheep.

Induction of acute bronchopneumonia in mice by intrabronchial inoculation of Pasteurella haemolytica serotype 1.

Dose dependent pulmonary lesions of acute bronchopneumonia were induced in male, outbred Swiss Webster mice by intrabronchial inoculation of Pasteurella haemolytica. Five exponential dilutions ranging from 5 x 10(4) to 5 x 10(8) colony forming units per mL (CFU/mL) of Pasteurella haemolytica serotype 1 were inoculated into five groups of mice. Mice were killed by cervical dislocation 24 hours postinoculation. Pulmonary lesions occurred in mice of all five groups, however, 5 x 10(7) CFU/mL was the minimal dose which consistently produced lesions. Focal parenchymal necrosis, suppurative bronchiolitis, and flooding of interalveolar septa and alveoli by edema fluid, fibrin, neutrophils and macrophages, were observed microscopically. We conclude that outbred Swiss Webster mice can be used as a model for the study of selected disease mechanisms of acute lung inflammation and that this model may be used to determine some of the pathogenic mechanisms involved in the development of pulmonary lesions in bovine pneumonic pasteurellosis.