John F. Long

Enzyme activities associated with the demyelinating phase of canine distemper

SummaryCanine distemper, a naturally occurring viral disease of dogs which often terminates in parainfectious demyelination, was used as a model to study the role of acid proteinase, neutral proteinase and beta-glucuronidase in demyelination. These enzymes were higher in cerebella of dogs with distemper-associated demyelination than in age-matched controls. The highest elevations corresponded with the most severely demyelinated cerebella. The source of the increased enzymes activities was apparently unrelated to the lymphocytes present in areas of demyelination.The direct effect of distemper virus and serum on these enzymes was tested in canine glial monolayers. Virus infection resulted in lower enzyme activities in cells concomitant with the appearance of cellular lesions. There was a relative increase of beta glucuronidase activity in the media suggesting that distemper virus released pre-formed lysosomal enzymes. Serum which was obtained from dogs with distemper-associated demyelination and had previously demyelinated cerebellar explants, also decreased activities of all 3 enzymesin vitro.The 3 enzymes were measured in gerbil brains at various time intervals following unilateral cerebral infarction to determine if processes other than demyelination also caused these enzymes to be increased. Uncomplicated ischemic necrosis (24 h post infarction) did not alter the activities of these enzymes. Invasion of macrophages to ingest and digest necrotic tissue 10 days after infarction resulted in greatly increased acid proteinase and beta-glucuronidase, but unchanged neutral proteinase, activities.It was concluded that the increased activities of acid proteinase and beta-glucuronidase in demyelinated tissue probably are derived from macrophages ingesting damaged tissue. Neutral proteinase may be more specifically involved in the demyelinating process since this is partially located within myelin and can degrade the basic protein of myelin.

The effect of iron on the biological activities of erionite and mordenite

Epidemiological data has demonstrated that environmental and/or occupational exposure to mineral particulates may result in the development of pulmonary fibrosis, bronchogenic carcinoma and malignant mesothelioma many years following exposure. It has been suggested that the genotoxic effects of fibrous particulates, such as asbestos, is due in part to the generation of reactive oxygen species (ROS) from iron associated with the particulates. However, the molecular mechanisms by which mineral particulates induce ROS that results in genotoxic damage remains unclear. The naturally occurring zeolites, erionite and mordenite share several physiochemical properties but they elicit very different biological responses, with erionite, a fibrous particulate, being highly toxic, and mordenite, a nonfibrous particulate, being relatively benign. We are using these natural zeolites as a model system to determine what physicochemical properties of these zeolites are responsible for their biological response(s) and to evaluate the parameters that influence these responses. The purpose of the present study was to determine the mutagenic potential of erionite and mordenite and to determine whether this mutagenic potential was modulated by iron. The results of this study using the Chinese hamster ovary cell line AS52 demonstrated that erionite was more cytotoxic than mordenite. However, the cytotoxicity of both zeolites was increased in the presence of physiological concentrations of ferrous chloride. Ferrous ions (5-20 microM) significantly (p<0.001) increased the cytotoxicity of mordenite, but only at the highest concentration (16 microg/cm(2)) of mordenite tested. Conversely, only the highest concentration (20 microM) of ferrous ion significantly (p<0.001) increased the cytotoxicity of erionite, but this enhanced cytotoxicity occurred over a wider concentration range (6-16 microg/cm(2)) of erionite. Mordenite was not mutagenic at any of the concentrations tested, and the mutagenic potential of mordenite was not enhanced by the addition of ferrous ion. Conversely, erionite was mutagenic in a dose-response manner at concentrations greater than 6 microg/cm(2) and the mutagenic potential of erionite was significantly enhanced by the addition of ferrous ions. These results suggest that while the cytotoxicity of mordenite and erionite may be related to the ability of these fibers to transport iron into a cell, the different coordination state of iron associated with the two fiber surfaces is critical for inducing genotoxic damage.

Comparison of ultrastructural cytotoxic effects of carbon and carbon/iron particulates on human monocyte-derived macrophages

In this study, we tested the hypothesis that the presence of iron in carbon particulates enhances ultrastructural perturbation in human monocyte-derived macrophages (MDMs) after phagocytosis. We used 1-μm synthetic carbon-based particulates, designed to simulate environmental particulates of mass median aerodynamic diameter ≤ 2.5 μm (PM2.5). Cultures of human MDMs or T-lymphocytes (as a nonphagocytic control) were exposed to carbon or carbon/iron particulates for various time periods and examined by transmission electron microscopy for ultrastructural changes. T-cells failed to internalize either of the particulates and showed no organelle or nuclear changes. Conversely, MDMs avidly phagocytized the particulates. MDMs treated with C particulates exhibited morphologic evidence of macrophage activation but no evidence of lysis of organelles. In contrast, MDMs treated with C/Fe particulates exhibited coalescence of particulate-containing lysosomes. This phenomenon was not observed in the case of C particulates. By 24 hr there was a tendency of the C/Fe particulates to agglomerate into loose or compact clusters. Surrounding the compact C/Fe agglomerates was a uniform zone of nearly total organelle lysis. The lytic changes diminished in proportion to the distance from the agglomerate. In such cells, the nucleus showed loss of chromatin. Although C particles induced no detectable oxidative burst on treated MDMs, C/Fe particles induced a nearly 5-fold increase in the extracellular oxidative burst by treated MDMs compared with untreated controls. Iron bound to C particles catalyzed the decomposition of hydrogen peroxide to generate hydroxyl radicals. Results of these studies suggest that, among particulates of similar size, biologic activity can vary profoundly as a function of particulate physicochemical properties.

Beta-Glucuronidase activity, and levels of protein and protein fractions in serum and cerebrospinal fluid of dogs with distemper associated demyelinating encephalopathy

Summaryβ-Glucuronidase activity in CSF was significantly elevated in dogs with distemper encephalitis compared with controls. Furthermore, there appeared to be a positive correlation between the degree of brain tissue destruction and the level of enzyme in the CSF. β-Glucuronidase may be an index of inflammation while albumin may be an index of alteration of the blood-brain barrier. There was a high degree of correlation between the β-glucuronidase and total protein, albumin and globulin values in the CSF.The possible diagnostic and prognostic use of the CSF β-glucuronidase determination in canine distemper is discussed.

Effects of a pathogenic canine herpesvirus on canine brain cell cultures and cerebellar explants

SummaryThe effects ofHerpes canis, an encephalitogenic agent of the canine species, on canine cerebellar explants and brain cell cultures were investigated. A progression of the infection from cell to cell in brain explant cultures could be followed resulting in degeneration and widespread cell death within 72 hours after inoculation. Astrocytes were recongized as the preferential cells for viral replication. Nerve cells were irreversibly damaged but manifested no definite inclusion bodies indicative of viral replication.The sequential effects ofHerpes canis on a neuroectodermal cell type (astrocytes) were studied in brain cell cultures by correlating the results of aridine orange cytochemistry and immunofluorescence with the findings of light and electron microscopy. The progression of lesions within astrocytes was divided into three phases. The early phase was characterized by viral entry and nucleolar segregation consisting of separation of the pars amorpha from the pars fibrosa which subsequently fragmented. Three forms of inclusion bodies appeared during the middle phase and viral antigen and viral particles in various stages of maturation were demonstrated within the nuclei of affected cells. The late phase consisted of cell degeneration and viral release.The twoin vitro systems (brain explants and cell cultures) offered an excellent opportunity for detailed and repeated observations of the interactions of a neuropathogenic herpes virus with the various neuroectodermal cellular elements obtained from the natural host. Many of the findings appear to be directly applicable to pathogenetic principles of canine herpes virus encephalitis.ZusammenfassungEs wurden die Auswirkungen vonHerpes canis, einem encephalitogenen Virus des Hundes, an Kleinhirnexplantaten vom Hund und an Hirzellkulturen untersucht. Ein Fortschreiten der Infektion von Zelle zu Zelle wurde an Hirnexplantatkulturen beobachtet mit dem Ergebnis einer cellulären Degeneration und ausgedehnter Nekrose innerhalb 72 Std nach der Inokulation. Astrocyten wurden als der bevorzugte Zelltyp für Virusvermehrung erkannt. Obwohl Nervenzellen unwiderruflich geschädigt waren, enthielten sie niemals eindeutig darstellbare Einschlußkörper, die auf Virusvermehrung hindeuten.Der progressive Effekt vonHerpes canis an einem neuroektodermalen Zelltyp (Astrocyten) wurde in Hirnzellkulturen untersucht, indem die Ergebnisse verschiedener Methoden, Acridinorange-Cytochemie, Immunofluorescenz sowie Licht-und Elektronenmikroskopie vergleichend ausgewertet wurden. Entsprechend dem Fortschreiten der Veränderungen in den Astrocyten wurden die Läsionen in drei Phasen eingeteilt: Die frühe Phase war durch virale Eintrittsstadien und nucleoläre Segregation gekennzeichnet, wobei sich die Pars amorpha erst von der Pars fibrosa separierte und beide Komponenten schließlich rhektisch zerfielen. Während der Mittelphase wurden drei Einschlußkörpertypen erkannt und sowohl Virusantigen als auch Viruspartikelchen in verschiedenen Stadien der Reifung in den Kernen betroffener Zellen nachgewiesen. Die späte Phase war durch Zelldegeneration und Virusentlassung in das Cytoplasma gekennzeichnet.Beidein vitro-Systeme (Hirnexplantate und Zellkulturen) boten eine ausgezeichnete Gelegenheit detaillierter und wiederholter Beobachtungen der Wechselbeziehungen eines neuropathogenen Herpesvirus mit verschiedenen neuroektodermalen Zelltypen, die dem natürlichen Wirt des Krankheitserregers entstammten. Viele Beobachtungen an den Gewebekulturen scheinen für die Pathogenese der Herpesencephalitis des Hundes von zutreffender Bedeutung zu sein.

Experimental Porcine Polioencephalomyelitis in Germfree Pigs A Silver Carbonate Study of Neuronal Degeneration and Glial Response

The neuronal changes and glial response in the spinal cord were studied by silver impregnation techniques in 23 germfree pigs orally infected with a porcine polioencephalomyelitis viras. By the sixth day swelling occurred in motor neurons. During the next 24 to 96 hours this progressed to diffuse chromatolysis, vesiculation, necrosis, and neuronophagia in massive areas of the ventral horns. Massive degeneration of axons in tracts originating in the ventral horns and from dorsal root ganglia correlated well with the extensive destruction of both motor and sensory neurons. The initial responses to necrosis of ganglion cells were infiltration and proliferation of microglial cells. As active neuronal destruction ceased (about two weeks following infection) the microglial reaction began to decline and proliferative astrocytosis became the predominant feature. The paucity of surviving neurons in the ventral horns, and the density of the mesh of astrocytic processes marked the chronic regressive phase of the disease.