Analysis of two matrix metalloproteinase inhibitors and their metabolites for induction of phospholipidosis in rat and human hepatocytes(1).

ABT-770 [(S)-N-[1-[[4'-trifluoromethoxy-[1,1'-biphenyl]-4-yl]oxy]methyl-2-(4,4-dimethyl-2,5-dioxo-1-imidazolidinyl)ethyl]-N-hydroxyformamide], a matrix metalloproteinase inhibitor (MMPI), produced generalized phospholipidosis in rats. Phospholipid accumulation was accompanied by retention of drug-related material and was associated with increased mortality. Generation of a successful drug candidate depended upon understanding the cause of the phospholipidosis and redesigning the chemical structure accordingly. ABT-770 and other MMPIs, plus several metabolites of each, were assayed for their ability to induce phospholipidosis in primary cultured rat and human hepatocytes. Phospholipid accumulation was detected by following the incorporation of a fluorescent phospholipid analogue into intracytoplasmic inclusion bodies characteristic of phospholipid storage disorders. At 24 and 48 hr, none of the parent compounds induced phospholipidosis in vitro in rat or human hepatocytes. Phospholipidosis was associated primarily with an amine metabolite of ABT-770. The amine metabolite of another MMPI, ABT-518 ([S-(R*,R*)]-N-[1-(2,2-dimethyl-1,3-dioxol-4-yl)-2-[[4-[4-(trifluoromethoxy)-phenoxy]phenyl]sulfonyl]ethyl]-N-hydroxyformamide), produced little phospholipidosis in rat and human hepatocytes even at concentrations up to 100 microM. The presence or absence of phospholipidosis in the in vitro assay correlated well with ultrastructural findings and drug accumulation in rat tissues. ABT-770, which produced phospholipidosis associated with its amine metabolite in vitro and in vivo, also generated a higher tissue to plasma distribution of metabolites particularly in tissues where phospholipidosis was observed. ABT-518 and its amine metabolite, however, produced low tissue to plasma ratios and induced little to no phospholipidosis in vitro or in vivo. These results demonstrate that the phospholipidosis observed for ABT-770 could be attributed to a cationic metabolite, and that altering the properties of such a metabolite, by modification of the parent compound, alleviated the disorder.

Tenascin synthesis, deposition, and isoforms in monocrotaline-induced pulmonary hypertensive rat lungs.

Monocrotaline (MCT)-induced pulmonary hypertension is characterized by alterations in vascular extracellular matrix and neomuscularization of small blood vessels. Tenascin (TN) is a matrix glycoprotein which modulates cellular attachment, proliferation, and migration. The present study used immunohistochemistry and Northern analyses to examine the hypothesis that treatment of rats with the potent pneumotoxin MCT induces temporal alterations in TN synthesis/deposition in the affected lungs. MCT produced progressive pathological alterations in the cardiopulmonary system, including increased dry lung weight, right ventricular hypertrophy, and pulmonary hypertension by days 7, 14, and 21, respectively. TN positive foci were first observed in the parenchyma surrounding small muscularized pulmonary arteries in MCT-treated rats at day 4; these foci became both more pronounced and frequent as the disease progressed. TN was also observed in the media of the intrapulmonary artery at day 21. Northern analysis demonstrated increases in TN transcripts in MCT-treated rats as early as day 1. Furthermore, a unique transcript, apparently lacking some fibronectin type III-like units, was observed in mRNA extracted from these rats. These data demonstrate alterations in TN synthetic capacity and focal increases in TN deposition in lungs from MCT-treated rats and suggest that TN may be associated with the pathogenesis of pulmonary hypertension.

Basic fibroblast growth factor alterations during development of monocrotaline-induced pulmonary hypertension in rats.

The chemical signaling pathways which orchestrate lung cell responses in hypertensive pulmonary vascular disease are poorly understood. The present study examined temporal alterations in lung basic Fibroblast Growth Factor (bFGF) in a well characterized rat model of monocrotaline (MCT)-induced pulmonary hypertension. By immunohistochemical analysis, there were progressive increases in bFGF in airway, vascular and gas exchange regions of MCT-treated rat lungs. Increases in bFGF preceded the onset of right ventricular hypertrophy at day 21 after MCT administration. Enhanced bFGF immunostaining was observed as early as day 4 in focal areas of the parenchyma, and by day 14 there was enhanced bFGF staining in alveolar macrophages, neutrophils and alveolar septa, which persisted through day 21. In conducting airways, there was elevated bFGF immunostaining in the smooth muscle cell (SMC) layer by days 4 and 7 and in the ciliated epithelium and its basement membrane at days 14 and 21. Cells morphologically similar to Clara cells in the luminal surfaces of bronchioles stained intensely on days 14 and 21. In the nucleus and cytoplasm of medial SMCs within pulmonary arteries, there was a progressive increase in bFGF staining starting at day 4. Lung bFGF mRNA was increased slightly at days 1, 4 and 7, while lung bFGF protein, as judged by western blot analysis, was increased at days 14 and 21 compared to controls. The present results, considered in teh light of teh documented roles of bFGF in vascular cell migration, growth and synthesis of extracellular matrix components, suggest that bFGF may contribute to the structural remodeling processes underlying the development of chronic pulmonary hypertension in MCT-treated rats.

Monocrotaline alters type II pneumocyte morphology and polyamine regulation.

Lungs from monocrotaline (MCT)-treated rats exhibit altered polyamine metabolism and content. One of the prominent morphological abnormalities in MCT-treated lungs is a decrease in population density of type II pneumocytes. Against this background, the present study tested the hypothesis that failure to maintain normal population density of type II pneumocytes is associated with MCT-induced derangements in polyamine biosynthesis and/or transmembrane polyamine transport. After a 24-hr treatment, cultured type II pneumocytes exhibited numerous vacuoles at the highest dose of 3.2 mM MCT but not at the lower dose of 1.6 mM MCT. Intracellular spermidine content was significantly reduced at the highest dose of MCT. Relative to controls, the abundance of mRNA for both ornithine decarboxylase, and S-adenosylmethionine decarboxylase, key regulatory enzymes in polyamine synthesis, was not altered. However, the activities of both of these enzymes were dramatically reduced. Increased mRNA for the catabolic polyamine enzyme, spermine/spermidine-N1-acetyltransferase (SAT), paralleled significant increases in SAT activity. MCT also caused a concentration-related inhibition of spermidine uptake in type II cells, characterized by a fourfold decrease in Vmax with little change in Km. These results show that MCT alters type II pneumocyte polyamine regulatory mechanisms and may help explain the decreased population density of type II pneumocytes in MCT-treated rats.

Uptake of ferritin and Bordetella avium in bronchus-associated lymphoid tissue of turkeys.

The uptake of macromolecular and particulate materials in bronchus-associated lymphoid tissue (BALT) in turkeys was examined using transmission electron microscopy. Tracer materials used were live and ultraviolet-killed (UV-killed) Bordetella avium and ferritin. Suspensions of bacteria and ferritin were instilled via intratracheal catheterization and allowed to remain in contact with the respiratory surfaces for 0, 10, 30, 60, 90, and 120 min. Ferritin and B. avium were taken up by both ciliated and non-ciliated cells of the epithelium overlying BALT (BALT epithelium). Ferritin was found in organelles associated with endocytosis (i.e. apical vesicles, endosomes, cytoplasmic vacuoles) and was apparently transported across epithelial cells, since it was also found in intercellular spaces. Bacteria were found in vacuoles within BALT epithelial cells, but not free in intercellular spaces. Some macrophages in BALT epithelium also contained bacteria. No differences were observed between uptake of live and UV-killed bacteria. We conclude that both ciliated and non-ciliated cells of BALT epithelium in turkeys are able to take up macromolecular and particulate materials. Bacteria are also accessible to intraepithelial macrophages, although whether they are taken up directly from the bronchial surface or whether they pass through epithelial cells first could not be determined. This evidence suggests that antigens, including respiratory pathogens, could gain access to cells of the avian immune system by transepithelial passage in BALT.

Morphogenesis of enteric lesions induced by group D rotavirus in ringneck pheasant chicks (Phasianus colchicus).

Eight day-old male and female ringneck pheasants (Phasianus colchicus) were inoculated with group D rotavirus and necropsied at 4, 7, and 11 days post-inoculation. The intestinal tracts were examined by light and electron microscopic and immunohistochemical methods. By 4 days post-inoculation, 2/3 (66%) inoculated birds were stunted and had diarrhea and dilated intestines. Intestinal villi were shortened, and many villous enterocytes were partially detached from the lamina propria. Crypts were hyperplastic, and the lamina propria contained a diffuse infiltrate of lymphocytes, plasma cells, and macrophages. Immunoreactivity to rotaviral antigen was localized to enterocytes on the tips of villi in the duodenum, jejunum, and proximal ileum. By 7 days post-inoculation, 3/3 (100%) inoculated birds had clinical signs and gross and microscopic changes similar to those at 4 days post-inoculation but more severe. Immunoreactivity was localized in enterocytes scattered along the sides of villi, in occasional crypt enterocytes, and within macrophages in the villous lamina propria. Ultrastructurally, infected enterocytes contained cytoplasmic aggregates of viroplasm with multiple viral core particles. Numerous mature virions (60-75 nm in diameter) were present within dilated components of the cytocavitary network. Macrophages within the lamina propria contained phagocytosed remnants of necrotic virus-infected cells. By 11 days post-inoculation, birds did not have gross lesions, but 1/2 (50%) had mild crypt hyperplasia and an infiltrate of leukocytes in the lamina propria. Occasional enterocytes along the sides of villi and macrophages in the lamina propria were immunoreactive for viral antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and development of bronchus-associated lymphoid tissue in conventionally reared broiler chickens.

The development of bronchus-associated lymphoid tissue (BALT) in conventionally reared broiler chickens of 1 day and 1, 2, 3, 4, 6, and 8 weeks of age was studied using light and electron microscopy (scanning and transmission). BALT in these chickens resembled other mucosa-associated lymphoid tissues (MALT) in that it was composed of an altered epithelium overlying a population of lymphocytes and contained potential antigen-presenting cells, such as macrophages and dendritic cells; high endothelial venules were also present. In contrast to other MALT, epithelial cells in chicken BALT were not of the M-cell type; i.e., they lacked large numbers of apical tubules and vesicles for specialized uptake of luminal antigens. There were age-related differences in size, number, and cellular composition of BALT nodules. Lymphoid nodules were progressively larger and more numerous with increasing age. Germinal centers were present in birds 2 weeks of age and older. BALT epithelium was primarily squamous and non-ciliated in 1-day and 1-week-old chicks, becoming progressively more columnar and ciliated in older chickens. Lymphocyte infiltration of the epithelium was extensive at 1 to 4 weeks of age; in older chickens, distinct epithelial and lymphocytic compartments were separated by connective tissue.

Distribution and quantitation of plasma cells, T lymphocyte subsets, and B lymphocytes in bronchus-associated lymphoid tissue of chickens: age-related differences.

Development of the lymphoid cell compartment in bronchus-associated lymphoid tissue (BALT) of specific pathogen free chickens was examined. Specifically, B lymphocytes, T cell subsets (CD4 and CD8), and IgA-, IgG-, and IgM-producing plasma cells were labeled using immunocytochemical methods. Immunoglobulin-producing cells (IgPC) were quantitated, and comparisons of IgPC numbers were made among chickens of different ages, among immunoglobulin isotypes, and between lymphoid (BALT) and nonlymphoid (non-BALT) areas in the primary bronchus. At hatching, BALT was devoid of IgPC, but by 2 weeks of age cells producing IgA, IgG, and IgM were present. Initially, there were approximately equal numbers of IgA-, IgG-, and IgM-PC; after 2 weeks of age, however, IgG- and IgM-PC outnumbered IgA-PC. At all ages, IgPC were more numerous in non-BALT regions of the primary bronchus than in BALT regions. Small numbers of T and B lymphocytes were present in BALT from 1-day old chickens, but substantial populations of these cells were not seen until 1-2 weeks of age. T helper (CD4+) cells were found near B cell regions in BALT lymphoid nodules, while T cytotoxic/suppressor (CD8+) cells were more evenly distributed throughout the nodules and in the epithelium. B lymphocytes predominated in germinal centers and also overlapped CD4+ populations adjacent to germinal centers. Lymphocyte cell types needed to initiate and regulate immune responses are present in chicken BALT and may be involved in protecting poultry from respiratory pathogens.

Long-segmented filamentous organisms observed in poults experimentally infected with stunting syndrome agent.

One-day-old turkeys were inoculated per os with material shown previously to induce stunting syndrome (SS). Weight gain and feed efficiency of inoculated poults from 1 to 13 days of age were impaired (P less than 0.01) compared with uninoculated poults. Examination of the jejunal mucosa by scanning and transmission electron microscopy showed the presence of long-segmented filamentous organisms (LSFOs) in poults that had been inoculated with SS. These organisms were not seen in jejuna of uninoculated poults. Further research is needed to characterize LSFOs and to determine their involvement, if any, in the adverse effects associated with SS.

A morphologic study of bronchus-associated lymphoid tissue in turkeys.

Bronchus-associated lymphoid tissue (BALT) in normal turkeys of ages 1 day and 1, 2, 3, 4, 8, and 18 weeks was examined by light microscopy and by scanning and transmission electron microscopy. Turkey BALT resembled other mucosa-associated lymphoid tissues; it was made up of a population of lymphocytes covered by a specialized epithelium different from typical pseudostratified ciliated columnar bronchial epithelium. There were distinct age-related differences in BALT structure. Bronchus-associated lymphoid nodules were larger and more numerous in older turkeys. In 1-day- to 2-week-old turkeys, the primary cell type of BALT epithelium was nonciliated cuboidal; in 2-week old turkeys it was squamous; and in turkeys older than 4-weeks of age, the epithelium was primarily ciliated columnar. In 1- to 4-week old turkeys, large numbers of intraepithelial lymphocytes disrupted the normal organization of the epithelium. In older turkeys, epithelial and lymphoid cells were in discrete compartments separated by connective tissue. Lymphocytes in 1-day-old turkeys were found in loose aggregates around venules and within the epithelium. In 1-week old turkeys, lymphocytes were organized into compartments of morphologically similar cells. By 3-weeks of age, lymphocytes were present in distinct germinal centers. Epithelial cells of BALT did not have large numbers of apical vesicles and thus were not structurally specialized for antigen uptake by endocytosis. However, the epithelial barrier appeared to be disrupted over lymphoid nodules, suggesting that antigen would be readily available to lymphocytes and phagocytes in BALT. Age-related differences in turkey BALT structure may have functional consequences with respect to the respiratory immune response.

Cultivation and partial characterization of bovine astrovirus.

Bovine astrovirus serotype 2 (US2) was adapted to primary neonatal kidney cell (NBK) cultures by the addition of 50 micrograms ml-1 of trypsin in the medium. Infectious virus was released from the cells within 7 days post-infection in early passages and within 3 days in later passages. In the absence of trypsin, neither passage of infected cells nor release of infectious virus occurred. The virus was shown to be similar to the fecal astrovirus by a neutralization test and by ultrastructural studies of infected cells. Primary embryo bovine kidney (EBK) and NBK cell cultures supported infection with both fecal and tissue culture adapted (TCA) astrovirus. The time-related development of infection, as studied by immunofluorescence, was similar for both fecal and TCA astrovirus and for both cell culture types. The first indication of viral infection and expression of viral antigens occurred at 7 h post-infection and was characterized by the appearance of a diffuse faint immunofluorescence (IF) of the cytoplasm. Soon after, two or three brilliant IF granules were observed in the nucleus, which appeared to involve the nucleoli. Subsequently, dense granular IF was seen in the perinuclear region of the cytoplasm, which later extended to involve all the cytoplasmic area. In both EBK and NBK cultures infected with either fecal or tissue culture adapted astrovirus, only a minority of cells became infected, even when the multiplicity of infection exceeded one. Occasionally 10-20% of cells were infected, but in most cultures the proportion did not exceed 2% and in NBK cultures, from 3/9 calves, no infected cells were observed. The virus did not infect bovine cell lines. Infectivity of the virus was not removed by treatment with chloroform, and iododeoxyuridine and actinomycin D when added to the medium, did not block replication. Masses of virions were observed by electron microscopy in discrete areas in the cytoplasm, with similar distributions as the viral antigen foci as seen by IF. The mean diameter of the virions was 34 nm. In conclusion, bovine astrovirus lacks both essential lipids and an envelope, probably has an RNA genome, may have a nuclear phase of replication involving the nucleoli which is not blocked by DNA inhibitors, and has a selective cell tropism.

A comparison of outer membrane proteins and surface characteristics of adhesive and non-adhesive phenotypes of Bordetella avium.

The outer membrane protein profiles of four adherent and one reduced-adherence mutant phenotype of Bordetella avium were compared; a non-adherent B. avium-like organism isolated from turkeys was also examined. The organisms were grown on brain-heart infusion agar at 35 C for 36 hours. In addition, one of the adherent phenotypes was grown at 18 C and 40 C. The outer membrane proteins were isolated by sonication and detergent extraction with Triton X-100. Surface characteristics of intact bacteria were examined using negative stain and transmission electron microscopy. The adherent phenotypes had identical protein profiles by electrophoresis. The non-adherent B. avium-like organism lacked at least five of the proteins present on the adherent strains. The non-adherent mutant phenotype had a protein profile similar to that of the adherent organisms, although several proteins were present in much lower concentrations. Fimbriae were found on both adherent and non-adherent organisms. By comparing protein profiles of adherent and non-adherent B. avium we were able to make a preliminary determination of the membrane proteins that lack adhesive properties.

Ultrastructural pathology of Bordetella avium infection in turkeys.

One-day-old turkeys were infected intranasally with Bordetella avium, and tracheas were examined by scanning and transmission electron microscopy at 1 to 5 weeks post-inoculation (PI). The predominant ultrastructural lesions were progressive loss of ciliated epithelium with replacement by nonciliated cells, bacterial colonization of ciliated cells, membrane-bound crystalline inclusions in cytoplasma of epithelial cells, depletion of mucous granules, and distortion of tracheal rings and the mucosal surface. Tracheal surface exudates consisted of mucus, necrotic cells, heterophils, and fibrin. Ciliated cells were replaced by immature cuboidal cells characterized by abundant rough endoplasmic reticulum with small numbers of electron-dense mucous granules in the apical cytoplasm. Bacterial surfaces were rough and contained numerous pleomorphic, knob-like structures, 20-50 nm in diameter. Other changes included enlarged mucosal gland openings, cell extrusion marks, pleomorphic microvilli, and cells with small numbers of short cilia.

Ultrastructure and stereology of leukocytes and platelets of normal foxes and a fox with a Chediak-Higashi-like syndrome.

Peripheral blood leukocytes and platelets from five normal foxes (Vulpes vulpes) and a fox with phenotypical characteristics of Chediak-Higashi syndrome (CHS) were examined by electron microscopy. Lymphocytes, monocytes, neutrophils, eosinophils, and platelets from the affected fox contained giant membrane-bound granules that resembled lysosomes. In eosinophils and neutrophils from the affected fox and a normal fox, relative cell volume occupied by granules and number of granules per unit area were calculated. Relative cell volume occupied by granules was the same in both foxes, but there were significantly fewer granules per unit area in the affected fox. This result is consistent with the idea that the giant granules arose from fusion of pre-existing, normal-sized granules, as occurs in CHS. In platelets from the affected fox, no osmiophilic granules were seen. Our findings agree with those from studies of CHS-affected blood cells in other species.

A morphological study of the replication of Breda virus (proposed family Toroviridae) in bovine intestinal cells.

The morphological aspects of Breda virus serotype 2 replication in intestinal cells of gnotobiotic calves were investigated by electron microscopy. Ultrastructural findings suggest a morphogenetic pathway involving cytoplasmic vesicles, the Golgi apparatus and the cell nucleus. Virus uptake probably occurs via a receptor-mediated endocytosis-like mechanism. Endocytotic vesicles then carry virus to an as yet undetermined site of uncoating. Masses of tubules having the same diameters as Breda virion cores are found in nuclei, suggesting a role for the cell nucleus in replication of nucleocapsids. Similar tubules, as well as complete virions, were found in the Golgi region, the apparent site of virus assembly. Virus-containing Golgi vesicles then presumably move to cell surfaces where they fuse with apical and baso-lateral cell membranes to release virions in a way that permits more than one viral replicative cycle to occur without damage to host cell integrity. Virions are elongated with rounded ends and measure 42 X 100.5 nm. The morphogenesis and replication of Breda virus most closely resembles that of Berne virus of the proposed family Toroviridae.

Astrovirus and Breda virus infections of dome cell epithelium of bovine ileum.

A bovine enteric virus antigenically related to the United Kingdom isolate of bovine astrovirus was isolated from diarrheic feces, also containing rotavirus, of a calf in Florida. The astrovirus infected cell cultures and the epithelial cells of domes in the ileum, and there was cross-immunofluorescence with antiserum to the United Kingdom astrovirus. Calves infected with astrovirus alone did not develop clinical disease, but when astrovirus was mixed with rotavirus or Breda virus 2, the calves developed severe diarrhea and more extensive astrovirus infection of the dome epithelium. The dome epithelial cells showed degeneration associated with astrovirus infection, and a few cells showed degeneration with Breda virus 2 infection. Virions with a 30-nm diameter were seen in astrovirus-infected dome cells, and Breda virus 2 virions were also observed either in separate cells or, on occasion, with both viruses in one cell.

Unusual psammoma bodies in an extracranial syncytial meningioma from a dog. A light and electron microscopic study.

An epidural neoplasm was removed from the right side of the third cervical vertebra of a Blue Tick Hound dog. By light microscopy the tumor was classified as a syncytial meningioma containing psammoma bodies that were histochemically positive for calcium salts, but negative for reticulum fibers and collagen. Ultrastructurally, the tumor cells had numerous processes and desmosomes. Cytoplasmic fibrils were sparse. Coated cytoplasmic vesicles frequently were fused with plasma membranes. The psammoma bodies were not within the cytoplasm, but were in spaces continous with the extracellular space. The bodies were composed of a medium electron-dense matrix; a crystalline, highly electron-dense center; and, sometimes, short fibrils embedded in the glanular matrix.