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Biomedical subjects

J A Ellis

Publications and source records attributed to J A Ellis.

At least 109 records · Page 6Linked to original sources
PubMed1994

Inhibition of apical but not basolateral endocytosis of ricin and folate in Caco-2 cells by cytochalasin D.

Apical and basolateral endocytic pathways in polarised Caco-2 cells were investigated by following the uptake, recycling and transcytosis of the galactose-binding protein toxin ricin, as a membrane marker. Differences in the extent and kinetics of lectin uptake, recycling and transcytosis were observed at the apical and basolateral domains and altered with the age of the cell monolayer. Treatment of polarised Caco-2 cells with cytochalasin D showed a domain-specific, concentration-dependent inhibition of apical endocytosis of ricin. Inhibition of apical endocytosis by cytochalasin D was not due to a gross change in brush border morphology, although actin stress fibres within the cell body were disrupted. It is not clear whether inhibition of apical endocytosis in polarized epithelial cells by cytochalasin D is caused simply by disruption of a mechanochemical motor involving microvillar actin filaments. The cytochalasin D effect was also observed when measuring uptake of folate, suggesting apical domain-specific inhibition of caveolar, as well as clathrin-mediated, endocytic routes.

Actin Cytoskeleton↗
PubMed1994

Dietary hydrated sodium calcium aluminosilicate reduction of aflatoxin M1 residue in dairy goat milk and effects on milk production and components.

Lactating dairy goats were exposed to aflatoxin (100 and 200 ppb) and hydrated sodium calcium aluminosilicate at 1, 2, and 4% in two separate experiments. Naturally occurring low levels of aflatoxin M1 (.009 ppb) were found in the milk of the control diet, whereas there were no detectable levels of aflatoxin M1 in the milk of diets containing hydrated sodium calcium aluminosilicate in both experiments. In Exp. 1, no treatment-related differences in clinical behavior or significant difference in the feed intake, milk production, or milk component analyses were observed with 200 ppb of aflatoxin and 4% hydrated sodium calcium aluminosilicate. However, 4% hydrated sodium calcium aluminosilicate was responsible for an 86.9% reduction of aflatoxin M1 residue in the milk of diary goats. In Exp. 2, the combination of 1% hydrated sodium calcium aluminosilicate and aflatoxin at 100 ppb resulted in an overall reduction of aflatoxin M1 residue by 51.9%, which represented a mean change of aflatoxin M1 from .553 to .266 ppb of aflatoxin M1 in the milk. The diet that contained 2% hydrated sodium calcium aluminosilicate and 100 ppb of aflatoxin further reduced aflatoxin residue by a mean change from .553 to .098 of ppb aflatoxin M1, which represents an 82.2% reduction of aflatoxin M1 residue in the milk. Analysis of the data by time indicated that there were no statistical differences between days of sampling. Information regarding the ability of hydrated sodium calcium aluminosilicate to prevent or reduce the level of aflatoxin M1 residues in milk is critically needed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin M1↗
PubMed1993

Capture immunoassay for ruminant tumor necrosis factor-alpha: comparison with bioassay.

Monoclonal antibodies and IgG purified from rabbit polyclonal antiserum, raised against recombinant bovine tumor necrosis factor-alpha (TNF-alpha), have been employed in ELISA procedures to quantitate bovine TNF-alpha. These antibodies were potent in neutralizing the biological activity of recombinant as well as natural bovine TNF-alpha. The monoclonal antibodies were used as capture antibodies and were either passively adsorbed or covalently linked to ELISA plates. Polyclonal rabbit anti-TNF IgG was used as the detecting antibody in combination with a biotinylated anti-rabbit serum and a streptavidin-horseradish peroxidase conjugate. The detection limit for recombinant TNF-alpha medium was 10 pg ml-1 and in bovine or ovine serum was 35 pg ml-1. A good correlation was found between the ELISA and the WEHI-164 Clone 13 biologic assay when TNF-alpha was measured in medium containing serum or in serum. This capture ELISA was also capable of detecting ovine, but not porcine. TNF in supernatants from cultures of lipopolysaccharide-stimulated pulmonary alveolar macrophages.

Animals↗
PubMed1993

A colorimetric assay for trypanosome viability and metabolic function.

We have adapted a tetrazolium salt (MTT) colorimetric cytotoxicity assay to the assessment of viability and metabolic function in cultured African trypanosomes. Trypomastigotes of Trypanosoma congolense and T. brucei rhodesianse were harvested from the blood of parasitemic rats and cultured under axenic conditions that support trypanosome viability and growth. Analysis of serial dilutions of these bloodstream forms indicated that the assay could detect 10(4) parasites. To assess the effect of lymphoid cytokines on trypomastigote viability, 10(5) freshly harvested parasites were cultured with a wide range of dilutions of human recombinant IL-1, IL-3, IL-6, interferon-gamma (IFN gamma) or tumor necrosis factor-alpha (TNF alpha), or bovine recombinant IFN gamma or TNF alpha for 24, 48 or 96 h. These cytokines had no apparent growth enhancing or inhibitory effect on the trypomastigotes compared with growth in supplemented medium alone. This assay has several advantages over traditional counting methods, including increased sensitivity and rapid, repeatable quantitation. This adaptation of the MTT colorimetric assay should be useful in screening drugs and host-derived factors for growth-modulating effects on trypanosomes and other extracellular protozoan parasites.

Animals↗
PubMed1993

Recollecting naturally-occurring intentions: a study of cognitive and affective factors.

This paper is concerned with the recall of naturally-occurring intentions during the interval between their formulation and their enactment, hours, days, or months later. When these intentions were spontaneously recalled, subjects recorded the prevailing cognitive and affective circumstances, and current events and actions. This task was undertaken for a period of five consecutive days. A subsequent task elicited similar information from the same subjects, on randomly sampled occasions that did not coincide with the recall of an intention, during a comparable time period. The results indicate that on occasions on which subjects report these spontaneous recollections, they often judge themselves to be concentrating less on a concurrent activity that requires less attention, compared to occasions on which behaviour is randomly sampled. The results suggest also that this prototypical 'recollective state' may vary with the type of intention that is recalled. The advantages and disadvantages of the methodology adopted in this study for research on prospective memory processes are considered.

Adult↗
PubMed1993

Pathogenesis of lymphoid interstitial pneumonia in natural and experimental ovine lentivirus infection.

Ovine lentivirus (OvLV), as a member of the lentivirinae subfamily of Retroviridae, shares morphological, genomic, and cytopathic features with human immunodeficiency virus (HIV). Although OvLV infection does not induce profound immune deficiency in sheep, it has many similarities with HIV infection, such as the capacity to infect macrophages, undergo antigenic variation in vivo, and induce slow progressive diseases involving the pulmonary, lymphoid, and central nervous systems. Studies of the pathogenesis of disease in sheep naturally or experimentally infected by OvLV are providing clues to the pathogenesis of HIV infection, including the significance of viral load, the emergence of cytopathic variants, the mechanisms and significance of viral antigenic variation, and viral neutralization, and mechanisms of lymphoproliferation and tissue destruction induced by the virus. Preliminary evidence suggests that infection by other microbial agents, including Mycoplasma species, may play a cofactor role in the pathogenesis of lentivirus-associated lymphoid interstitial pneumonia in sheep, but further studies are required to address this issue.

Animals↗
PubMed1993

Prevalence of bluetongue virus expression in leukocytes from experimentally infected ruminants.

Replication of bluetongue virus (BTV) in leukocytes from the blood of sheep, cattle, elk, and mule deer inoculated with BTV serotype 10 or 17 was assessed by immunocytochemical staining and dot blot northern hybridization to determine if differences in the prevalence of infection in this blood fraction might account for the differences in clinical disease among these species. Viremia was confirmed by virus isolation in all inoculated animals. Analysis of leukocytes with monoclonal antibodies specific for BTV proteins revealed low numbers of infected leukocytes in only 2 sheep 8 days after inoculation with BTV serotype 10. Most of the cells expressing BTV were identified morphologically as monocytes; approximately 10% of infected cells were lymphocytes. Bluetongue virus was not detected by use of dot-blot hybridization on samples of blood. Our results suggest that differential infection of leukocytes does not account for the pronounced differences in clinical signs and pathologic changes among ruminants.

Animals↗
PubMed1993

Cryopreservation of bovine buffy coat leukocytes for use in immunologic studies.

A simple cryogenic technique for preserving bovine buffy coat leukocytes was developed. This was coupled with a variation of the standard discontinuous gradient technique to purify mononuclear cells that retained immunologic function. The total number of mononuclear cells recovered from cyropreserved samples were only 87 to 42% of those recovered from freshly obtained blood samples. However, the functional capabilities of mononuclear cells from cyopreserved buffy coat preparations were retained. Polyclonal proliferative responses to 3 mitogens were measured, using a titration of mitogen concentrations, and were found to be normal, compared with those of cells isolated from fresh blood. Blood samples collected after vaccination with Brucella abortus contained leukocytes that responded to irradiated B abortus. These antigen-specific responses were also retained through cyopreservation. Cell surface expression of T-lymphocyte antigens, CD2, CD4, and CD8, and cell-surface IgM on B lymphocytes was also evaluated. Flow cytometric analysis of fresh and cryopreserved mononuclear cell preparations indicated that the relative proportions of different subpopulations were not altered. The technical simplicity of our cryopreservation system will allow processing of large numbers of samples with the ability to assay various immune functions at a later time.

Animals↗
PubMed1992

The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells.

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5'-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.

Adenocarcinoma↗
PubMed1992

Bovine respiratory syncytial virus-specific immune responses in cattle following immunization with modified-live and inactivated vaccines. Analysis of proliferation and secretion of lymphokines by leukocytes in vitro.

Cattle were immunized with vaccines containing modified-live or inactivated bovine respiratory syncytial virus (BRSV) and lymphocyte proliferative responses and cytokine secretion were monitored sequentially. Compared to pre-inoculated values, significant increases in proliferative responses to modified-live BRSV were detectable by Day 7 after the primary immunization with the vaccine containing inactivated BRSV, and by 7 days after the second immunization with modified-live virus. After a third immunization with the respective vaccines, proliferative responses to live BRSV were significantly higher in the group that received modified-live vaccine compared to the group that received inactivated vaccine. Proliferative responses to live BRSV corresponded with the presence of interleukin-2 (IL-2) in the supernatants from BRSV-stimulated leukocyte cultures and there were significantly higher levels of IL-2 in cultures from the group that received modified-live BRSV. An interferon species with the characteristics of interferon-alpha was also present in the supernatants from leukocyte cultures and there were no significant differences between the groups of vaccines. The predominant phenotype of proliferating cells in BRSV-stimulated leukocyte cultures derived from both groups of bovine vaccines was a BoCD4+ T-lymphocyte. These in vitro data suggest that both types of vaccines are capable of stimulating cell-mediated immune responses to BRSV in cattle.

Animals↗
PubMed1992

Bovine respiratory syncytial virus-specific immune responses in cattle following immunization with modified-live and inactivated vaccines. Analysis of the specificity and activity of serum antibodies.

Cattle were immunized with vaccines containing modified-live or inactivated bovine respiratory syncytial virus (BRSV) and serum antibody responses were analyzed. Compared with preinculation values, at Day 14 after two biweekly immunizations with modified-live or inactivated vaccines there were significant increases in BRSV-specific titers in the sera of cattle that received both types of vaccines, as determined by a whole cell ELISA. Using a blocking ELISA and radioimmune precipitation it was determined that there was recognition of the fusion (F) protein by antibodies from cattle that received both types of BRSV antigens: however, virus neutralization assays revealed that only cattle that received modified live virus, either in monovalent or polyvalent vaccines, developed neutralizing antibodies to BRSV after two immunizations. These results indicate that inactivation of BRSV can lead to a dissociation between serological recognition of the F protein and virus neutralization in vaccinated cattle.

Animals↗
PubMed1992

The detection of intracellular bluetongue virus particles within ovine erythrocytes.

1. We report here a simplified method for detecting viruses and other antigenic agents in red blood cells (RBCs). Using a nonionic detergent to prepare cytoskeletons, the interior of RBCs can be scanned rapidly using immunoelectron microscopy. 2. In this study, RBCs from bluetongue (BLU) virus-infected sheep were adsorbed directly onto Formvar-coated, gold electron microscope grids. 3. Cytoskeletons were prepared and then probed using a monoclonal antibody to VP 7, a structural BLU-virus protein and Protein-A gold. 4. Of the ca 32,000 RBCs that were examined from BLU virus-infected sheep, 34 (0.106%) contained labelled BLU virus particles. 5. No labelled particles were observed in any of ca 8000 RBCs taken prior to BLU virus inoculation of sheep. 6. If the antigenic BLU virus particles (which may be viral cores) are in fact infectious, this method of sequestration of virus within RBCs could contribute to the prolonged viremia typical of this arboviral disease, which is known to occur concurrently with circulating neutralizing antibody.

Animals↗
PubMed1992

Detection and neutralization of bovine tumor necrosis factor by a monoclonal antibody.

Monoclonal antibodies (MAbs) have been produced which are specific for bovine tumor necrosis factor (TNF). MAb BC9 detects bovine TNF in a radioimmunoassay with a detection limit of 24 pg/ml. BC9 also neutralizes the in vitro biological function of bovine recombinant TNF. The activity of 250 ng TNF/ml was entirely neutralized by 1% ascitic fluid. When ascites was added at a saturating concentration (10% ascitic fluid), up to 25 micrograms TNF per ml was neutralized. The neutralizing effect of BC9 was seen in cytotoxic assays using L929 cells and WEHI 164 clone 13 cells. The cytotoxic activity of supernatants from in vitro activated bovine monocytes was entirely blocked by BC9.

Animals↗
PubMed1992

Effects of virus load in the pathogenesis of lentivirus-induced lymphoid interstitial pneumonia.

To better define the relationship between ovine lentivirus (OvLV) infection and respiratory disease, pulmonary leukocytes and postmortem lung specimens from 42 sheep seropositive or at risk for OvLV infection were obtained. The lungs were examined for lesions of lymphoid interstitial pneumonia (LIP), and animals were categorized into five groups by severity of LIP and OvLV serologic status. The presence of OvLV in alveolar macrophages was established by proviral DNA amplification using the polymerase chain reaction (PCR), and the proportion of infected cells was determined by a quantitative focal immunoassay (FIA) and by immunohistochemistry. The concentration of OvLV p25 in serum was measured by capture ELISA. In contrast to animals with mild or no pulmonary lesions, sheep with moderate or severe LIP (17/42) were all seropositive, 71% had antigenemia (greater than 2 ng/mL), and 82% had proviral DNA in 1.5 x 10(5) alveolar macrophages. Of sheep positive by PCR, those with moderate or severe LIP (79%) had an average of 3 infected cells/10(3) alveolar macrophages by FIA. These results implicate alveolar macrophages as important target cells in the pathogenesis of OvLV-induced respiratory diseases.

Animals↗
PubMed1991

Antigen specificity of antibody responses to Corynebacterium pseudotuberculosis in naturally infected sheep with caseous lymphadenitis.

Constituents of Corynebacterium pseudotuberculosis were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis of sonicated whole bacterial cells and ether-extracted cells revealed more than 35 bands in silver-stained gels. SDS-PAGE analysis of concentrated culture filtrates with exotoxin activity demonstrated more than 15 bands. Sera from sheep with C. pseudotuberculosis-induced disease of variable severity were used to probe immunoblots of electrophoresed ether-extracted cells and culture filtrates. Twenty or more corynebacterial molecules, ranging in molecular weight from 20 to 112 kDa, in ether-extracted cells were recognized by antibodies in the sera of naturally exposed sheep with positive ELISA titers. These sera also recognized up to six molecules, ranging from, 20 to 68.1 kDa, on immunoblots of ammonium sulfate-concentrated culture filtrate. There was no apparent relationship between the stage of disease and the response to specific corynebacterial antigens in these animals.

Analysis of Variance↗