Biomedical subjects

K C McCullough

Publications and source records attributed to K C McCullough.

At least 37 records · Page 2Linked to original sources

PubMed1999

Intermediate stages in monocyte-macrophage differentiation modulate phenotype and susceptibility to virus infection.

The kinetics of monocyte-macrophage differentiation was analysed using two Swine Workshop Cluster (SWC) CD molecules: SWC1 and SWC9. Myeloid cells were selected by labelling for the common myeloid antigen, SWC3. Confirmation of macrophage identification used acid phosphatase and phagocytosis activities. During differentiation, SWC1 was gradually lost. SWC9 was absent on monocytes but up-regulated early. Consequently, monocytes were SWC1+ SWC9- and macrophages were SWC1- SWC9+. An additional, intermediate, cell population was identified as SWC1+ SWC9+. Size and granularity characteristics mirrored the monocyte, macrophage and intermediate-cell phenotypes. Overall, SWC9 up-regulation was central in macrophage differentiation and dependent on plasma factors. The concomitant loss of SWC1 was independent of these factors, but always associated with mature macrophages. Upon up-regulation of SWC9, the SWC1+ SWC9+ intermediate monocytic cells became susceptible to African swine fever virus infection. These results demonstrate the heterogeneity of monocytic cell differentiation and the importance of these characteristics for interaction with monocytotropic viruses.

African Swine Fever↗

PubMed1999

Rapidity of specific antibody-antigen interactions.

Analysis of humoral immune responses against viruses has concentrated on studies with serum dilutions, which reflect characteristics pertaining to the diluent buffer but not the serum environment. The majority of virus-specific antibody in serum from foot-and-mouth disease virus (FMDV)-vaccinated cattle bound to antigen within 10-60 s, whereas aspecific reactions evolved more slowly. Upon dilution of sera, the reaction characteristics no longer related to those obtained with the serum, particularly when individual animals were compared. Diagnostic enzyme-linked immunosorbent assay (ELISA) - employing diluted serum - identified little variation in the reactivity of serum samples from different vaccinated cattle. This related to previous analyses showing similar specific antibody titres. In contrast, analyses of serum reactivities over a 10- and 60-s incubation period demonstrated high variation between individual animals. Furthermore, when a challenge infection was performed on vaccinated animals, only those with the higher serum reactivities over a 10- and 60-s incubation were protected. These results demonstrate the importance of the specific serum antibody reactions, which will occur within seconds. Moreover, such qualitative characteristics would be overlooked when employing conventional assays with diluted sera and long incubation periods.

Animals↗

PubMed1998

Low density blood granulocytic cells induced during classical swine fever are targets for virus infection.

Classical swine fever virus infection of pigs causes a severe leukopenia and immunosuppression. In the present study, the kinetics of virus infection, and identification of target cells for the virus in peripheral blood were analysed. Virus infection was often not detectable before 5-7 days p.i. A minority of animals yielded detectable infected cells at 3 days p.i., but < 5% PBMC. It was not until 10 days p.i. that this figure increased-to 35-70% PBMC depending on the animal. Detailed analysis of Ficoll-Hypaque-purified PBMC identified the major population to be SWC3+SWC8+CD14+MHCII- granulocytic cells. Microscopic observations determined that these low density granulocytic cells in the PBMC from CSFV infected animals were indeed immature cells. Both the low density granulocytic cells and monocytes were major targets for CSFV infection in the peripheral blood. This is the first demonstration that low density granulocytic cells dominate the blood leukocyte population during CSF, and that such cells are targets for virus infection. The present work also demonstrates that the leukocyte population changes, such as B lymphocyte depletion and the relative dominance of myeloid cells in the blood during CSF, occur before virus infection of the affected cells. Thus, the pathological mechanism therein is not a direct consequence of virus infection.

Animals↗

PubMed1998

Macrophage culture: influence of species-specific incubation temperature.

Cultured mammalian cells are traditionally maintained at 37 degrees C, despite the fact that core body temperatures differ considerably among mammals. Considering the body temperature of the adult pig, comparison was made of porcine macrophage cultures maintained at 37 degrees C and 39.2 degrees C. Examination of the cells showed that granularity was higher in macrophages maintained at 39.2 degrees C, although no differences in cell size were observed. The density of MHC Class I and II expression was higher on cells maintained at 39.2 degrees C, as was the percentage of MHC Class II positive cells. In contrast, expression of CD44 and CD11a/18 remained unchanged. Following stimulation with lipopolysaccharide, only cells maintained at 39.2 degrees C produced detectable levels of TNF-alpha. As a final reference criterion, replication of the macrophage tropic African swine fever virus was monitored. At 39.2 degrees C, virus antigen production was less efficient, and virus isolate-related differences in the replication kinetics were observed. Infectious virus production was not different at the two temperatures, implying that virus maturation may have been more efficient at the higher temperature. These results indicate that incubation of cultured cells at the temperature of their donor species has an important influence on their characteristics.

African Swine Fever Virus↗

PubMed1998

Macrophage cytoplasmic vesicle pH gradients and vacuolar H+-ATPase activities relative to virus infection.

A number of viruses replicate in macrophages, some having an obligate requirement for a macrophage host. This raised the question concerning the role of the macrophage endosomal/lysosomal compartment during such infections. Both lysosomotropic weak bases, amantadine and chloroquine, which interfere with endosomal/lysosomal pH gradients, and the macrolide antibiotic bafilomycin A1, which interferes with vacuolar H+-ATPase, inhibited African swine fever (ASF) virus replication in porcine macrophages. This inhibition was reversible: replenishment of bafilomycin, but not amantadine or chloroquine, maintained the inhibition. The characteristics of the inhibition, and the capacity of virus to escape and re-commence replication, related to the capacity of each drug to interfere with the endosomal/lysosomal proton pump. These results demonstrate that the virus actually requires macrophage endosomal/lysosomal activity for its replication. Therein, vacuolar H+-ATPase activity is particularly critical for successful virus replication, which is interesting considering the importance of this for endosomal/lysosomal activity and macrophage function.

Acridine Orange↗

PubMed1998

Differential adhesion molecule expression on porcine mononuclear cell populations.

Adhesion molecule expression was analysed on porcine blood and lymphoid organ CD4+ CD8 naive T helper (Th) lymphocytes, CD4+CD8+ memory Th lymphocytes (particular to the pig), CD4-CD8high cytotoxic T (Tc) lymphocytes, CD4 CD8low NK cells (CD3- in the pig), CD4-CD8- T-cell receptor-gammadelta-positive (TCRgammadelta+) lymphocytes, B lymphocytes and monocytes. While CD44 expression was relatively homogeneous amongst mononuclear cells, differences were noted for the integrins. Blood naive Th lymphocytes were CD49d(low)CD11a(low), as were splenic naive Th cells; blood memory Th lymphocytes were CD49d(high)CD11a(low), splenic memory Th cells were CD49d(high)CD11a(high) with a CD49d(high)CD11a(low) subpopulation; blood Tc lymphocytes were mainly CD49d(low)CD11a(low), and splenic cells were CD49d(high) CD11a(high). Lymph node lymphocytes were more homogeneous in their integrin expression. These were relatively CD49d(low)CD11a(low), except the memory Th lymphocytes which had higher integrin expression. B lymphocytes related to the majority of integrin(low) T cells, while monocytes and NK cells were CD49d(high) CD11a(high); gammadelta T lymphocytes showed variable CD49d expression but a CD11a(high) phenotype. CD49d(high) CD11a(high) co-expression was found, and this phenotype was typical of, but not exclusive to, CD25+ (activated) lymphocytes. These results demonstrated that porcine memory Th lymphocytes and NK cells, as well as activated cells, would have increased integrin-dependent activities compared with naive Th lymphocytes, and integrin-dependent reactions would probably vary between blood and lymphoid organ cells.

Animals↗

PubMed1998

Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death.

Infection of pigs with classical swine fever virus (CSFV), a member of the Flaviviridae family, causes a severe leukopenia, particularly notable with the lymphocytes. The goal of this study was to analyze mechanisms behind this CSFV-induced lymphopenia. To this end, the kinetics of leukocyte depletion, the appearance of apoptotic cells, and virus infection of leukocytes after infection of pigs with the virulent CSFV strain Brescia were analyzed. Depletion of B and T lymphocytes was noted as early as 1 day postinfection (p.i.). Circulating viable lymphocytes with reduced mitochondrial transmembrane potential--a particular early marker for apoptosis--were also detectable as early as 1 day p.i. When isolated peripheral blood mononuclear cells were cultured for 6 h, significantly more sub-G1 cells with reduced DNA content were detected among the lymphocytes from CSFV-infected animals, again as early as 1 to 3 days p.i. The first time virus was first found in the plasma, as well as infection of leukocytes, was 3 days p.i. However, throughout the observation time of 1 week, <3% of the circulating leukocytes and no lymphocytes contained virus or viral antigen. Further analysis of the T lymphocytes from infected animals demonstrated an increase in CD49d, major histocompatibility complex class II, and Fas expression. An increased susceptibility to apoptosis in vitro was also observed, particularly after addition of concanavalin A as well as apoptosis-inducing anti-Fas antibody to the cultures. Taken together, these results imply that activation-induced programmed cell death was the mechanism behind lymphopenia during classical swine fever.

Animals↗

PubMed1997

Phenotype of porcine monocytic cells: modulation of surface molecule expression upon monocyte differentiation into macrophages.

When porcine blood monocytes differentiate in vitro into macrophages, their morphology, as well as side scatter and forward scatter measured by flow cytometry, changed in a manner similar to that with human cells. During this differentiation, the initial high expression of CD molecules on porcine monocytes was down-regulated, with one exception--SWC9. Freshly isolated blood monocytes were SWC9-, but after culture the cells had become SWC9+. Thus, porcine monocytes were characterised as SWC3+SWC9-CD14high; macrophages were SWC3+SWC9+CD14low, the latter also displaying a down-regulation of CD11a/18, and, to a lesser degree, CD44. Both SWC9-CD14high monocytes and SWC9+CD14low macrophages were identifiable in freshly prepared monocytic cells from the spleen. Alveolar macrophages, on the other hand, were dominated by SWC9+CD14low cells, similar in phenotype to in vitro monocyte-derived macrophages. The consequences which these results have for studies on virus infections of porcine monocytic cells are discussed.

Animals↗

PubMed1997

Porcine bone marrow myeloid cells: phenotype and adhesion molecule expression.

The purpose of this study was the discrimination of porcine bone marrow hematopoietic cell (BMHC) subpopulations and cell maturation stages by using cluster of differentiation (CD) and Swine Workshop Cluster (SWC) molecules, combined with cell size, granularity, and the capacity to generate an oxidative burst. SWC3 was the earliest myeloid marker identified in the differentiation pathways. Through SWC3/SWC8 double labeling, three populations committed to the myeloid lineage were discriminated: early myeloid progenitors, which were SWC3lowSWC8-; cells committed to the granulocytic lineage, which were SWC3+SWC8+; monocytic cells, which were SWC3+(-)SWC8-. The SWC3lowSWC8- myeloid progenitors expressed high levels of CD44 and CD49d, but no CD14. Among the SWC3+SWC8+ granulocytic cells, the most immature were CD14-CD11a/18-CD44low, an intermediate stage was CD14+CD11a/18lowCD44-, and the most mature were CD14+CD11a/18high-CD44high. Only the latter could generate a rapid oxidative burst. Moreover, during granulocytic maturation, a constant decrease in CD49d/29 expression was observed. In contrast, the SWC3+SWC8- monocytic cells were mainly CD14+ and expressed high levels of the adhesion molecules. These results define porcine hematopoietic differentiation and maturation stages. Similarities in human and porcine adhesion molecule expression have been identified, suggesting common functionalities in the regulation of hematopoiesis and the potential of the pig as a model for hematopoietic studies.

Animals↗

PubMed1997

Rinderpest virus isolates of different virulence vary in their capacity to infect bovine monocytes and macrophages.

Three isolates of rinderpest virus (RPV) with different in vivo virulence were able to infect and productively replicate in bovine monocytic cells. They differed in their kinetics of replication and the morphological changes induced in infected cultures. The highly virulent RPV-Saudi infected > 80% of cells within 6 days p.i. (m.o.i. = 0.1 TCID50 per cell). Under identical conditions, > 50% of cells were infected by the 'mild' (causes minimal mortality in vivo) isolate RPV-Egypt, whereas only 25% were infected by the avirulent RPV-RBOK. Infection by all three viruses produced infectious progeny, induced the formation of syncytia and stellate cells with long processes, and down-regulated MHC class II expression; there was no apparent effect on MHC class I nor LFA-1. RPV-Saudi was the most efficient at generating progeny virus and producing syncytia. While RPV-RBOK was the least efficient at inducing syncytia, RPV-Egypt was the least efficient for progeny virus production. In contrast, RPV-Egypt was particularly efficient at inducing stellate cell formation and down-regulating MHC class II expression. These results indicate a relationship between in vivo virulence and the characteristics of replication and induced morphological changes in monocytes/macrophages. The down-regulation of MHC class II expression would offer a means by which the virus could evade immune recognition. This would be particularly useful for the more cell-associated, but less efficient at maturing, RPV-Egypt.

Animals↗

PubMed1996

Relative ability of different bovine leukocyte populations to support active replication of rinderpest virus.

Bovine peripheral blood mononuclear cells (PBMC) were infected with the pathogenic Saudi isolate of rinderpest virus (RPV) in order to identify the cell subpopulation(s) susceptible to active replication of this virus. Flow cytometry analysis, using a monoclonal antibody recognizing the H glycoprotein of RPV, showed that monocytes were the main subpopulation in which the virus replicated, whereas <2% of lymphocytes expressed viral antigen. The activation of PBMC with concanavalin A before infection resulted in an increase in the capacity of lymphocytes to support RPV replication; >90% of CD4+ and CD8+ T lymphocytes expressed viral antigen at 3 days postinfection, although < or = 40% of gamma/delta T cells were productively infected. B-lymphocyte activation with pokeweed mitogen also resulted in increased replication of this virus in these cells, involving up to 40% of B lymphocytes. An enhancement of lymphocyte susceptibility to infection and active replication by RPV was observed upon coculture of RPV-infected PBMC on bovine endothelial cells. Such enhancement was most marked with the B-cell and CD4+ T-cell subpopulations. Contact between lymphocytes and extracellular matrix components did not alter the capacity of RPV to replicate in lymphocytes. This intercellular contact with endothelial cells increased the viability of certain lymphocyte subpopulations, but it alone could not explain the increased sensitivity to RPV. Intercellular signalling, which resulted in interleukin-2 receptor upregulation, probably played a role. In summary, monocytes are the main target for active, productive infection by RPV. Similar replication in lymphocytes depends on their activation state and on contact with accessory cells such as endothelial cells. These characteristics have important implications for virus traffic in vivo and the pathogenesis of this disease.

Animals↗

PubMed1995

Influence of antigen presentation and exogenous cytokine activity during in vitro primary immunizations employed for the generation of monoclonal antibodies.

Hybridomas secreting monoclonal antibodies (MAbs) against African horse sickness virus (AHSV) were generated using different AHSV antigen preparations (inactivated AHSV, semi-purified virus, and a preparation of nonstructural viral proteins) in one of three different in vitro primary immunization systems: (i) the Cel-prime kit, a method using immunization of splenocytes aided by antigen-primed support cells; (ii) a system based on a cytokine soup derived from a mixed lymphocyte reaction plus stimulated EL4-IL-2 cells; (iii) a system based on a cytokine soup derived from splenocytes stimulated by pokeweed mitogen in order to obtain a mixture of cytokines enriched for Th2 lymphokines. The viability of immunized BALB/c mouse splenocytes, immunoglobulin production by the subsequently generated hybridomas, and the specificity of the MAbs were compared. The most efficient in vitro primary immunization system was the Cel-prime system employing semi-purified antigen. This efficiency was manifest in terms of a greater viability of the splenocytes in the immunization, as well as a higher number of specific antibody-secreting hybridomas. It seems probable that the support cells of the Cel-prime system have an accessory function such as that attributed to antigen-presenting cells. Such a function would result in impairment of apoptosis, and thus increase the viability of the splenocytes in the in vitro primary immunization system, as well as enhancing stimulation of the immune response against the antigen used. The presence of cytokines at the beginning of the in vitro primary immunization did have an influence, but this was secondary to what appeared to be the major event of cellular interaction associated with the accessory cell function of the support cells.

African Horse Sickness Virus↗

PubMed1995

Rinderpest virus infection of bovine peripheral blood monocytes.

The ability of rinderpest virus (RPV) to replicate in vitro in adherent peripheral blood monocytes and monocyte-derived macrophages under non-stimulation conditions was investigated. When flow cytometry analysis on bovine peripheral blood mononuclear cells (PBMC) was performed, monocytic cells were seen to be targets for infection by the cell culture-attenuated RBOK vaccine strain of RPV. Viral glycoprotein (H) and nucleoprotein (N) expression in adherent blood monocytes and monocyte-derived macrophages was compared with the infection in Vero cells, in which a productive infection typical of morbilliviruses is obtained. In both cell types, the infection was m.o.i.-dependent, but the rate of viral protein accumulation was slower in monocytes/macrophages. Double-labelling experiments with monoclonal antibodies against RPV and the myeloid marker CD14 confirmed that the infected blood adherent cells were monocytes and macrophages. Productive infection of monocytes was confirmed by progeny virus titration. Permissiveness to infection was not dependent on macrophage differentiation: in vitro maturation of monocytes to macrophages before infection, did not increase the susceptibility of these cells to RPV infection. With the virulent Saudi RPV isolate, similar results were obtained, although the Saudi virus apparently had a higher rate of replication compared to the attenuated virus. These observations demonstrate clearly that bovine blood monocytes and monocyte-derived macrophages serve as hosts for a relatively slow but productive infection by rinderpest virus.

Animals↗

PubMed1993

Rapid generation of monoclonal antibody-secreting hybridomas against African horse sickness virus by in vitro immunization and the fusion/cloning technique.

Splenocytes from non-immune mice were stimulated in vitro using an equimolar mixture of factors from mixed lymphocyte reaction (MLR) and from phorbol-12-myristate 13-acetate (PMA) stimulated EL-4 cells, and concomitantly immunized with inactivated African horse sickness virus (AHSV) antigen serotype 4 or viral proteins 2 and 5 from AHSV serotype 9. Fusion with NSO myeloma cells was performed five days after primary or secondary stimulation/immunization. The record of hybridoma growth after a standard method of fusion, expansion of cells and subsequent cloning was compared with a fusion/cloning method in which cells were cloned within 2 to 3 days of the fusion event. Detection of antigen specific antibodies in the hybridoma culture supernatants was successful only with cells derived from primary stimulation/immunizations. Antibodies were detected using an indirect ELISA with the immunizing antigen coated on to the surface of the plates. Monoclonal hybridomas were isolated within 2 to 3 weeks using the fusion/cloning method, compared with the standard method, where it took 4 to 5 weeks. Although the total number of clones isolated from the fusion/cloning method was less than that obtained through the standard method, the yield of specific antibody-producing hybridomas as a percentage of the total picked was often more efficient with the fusion/cloning method. With respect to the immunoglobulin isotype produced, not all of the antibodies could be classified by the ELISA system used; 14% of anti-AHSV positive clones were identified as IgG-secreting cells, 25% as IgM-secreting, 18% were cross-reacting with IgG and IgM, and 43% could not be classified. Similar results in all aspects of the work were obtained whether a crude infected cell extract or purified outer capsid polypeptides VP2/5, from serotype 4 and serotype 9 respectively, were used.

African Horse Sickness Virus↗

PubMed1993

The relative density of CD44-positive porcine monocytic cell populations varies between isolations and upon culture and influences susceptibility to infection by African swine fever virus.

African swine fever (ASF) virus has been reported to infect cells of the monocyte family, probably macrophage-like cells, but there is variation in the apparent susceptibility of these cells. We have demonstrated that the phenotype and activity of porcine monocytic cells varies between different isolations and also upon culture. The variation during culture is dependent upon the phenotype of the cells at the time of isolation. As for the susceptibility of porcine monocytes/macrophages to infection by ASF virus, it was seen that this could be related to the variation in cell phenotype and activity. The susceptibility was determined by the relative density of particular subpopulations of cells present. Whilst inflammatory macrophages did not have an apparent role to play, phagocytic activity was influential. Furthermore, the expression of CD44 and the DH59 myeloid cell marker was important, whereas the relevance of MHC Class II expression was variable. Overall, it was concluded that susceptibility to infection required that a culture be dominated by CD44-positive cells which were non-inflammatory, of low phagocytic activity, and characterizable as being of the myeloid (DH59-positive) lineage.

African Swine Fever↗

PubMed1993

Identification of epitope expression on the internal proteins of rinderpest virus which is dependent upon virion maturation events.

Monoclonal antibodies (MAb) identified the existence of both maturation-dependent and maturation-independent epitopes on rinderpest virus antigens. The former were divided into (i) post-maturation antigenic determinants, which were dependent upon the maturation of viral antigen into complete virions; and (ii) pre-maturation antigenic determinants, which were only expressed on what appeared to be immature particles before 'budding' into the extracellular environment. Epitope expression could be related to the kinetics of virus production, with the 'post-maturation' sites requiring the production of mature/infectious virions, but the 'pre-maturation' sites being lost when mature virus was formed (these 'pre-maturation' determinants were strongly cell-associated). MAb against the different virion proteins of measles virus, when reacting with rinderpest virus did not demonstrate the same relationship to virion maturation as did the anti-rinderpest virus MAb: the anti-measles virus MAb detected maturation-independent epitopes. This work demonstrates the caution which should be taken when preparing antigens for diagnostic and epidemiological purposes, especially when MAb are being used to identify antigenic differences between isolates, and/or to compare antigenically isolates with vaccine viruses.

Animals↗

PubMed1993

Seroepidemiological study of African horse sickness virus in The Gambia.

An indirect enzyme-linked immunosorbent assay was used for the screening of horse sera from The Gambia for antibodies against African horse sickness virus (AHSV). The AHSV antigen used for coating was semipurified according to the method of Manning and Chen (Curr. Microbiol. 4:381, 1980); control mock-infected Vero cell antigen was treated in the same manner. A total of 459 horse serum samples were assayed at a single dilution (1:10), and their reactivities were compared with those of reference positive anti-AHSV and reference negative horse sera. A total of 81% of the horse serum samples clearly contained antibodies against AHSV; this consisted of 18% (of the total number of serum samples) strongly positive, 46.5% moderately positive, and 16.5% weakly but still clearly positive. Such results suggest a high prevalence of AHSV in the regions from whence the samples originated. Reports from investigations in other countries in this area of West Africa have also shown a high prevalence for anti-AHSV antibodies in equids. The question is raised as to how the animals became seropositive and whether the observations represent an increased resistance of horses living in a region in which AHS is enzootic.

African Horse Sickness↗

PubMed1993

The application of biotechnology to the diagnosis and control of animal diseases.

Methodologies for animal disease diagnosis and the analysis and control of animal health currently make much use of immunoassay techniques, in particular ELISA, and tests based on nucleic acid measurement (nucleic acid hybridisation, polymerase chain reaction [PCR]). In the latter area, the recently developed PCRs have become most useful. Recombinant technologies have yielded defined and extremely pure antigens, which can give more accurate interpretations of results obtained using diagnostic tests; in addition, such antigens obviate the need to handle live pathogens, except where these may be contained within the sample for diagnosis. The application of monoclonal antibodies (MAbs) to many tests, in particular those which use immunoassay bases or perform molecular fingerprinting of isolates, has increased the specificity and usefulness of these methodologies. Modern technology has permitted a considerable reduction in the use of animals for MAb generation through in vitro immunisation methods and the application of serum-free media with bio-reactors. These in vitro immunisation procedures have an additional application in the analysis of vaccine efficacy without recourse to animal experimentation. Furthermore, in vitro methods can be used to monitor the immune response in an animal and, in particular, to measure the development of immunological memory. It is now possible to estimate more accurately the quality and duration of an induced immune response without using animals for testing. Not only are the assay systems developed through biotechnology more sensitive and less hazardous than previous techniques (especially with regard to the necessity of handling live pathogens), but they can also determine more accurately the immune responsiveness or immune status of the animals under study.

Animal Diseases↗