Meeting report: Spontaneous lesions and diseases in wild, captive-bred, and zoo-housed nonhuman primates and in nonhuman primate species used in drug safety studies.

The combination of loss of habitat, human population encroachment, and increased demand of select nonhuman primates for biomedical research has significantly affected populations. There remains a need for knowledge and expertise in understanding background findings as related to the age, source, strain, and disease status of nonhuman primates. In particular, for safety/biomedical studies, a broader understanding and documentation of lesions would help clarify background from drug-related findings. A workshop and a minisymposium on spontaneous lesions and diseases in nonhuman primates were sponsored by the concurrent Annual Meetings of the American College of Veterinary Pathologists and the American Society for Veterinary Clinical Pathology held December 3-4, 2011, in Nashville, Tennessee. The first session had presentations from Drs Lowenstine and Montali, pathologists with extensive experience in wild and zoo populations of nonhuman primates, which was followed by presentations of 20 unique case reports of rare or newly observed spontaneous lesions in nonhuman primates (see online files for access to digital whole-slide images corresponding to each case report at http://www.scanscope.com/ACVP%20Slide%20Seminars/2011/Primate%20Pathology/view.apml). The minisymposium was composed of 5 nonhuman-primate researchers (Drs Bradley, Cline, Sasseville, Miller, Hutto) who concentrated on background and spontaneous lesions in nonhuman primates used in drug safety studies. Cynomolgus and rhesus macaques were emphasized, with some material presented on common marmosets. Congenital, acquired, inflammatory, and neoplastic changes were highlighed with a focus on clinical, macroscopic, and histopathologic findings that could confound the interpretation of drug safety studies.

Systemic proliferative changes and clinical signs in cynomolgus monkeys administered a recombinant derivative of human epidermal growth factor.

Epidermal growth factor (EGF) effects have been explored extensively in vivo in rodents, but little is known about trophic responses in nonhuman primates. A previous publication reports the hyperplastic epithelial/parenchymal changes noted in the digestive tract (tongue, esophagus, stomach, intestine, liver, gallbladder, pancreas, and salivary glands) of adult cynomolgus monkeys treated with recombinant human EGF(1-48) (rhEGF(1-48)). This report documents clinical findings and structural effects in the remaining epithelium-containing tissues of these animals. Two monkeys/sex/dose received rhEGF(1-48) by intravenous bolus at 0 (vehicle), 10, 100, 500 (females only), or 1,000 microg/kg/day (males only) daily for up to 2 weeks. Treatment- and dose-related clinical findings included emesis, fecal alterations (soft feces and diarrhea), lacrimation, nasal discharge, hypoactivity, transient hypotension, and salivation after dosing. Male monkeys administered 1,000 microg/kg became moribund after 5 days of treatment and were necropsied. All other monkeys completed the 2-week treatment period. Necropsy findings in nongastrointestinal tissues were: enlarged, pale kidneys at 100 microg/kg and greater; small thymuses seen sporadically at all doses; and enlarged adrenals and small thyroids in males at 1,000 microqg/kg. Respective organ-to-brain weight ratios at 500 and 1,000 microg/kg for kidneys were 1.5- and 2.6-fold greater and for heart were 1.7- and 1.3-fold greater than controls. Microscopically, pronounced dose-related epithelial hypertrophy and hyperplasia were evident in kidney, urinary bladder, skin (epidermis and adnexa), mammary gland, prostate, seminal vesicles, epididymis, uterus, cervix, vagina, thyroid, thymus, tonsillar crypts, cornea, trachea, and pulmonary airways. Epitheliotrophic effects were conspicuous in many tissues at 100 to 1,000 microg/kg. Changes to renal collecting ducts were present at 10 microg/kg, suggesting that kidneys were a relatively sensitive target. Proliferative alterations were not apparent in testes, intraocular structures, brain ependyma and choroid plexus at any dose. Aside from the noted exceptions, rhEGF(1-48) was a pantrophic epithelial mitogen in cynomolgus monkeys when used intravenously at suprapharmacologic doses.

Growth and development in rats given recombinant human epidermal growth factor(1-48) as neonates.

To assess effects of supraphysiologic doses of human recombinant epidermal growth factor(1-48) (rhEGF(1-48)) on neonatal rats, 10 litters of Wistar rats/treatment group were given 0 (formulated vehicle), 10, 100, or 1000 microg/kg daily by subcutaneous injection on postnatal days (PND) 1 through 6. Clinical signs, body weight, acquisition of developmental landmarks and reflexes, and behavior were monitored during treatment and for 5 weeks thereafter (to PND 42). A subset of animals was euthanized weekly from PND 7-28 and necropsied. Selected tissues were examined microscopically. Body weight gain at 1000 microg/kg during treatment was significantly less than control. Precocious incisor eruption, eye opening, vaginal opening, and preputial separation occurred at 100 and/or 1000 microg/kg. Acquisition of reflexes (negative geotaxis, wire maneuver, acoustic startle reflex, and visual placing) was delayed at 1000 microg/kg. Acquisition of adult locomotion was also delayed at 1000 microg/kg. These effects were transient, as locomotor activity at PND 28 and 42 did not differ from control. Effects on acoustic-startle responding persisted in females to final assessment on PND 42. Habituation to repeated acoustic stimuli was impaired, as well as response inhibition following a prepulse acoustic stimulus. rhEGF(1-48) induced structural changes in the skin, retina, kidney, oral and nasal mucosa, lung, and liver. Many of these changes were consistent with the expected mitogenic activity of rhEGF(1-48) and were transient in nature, as severity and incidence diminished with time. An exception was changes observed in the retina at 1000 microg/kg (rosettes/folds and focal defects in the outer nuclear/photoreceptor layers) that were still present 3 weeks after termination of treatment. Acceleration of developmental landmarks; suppression of reflexes, behavior, and somatic growth; and mitogenic responses in epidermal tissues have been reported in rodents treated with epidermal growth factor (EGF) derived from various mammalian species. These results demonstrate that a 48-amino acid fragment of human EGF produced by recombinant technology also induces such effects.

RNA expression in the early characterization of hepatotoxicants in Wistar rats by high-density DNA microarrays.

High-density microarrays are useful tools to study gene expression for the purpose of characterizing functional tissue changes in response to the action of drugs and chemicals. To test whether high-density expression data can identify mechanisms of toxicity and to identify an unknown sample through its RNA expression pattern, groups of male Wistar rats were administered 6 hepatotoxicants. The compounds chosen for this study were microcystin-LR (MLR), phenobarbital (PB), lipopolysaccharide (LPS), carbon tetrachloride (CT), thioacetamide (THA), and cyproterone acetate (CPA). These hepatotoxicants are known to induce adverse liver effects through different mechanisms. Liver mRNA was isolated and used to generate biotinylated cRNA for hybridization to a custom 1,600-rat gene DNA microarray. Treatment correlation matrices analyzed hybridization data from a hepatotoxicant-blinded sample, with gene expression coefficients (GEC) evaluated by means of hierarchical cluster analysis and visual representation as dendrograms. The experimental liver toxicity from the different treatments was confirmed by means of concurrent histopathology, liver enzymes, and bilirubin assays. This toxico genomic analysis identified multiple genes and groups of genes that were affected by the hepatotoxicants on study, indicating that high-density microarray expression data are useful to identify groups of genes involved in toxicity. In addition, the mRNA expression profile of an unidentified sample can be accurately identified when compared with the expression profiles resident in the data set. This study supports the use of gene expression-profiling technology to determine or to predict toxic liver effects.

Spontaneously occurring hepatocellular neoplasia in adolescent cynomolgus monkeys (Macaca fascicularis).

Spontaneous hepatic neoplasms were identified in two adolescent (<5 years of age) male cynomolgus monkeys (Macaca fascicularis). Monkey No. 1 had a solitary hepatocellular carcinoma (HCC). Monkey No. 2 had multiple discrete tumors consisting of several poorly circumscribed HCCs and a mixed hepatocholangiocellular carcinoma (MHC). Metastases were not evident in either monkey. Histochemical and immunohistochemical stains were used to assess phenotypic alterations in the tumors. Many or most neoplastic hepatocytes (NHs) of both monkeys stained positive for low-molecular-weight cytokeratin (LMWCK), cytokeratin (CK) 8, and CK 18. In monkey No. 1, small aggregates of NHs were positive for carcinoembryonic antigen (CEA), glutathione S-transferase-pi (GST), and alpha-fetoprotein (AFP), but NHs were uniformly negative for CK 7. NHs in monkey No. 2 were negative for CEA and AFP but were multifocally positive for GST and CK 7. Broad-spectrum cytokeratin (BSCK), high-molecular-weight cytokeratin (HMWCK), and CK 19 did not react with NHs of either animal. Neoplastic cells forming ductlike structures in the MHC of monkey No. 2 stained with LMWCK, CK 7, CK8, CK 18, BSCK, and GST but not with HMWCK or CK 19. Tumors in both monkeys had enhanced pericellular fibronectin staining. Nonneoplastic parenchyma of both monkeys contained multiple discrete foci of cellular alteration and scattered aggregates of hepatocytes with strong cytoplasmic staining for fibronectin. Staining patterns of these tumors demonstrate immunophenotypic heterogeneity of the neoplastic cells within individual tumors and variability among tumors. This information may serve as a useful reference for others encountering similar lesions in primates.

An epizootic of lymphoplasmacytic gastritis attributed to Helicobacter pylori infection in cynomolgus monkeys (Macaca fascicularis).

An epizootic of subclinical lymphoplasmacytic gastritis occurred in cynomolgus monkeys maintained at our research facility. Gastric pathology data and histologic sections of 63 adolescent monkeys (2.5-3.5 years old) sacrificed during the epizootic were reviewed. Localized to multifocal reddening of the gastric mucosa was noted grossly in 7 of 44 (16%) monkeys harboring Helicobacter pylori, but not in any of 19 monkeys in which these bacteria were not seen. Gastritis, characterized by accentuation of lymphoplasmacytic infiltrates in antral and to a lesser degree cardiac mucosa, occurred in 42 of 63 (67%) monkeys evaluated and in 42 of 44 (93%) monkeys in which H. pylori was observed microscopically. Two monkeys with H. pylori infection had infiltrate scores that overlapped with the upper limit of scores of H. pylori-negative animals. Coincident with accentuated infiltrates were gastric gland epithelial hyperplasia, reduction in mucin content of surface and gland epithelia, and comparatively minor infiltrates of neutrophils in superficial lamina propria and gastric glands. Antral mucosa thickness often exceeded 1.5 to 2 times normal. Antral mucosal erosions occurred in 7 of 44 (16%) monkeys with H. pylori. Argyrophilic bacteria morphologically consistent with H. pylori were present in antral and less commonly cardiac mucosal glands. Intensity of bacterial colonization correlated with lymphoplasmacytic infiltrates (r = 0.754) and hyperplasia (r = 0.700), although responses were quite variable. These bacteria were not detected in fundic mucosa except in instances where parietal cells were substantially depleted in glands coincident with localized increases in lamina propria inflammatory cell infiltrates. Helicobacter heilmannii-like organisms (HHLOs) were present in fundic glands of all 63 monkeys; colonization was often pronounced. Scores for fundic mucosal inflammation did not correlate with presence or intensity of colonization with HHLOs (r = 0.005). Rather, fundic inflammation scores positively correlated with the antral inflammation scores (r = 0.548). Bacteria morphologically, biochemically, and genetically consistent with H. pylori were cultured from gastric mucosal specimens confirming bacterial identification. These findings demonstrate that adolescent cynomolgus monkeys are susceptible to natural infection with H. pylori and develop many morphologic hallmarks of H. pylori-related gastritis in humans.

alpha-Substituted malonester amides: tools to define the relationship between ACAT inhibition and adrenal toxicity.

We prepared a series of alpha-substituted malonester amides that were evaluated for their ability to inhibit acyl-CoA:cholesterol O-acyl transferase activity in vitro and to lower plasma total cholesterol levels in a variety of cholesterol-fed animal models. Compounds of this series were also useful in examining the relationship between adrenal toxicity and ACAT inhibition. One compound from this series, 9f, was a potent inhibitor of ACAT in both the microsomal and cellular assays. It was also bioavailable as determined by both a bioassay and a HPLC-UV assay. This compound was evaluated in both guinea pig and dog models of adrenal toxicity and compared to tetrazole amide 15. In the most sensitive species, the dog, both of these compounds achieved good plasma levels; however, compound 9f caused adrenal necrosis, whereas compound 15 had no effect on the adrenal gland. This adds to the growing body of evidence that the adrenal toxicity observed with ACAT inhibitors may not be mechanism related.

Recombinant human epidermal growth factor1-48-induced structural changes in the digestive tract of cynomolgus monkeys (Macaca fascicularis).

To determine the cellular effects and potential toxicity of exogenously administered recombinant human epidermal growth factor1-48 (EGF1-48) in primates, intravenous bolus injections were given to 2 cynomolgus monkeys per sex at 0 (vehicle control). 10, 100, 500 (females only), and 1,000 micrograms/kg/day (males only) for up to 2 wk. Males given the suprapharmacologic dose of 1,000 micrograms/kg did not tolerate treatment and were necropsied after 5 days of dosing. All other monkeys completed the 2-wk study. Necropsy findings included enlarged, discolored, pale tan livers at 500 micrograms/kg and greater, firm, thickened pancreata in 500-micrograms/kg females, and enlarged salivary glands at all doses. Relative liver weights were increased at 500 and 1,000 micrograms/kg: mean salivary gland weights in all dose groups were greater than in controls. Histopathologic changes were primarily those of diffuse epithelial cell hypertrophy and hyperplasia in liver (hepatocytes and biliary tract), pancreas, salivary glands, tongue, esophagus, stomach, small and large intestine, and gallbladder. Alterations were dose-related in intensity and occurred in at last some tissues at the lowest dose. In gastric glands, colon crypts, pancreatic ducts, biliary tract, and salivary glands, differentiated epithelial cells were replaced by cells of less differentiated phenotype. These morphologic alterations were consistent with exuberant proliferation induced by this epithelial mitogen. The extent of the proliferative response in tissues of the digestive tract attests to the potency of this fragment of human EGF1-53 in primates. Furthermore, the epithelial proliferation was significantly greater than that reported previously in EGF-treated rodents.

Heterocyclic amides: inhibitors of acyl-CoA:cholesterol O-acyl transferase with hypocholesterolemic activity in several species and antiatherosclerotic activity in the rabbit.

A series of heterocyclic amides were synthesized and evaluated as inhibitors of acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and for cholesterol lowering in cholesterol-fed rats. Compounds were evaluated for cell-based macrophage ACAT inhibition, bioactivity, and adrenal toxicity. Candidates were selected for evaluation in cholesterol-fed dogs and, ultimately, the injured cholesterol-fed rabbit model of atherosclerosis. The heterocyclic amides potently inhibited rabbit liver ACAT (IC50's = 0.014-0.11 microM), and the majority of compounds significantly lowered plasma cholesterol (42-68%) in an acute cholesterol-fed rat model at 3 mg/kg. The most efficacious compounds in the rat were evaluated for bioactivity in vivo and arterial ACAT inhibition in a cell-based macrophage ACAT assay. Two highly bioactive analogs, (+/-)-2-(3-dodecylisoxazol-5-yl)-2-phenyl-N-(2,4,6-trimethoxypheny l) acetamide (13a) and (+/-)-2-(5-dodecylisoxazol-3-yl)-2-phenyl-N-(2,4,6-trimethoxypheny l) acetamide (16a), were selected for further study and were found to be nontoxic in a guinea pig model of adrenal toxicity. Compounds 13a and 16a lowered total cholesterol in the cholesterol-fed rat, rabbit, and dog models of pre-established hypercholesterolemia. Compound 13a in the injured cholesterol-fed rabbit model of atherosclerosis was effective in slowing the development of cholesteryl ester-rich thoracic aortic lesions, reducing lesion coverage by 53% at a dose of 1 mg/kg.

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. synthesis and pharmacological activity of (+/-)-2-dodecyl-alpha-phenyl-N-(2,4,6-trimethoxyphenyl)-2H-tetrazole-5- acetamide and structurally related tetrazole amide derivatives.

A series of tetrazole amide derivatives of (+/-)-2-dodecyl-alpha-phenyl-N-(2,4,6-trimethoxyphenyl)-2H-tetrazole-5- acetamide (1) was prepared and evaluated for their ability to inhibit acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in vivo. For this series of compounds, our objective was to systematically replace substituents appended to the amide and tetrazole moieties of 1 with structurally diverse functionalities and assess the effect that these changes have on biological activity. The ensuing structure-activity relationship (SAR) studies identified aryl (7b) and heteroaryl (7f,g) replacements for 2,4,6-trimethoxyphenyl that potently inhibit liver microsomal and macrophage ACAT in vitro and exhibit good cholesterol lowering activity (56-66% decreases in plasma total cholesterol at 30 mg/kg), relative to 1, when compared in the acute rat model of hypercholesterolemia. Replacement of the alpha-phenyl moiety with electron-withdrawing substituents (13e-h), however, significantly reduced liver microsomal ACAT inhibitory activity (IC50 > 1 microM). This is in contrast to electron-donating substituents (13ij,m-q), which produce IC50 values ranging from 5 to 75 nM in the hepatic microsomal assay. For selected tetrazole amides (1, 7b, 13n,o), reversing the order of substituents appended to the 2- and 5-positions in the tetrazole ring (36a-d), in general, improved macrophage ACAT inhibitory activity and provided excellent cholesterol-lowering activity (ranging from 65% to 77% decreases in plasma total cholesterol at 30 mg/kg) in the acute rat screen. The most potent isomeric pair in this set of unsubstituted methylene derivatives (13n and 36a) caused adrenocortical cell degeneration in guinea pigs treated with these inhibitors. In contrast, adrenal glands taken from guinea pigs treated with the corresponding alpha-phenyl-substituted analogs (7b and 36c) were essentially unchanged compared to untreated controls. Subsequent evaluation of 7b and 36c in a rabbit bioassay showed that both compounds and/or their metabolities were present in plasma after oral dosing. Unlike 7b and 36c, compound 1 and related 2,4,6-trimethoxyanilides (13j, 30c,d) showed poor oral activity in the rabbit bioassay. Nevertheless, in cholesterol-fed rabbits, both systemically available (7b, 36c) and poorly absorbed inhibitors (1, 36d) were more effective in lowering plasma total cholesterol than the fatty acid amide CI-976.

Cellular hyperplasia in rats following continuous intravenous infusion of recombinant human epidermal growth factor.

In this study, we determined in vivo morphologic effects of continuous intravenous infusion of recombinant human epidermal growth factor (EGF) in adult Wistar rats. The EGF used consisted of the amino acid residues 1-48 of the human 53-amino-acid EGF molecule, purified from transfected Escherichia coli. Doses of 25, 100, or 250 micrograms/kg body weight were administered using Harvard digital syringe infusion pumps for 4 weeks. At necropsy, the submandibular salivary glands, Harderian glands, liver, kidneys (females only), and ovaries were enlarged and urinary bladders were thickened in 100- and 250-micrograms/kg rats. Numerous tissues of the 100- and 250-micrograms/kg rats contained hyperplastic epithelial cells, and selected organs also had mesenchymal cell proliferation. Epithelial proliferation was most pronounced in the trachea, nasal cavity, nasolacrimal duct, tongue, stomach, small intestine, large intestine, urinary tract, salivary gland ducts, and Harderian gland. Periportal hepatocytes were hypertrophic, correlating with increased liver weight. In addition, mesenchymal cell proliferation was evident in the gastric mucosa lamina propria and in heart valves in 100- and 250-micrograms/kg rats. Increased ovarian weight correlated with increased number and size of corpora lutea and an increased incidence of luteal cysts. Continuous systemic exposure of adult Wistar rats to high doses of EGF resulted in generalized epithelial hyperplasia and tissue-selective mesenchymal proliferation.

Toxicologic effects of a novel acyl-CoA:cholesterol acyltransferase inhibitor in cynomolgus monkeys.

PD 132301-2, an acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor, was administered orally to cynomolgus monkeys for 2 wk at doses of 25, 50, 100, and 200 mg/kg to assess potential subacute toxicity. Sporadic episodes of soft feces and diarrhea increased in incidence from 100 to 200 mg/kg. Histopathologic alterations in adrenocortical cells of treated monkeys consisted of a dose-related decrease in cytoplasmic fine vacuolation and an increase in cytoplasmic eosinophilia most conspicuous in the zona fasciculata and reticularis. At 50, 100, and 200 mg/kg, a narrow discontinuous zone of cytotoxic cortical cell degeneration occurred in the outer zona fasciculata. Decreased fine vacuolation of cortical cells correlated ultrastructurally with reduced size and number of intracellular lipid vacuoles and biochemically with a dose-related decrease in adrenal total cholesterol (from 56 to 13% of control) and cholesteryl ester (from 51 to 3% of control) concentrations. Other ultrastructural changes noted in zona fasciculata cortical cells at all doses were an apparent increase in both smooth endoplasmic reticulum and variably sized autophagic vacuoles. Ovarian corpora lutea in some females at all doses had increased coarse vacuolation of luteal cells, foci of cellular degeneration, increased numbers of cholesterol clefts, and slight infiltrates of mononuclear cells. Sebaceous glands were atrophic in all treated monkeys due largely to a reduction in size and number of differentiated foam cells. Sebaceous gland reserve cells were hypertrophic and hyperplastic. Toxicity data from this study indicated that PD 132301-2 at 25-200 mg/kg targeted cholesterol-rich cells of the adrenals, ovaries, and skin adnexa.

Subacute toxicity of a novel inhibitor of acyl-CoA: cholesterol acyltransferase in beagle dogs.

PD 132301-2 is a substituted urea hypolipidemic and antiatherosclerotic agent that is a potent inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). To determine its subacute toxicity, PD 132301-2 was administered orally to beagle dogs at 0, 6, 12, 25, 50, 200, 400, or 800 mg/kg/day for 2 weeks. Clinico-pathologic evaluations were completed on all dogs. Liver and adrenal total and esterified cholesterol concentrations, adrenocorticotrophic hormone (ACTH) responsiveness, and adrenal ultrastructure were determined at 0, 6, 12, and 25 mg/kg. At 12 mg/kg or greater, salivation, epiphora, conjunctivitis, emesis, anorexia or decreased food consumption, and soft to mucoid feces and/or diarrhea were noted. Suppression of ACTH response occurred by Day 6 at all doses. Adrenocortical degeneration and/or necrosis in zona fasciculata and reticularis was seen at all doses; adrenal free and esterified cholesterol were normal at 6 mg/kg and decreased at 12 and 25 mg/kg. Increases in serum alanine aminotransferase (2- to 15-fold), aspartate aminotransferase (2- to 12-fold), and alkaline phosphatase (2- to 7-fold) were noted at 50 mg/kg or greater. Periportal hepatocellular hypertrophy and hypereosinophilia occurred at 50 mg/kg or greater; hepatic cholesterol values were not significantly affected by treatment. Dose-dependent ultrastructural alterations in adrenocortical cells included decreased numbers of mitochondria and smooth endoplasmic reticulum profiles, qualitative and quantitative changes in lipid globules, and increased numbers of autolysosomes. PD 132301-2 or one of its metabolites has potent adrenocorticolytic properties and limited hepatotoxic properties by mechanism(s) that are likely independent of systemic ACAT inhibition.

Mesenteric lymph node hemangiomas of Wistar rats.

Vascular tumors in rodent mesenteric lymph nodes are uncommon. Fifty-seven of these neoplasms were identified in control and treated Wistar rats from 6 tumor bioassays. Tumor incidence ranged from 0.75% to 5.50% and was higher in males than females (2:1). Lesions, noted as incidental necropsy findings or in routine histologic sections, were typically solitary and restricted to nodal and perinodal tissue. Additional solitary vascular tumors were identified in skin of 3 rats and pararenal lymph node of 1 rat. Distinct metastases were not evident. When apparent grossly, affected nodes were red to purple, hemorrhagic, and/or enlarged. Histologically, all tumors were composed of variably sized, endothelial-cell-lined, blood-filled spaces separated by variable amounts of poorly cellular stroma. Nodal effacement was common in larger tumors. Approximately half of the tumors had features of typical cavernous hemangiomas. The remaining tumors had slightly more aggressive features consisting of single or multiple foci of lymph node capsule invasion, presence of tumor cells in muscular blood vessels, or cellular atypia with variable mitotic activity. Death due to tumor rupture and consequent hemoperitoneum occurred in 1 rat only.

Comparison of response of bovine and porcine pulmonary arterial endothelial cells to monocrotaline pyrrole.

Monocrotaline (MCT)-induced vascular injury in liver and lung may be caused by interaction of MCT metabolites such as monocrotaline pyrrole (MCTP) with vascular cells. Responses of bovine and porcine pulmonary artery endothelial cells (BECs and PECs, respectively) to a single administration of MCTP were compared. MCTP caused a delayed and progressive release of lactate dehydrogenase (LDH) activity from BECs and a gradual decrease in monolayer cellularity. Surviving cells became markedly hypertrophic. PECs were less sensitive to the cytolytic effects of MCTP, showing minimal cell detachment and little release of LDH activity. However, monolayer cellularity, as assessed by PEC enumeration, decreased in a dose-dependent manner. Hypertrophy of surviving PECs was less pronounced than in BECs. MCTP caused enhanced release of prostacyclin from monolayers of BECs and PECs exposed to 10 micrograms MCTP/ml, and concentrations of 0.5 microgram/ml or greater caused equivalent reduction in colony-forming efficiency in both cell types. In summary, whereas BECs were more susceptible to the cytolytic and hypertrophic effects of MCTP, BECs and PECs responded similarly with regard to prostacyclin release and were equally sensitive to the cytostatic effects of this compound.

Experimental reproduction of respiratory tract disease with bovine respiratory syncytial virus.

An experiment was conducted to reproduce respiratory tract disease with bovine respiratory syncytial virus (BRSV) in one-month-old, colostrum-fed calves. The hypothesized role of viral hypersensitivity and persistent infection in the pathogenesis of BRSV pneumonia was also investigated. For BRSV inoculation a field isolate of BRSV, at the fifth passage level in cell culture, was administered by a combined respiratory tract route (intranasal and intratracheal) for four consecutive days. Four groups of calves were utilized as follows: Group I, 6 calves sham inoculated with uninfected tissue culture fluid and necropsied 21 days after the last inoculation; Group II, 6 calves inoculated with BRSV and necropsied at the time of maximal clinical response (4-6 days after the last inoculation); Group III, 6 calves inoculated with BRSV and necropsied at 21 days after the last inoculation; Group IV, 6 calves inoculated with BRSV, rechallenged with BRSV 10 days after initial exposure, and necropsied at 21 days after the initial inoculation. Clinical response was evaluated by daily monitoring of body temperature, heart rate, respiratory rate, arterial blood gas tensions, hematocrit, total protein, white blood cell count, and fibrinogen. Calves were necropsied and pulmonary surface lesions were quantitated by computer digitization. Viral pneumonia was reporduced in each principal group. Lesions were most extensive in Group II. Disease was not apparent in Group I (controls). Significant differences (p less than 0.05) in body temperature, heart rate, respiratory rate, arterial oxygen tension, and pneumonic surface area were demonstrated between control and infected calves. Results indicate that severe disease and lesions can be induced by BRSV in one-month-old calves that were colostrum-fed and seropositive to BRSV. BRSV rechallenge had minimal effect on disease progression. Based on clinical and pathological response, results did not support viral hypersensitivity or persistent infection as pathogenetic mechanisms of BRSV pneumonia.

The effects of monocrotaline pyrrole on cultured bovine pulmonary artery endothelial and smooth muscle cells.

Monocrotaline pyrrole (MCTP), a reactive electrophile, induces delayed and progressive pulmonary edema, vascular remodeling, and pulmonary hypertension after a single intravenous administration to rats. The effects of a single exposure of cultured bovine pulmonary artery endothelial cells (BEC) and bovine pulmonary artery smooth muscle cells (BSMC) to MCTP were examined. Monocrotaline pyrrole caused a dose-dependent, delayed, and progressive cell detachment and release of lactate dehydrogenase activity from monolayers of BECs but not BSMCs. Monolayers of BECs also released increased concentrations of 6-keto-prostaglandin F1 degrees, the stable metabolite of prostacyclin, as the post-treatment interval increased. Progressive and marked endothelial cell hypertrophy occurred after exposure to a nominal concentration of 5 or 50 micrograms/ml of MCTP but not after 0.5 micrograms/ml. Morphologic changes in monolayers of BSMCs were minimal, even up to 2 weeks after exposure. Ultrastructurally the hypertrophic, MCTP-treated BECs had enlarged cell profiles with enlarged nuclei. The nucleoli were prominent, occasionally multiple, and had separation of granular and fibrillar components. Cytoplasmic microtubules and perinuclear intermediate filaments were prominent in some cells, as were the golgi apparatus and endoplasmic reticulum. Degenerative changes were not prominent in cells that remained in the monolayer. Monocrotaline pyrrole inhibited proliferation of both cell types at concentrations (0.5 micrograms/ml) that were not cytotoxic. These findings indicate that MCTP induces direct, dose-dependent injury to cells in culture that is delayed and progressive, and the expression of this injury depends, in part, on the cell type.

Comparison of proteoglycans synthesized by porcine normal and polycystic renal tubular epithelial cells in vitro.

Newly synthesized porcine tubular epithelial cell proteoglycans were labeled in vitro with Na2[35S]SO4. At the beginning of the labeling period (24 h) [35S] sulfate incorporated into macromolecules was measured following PD-10 chromatography. There was a significant reduction in the amount of 35S-labeled macromolecules isolated from polycystic cells compared to that from normal cells. The distribution of recovered radiolabeled material among the medium, cell surface, and intracellular fractions was similar for both normal and polycystic cells. Analysis of the proteoglycans in polycystic cells demonstrated that 86 and 73% of 35S-labeled macromolecules were of the heparan sulfate type in polycystic and normal cells, respectively. The remainder was chondroitin sulfate. Proteoglycans were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC, heparitinase, and nitrous acid digestion followed by Sepharose CL-4B gel permeation chromatography. The majority of radiolabeled material in the medium, cell surface, and intracellular fractions eluted between 0.35 and 0.39 M NaCl. However, a second peak (peak II) that eluted at 0.25 M NaCl was found in the medium from polycystic cells. This peak accounted for 27% of the total macromolecules secreted into the medium. Proteoglycans in the major peak were susceptible to nitrous acid and chondroitinase ABC digestion. A similar proportion of peak II was degraded by chondroitinase ABC. However, the remainder was only slightly susceptible to treatment with nitrous acid or heparitase. In normal cells a small amount of material eluted at a similar low charge; the proteoglycans were the same as those found in the major peak and appeared as a shoulder on this peak.

Lung vascular injury from monocrotaline pyrrole, a putative hepatic metabolite.

The pyrrolizidine alkaloid, monocrotaline (MCT), is a plant toxin that causes injury to the vasculature of the lungs and pulmonary hypertension in animals. To produce lung injury, MCT is bioactivated in the liver by cytochrome P450 monooxygenases to pyrrolic metabolites which travel via the circulation to the lungs, where they cause injury by unknown mechanisms. One putative metabolite of MCT is monocrotaline pyrrole (dehydromonocrotaline, MCTP), a moderately reactive, bifunctional alkylating agent. A single, iv injection of chemically synthesized MCTP into rats causes delayed and progressive lung vascular injury and pulmonary hypertension similar to that caused by MCT itself. Since pulmonary vascular endothelium is likely an important target of MCTP in vivo, the effects of MCTP on cultured endothelium were studied. A single application of MCTP to confluent monolayers of cultured endothelium from bovine pulmonary artery results in release of lactate dehydrogenase, some cell detachment from the growth surface and markedly altered morphology of remaining viable cells. These effects are dose-dependent and, as in vivo, are delayed in onset (1-2 days) and progressive. In endothelial cells of porcine origin, these particular responses to MCTP are also apparent but much less pronounced. Inhibition of proliferation of cells plated at low density occurred in both cell types at nominal MCTP concentrations (0.5 micrograms/ml) that were not overtly cytotoxic. These results indicate that MCTP causes a direct, dose-dependent injury to pulmonary vascular endothelium in culture that is delayed and progressive and suggest a mechanism by which MCT may act in vivo to cause lung injury and pulmonary hypertension.

Development of morphologic, hemodynamic, and biochemical changes in lungs of rats given monocrotaline pyrrole.

A single, intravenous administration of a low dose of monocrotaline pyrrole (MCTP), a derivative of the pyrrolizidine alkaloid monocrotaline (MCT), induces progressive pulmonary hypertension and right ventricular hypertrophy (RVH) in rats. The temporal relationship between morphologic alterations, biochemical markers of lung injury, and the development of pulmonary hypertension was determined during the developing pulmonary disease. Three days after a single iv injection of 3.5 mg/kg MCTP, small increases in bronchoalveolar lavage (BAL) fluid lactate dehydrogenase (LDH) activity and accumulation in the lungs of intravenously administered 125I-bovine serum albumin (BSA) were associated with minimal to mild interstitial edema around large airways and blood vessels. By Day 5, BAL fluid LDH activity and 125I-BSA accumulation had increased further, and lung weight/body weight ratio and BAL fluid protein concentration were greater than those of control. Interstitial edema was more pronounced and involved patches of alveolar septal walls. A mild increase in numbers of mononuclear cells, including hypertrophied interstitial cells, was evident in these areas. Walls of pulmonary arteries less than 60 microns in diameter were mildly thickened. By Day 8, scattered clusters of alveolar sacs contained serous exudate, and interstitial mononuclear infiltrates were more pronounced. Mild to moderate thickening of arterial walls was apparent in small and large vessels. By Day 14, pulmonary arterial pressure was elevated and RVH was evident. Arterial walls were thickened and had hypertrophy of medial smooth muscle cells and intercellular edema, which was particularly prominent in areas with perivascular interstitial inflammation. Large patches of lung interstitium and alveolar lumens contained serous or fibrinous exudate. In summary, a single, intravenous administration of MCTP induced a delayed and progressive pulmonary microvascular leak, interstitial inflammation, and alterations in muscular blood vessels which resulted in pulmonary hypertension within 14 days. These morphologic, biochemical, and hemodynamic changes are nearly identical to alterations induced by the parent alkaloid, MCT.