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Biomedical subjects

A L Ford

Publications and source records attributed to A L Ford.

17 recordsLinked to original sources
PubMed1998

Central nervous system microglial cell activation and proliferation follows direct interaction with tissue-infiltrating T cell blasts.

Central nervous system (CNS)-resident macrophages (microglia) normally express negligible or low level MHC class II, but this is up-regulated in graft-vs-host disease (GvHD), in which a sparse CNS T cell infiltrate is observed. Relative to microglia from the normal CNS, those from the GvHD-affected CNS exhibited a 5-fold up-regulation of characteristically low CD45, MHC class II expression was increased 10- to 20-fold, and microglial cell recoveries were enhanced substantially. Immunohistologic analysis revealed CD4+ alphabetaTCR+CD2+ T cells scattered infrequently throughout the CNS parenchyme, 90% of which were blast cells of donor origin. An unusual clustering of activated microglia expressing strongly enhanced levels of CD11b/c and MHC class II was a feature of the GvHD-affected CNS, and despite the paucity of T lymphocytes present, activated microglial cell clusters were invariably intimately associated with these T cells. Moreover, 70% of T cells in the CNS were associated with single or clustered MHC class II+ microglia, and interacting cells were predominantly deep within the tissue parenchyme. Approximately 3.7% of the microglia that were freshly isolated from the GvHD-affected CNS were cycling, and proliferating cell nuclear Ag-positive microglia were detected in situ. Microglia from GvHD-affected animals sorted to purity by flow cytometry and cultured, extended long complex processes, exhibited spineous processes, and were phagocytic and highly motile. These outcomes are consistent with direct tissue macrophage-T cell interactions in situ that lead to activation, proliferation, and expansion of the responding tissue-resident cell.

Animals↗
PubMed1998

Anti-centromere autoantibody in a patient evolving from a lupus/Sjögren's overlap to the CREST variant of scleroderma.

We characterized the development of the anti-centromere antibody in a patient prior to the development of CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) symptoms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting (IgG and IgM) of cellular extracts enriched for centromere antigens and indirect immunofluorescence were used to study the anti-centromere immune response. The sera recognized 3 centromere antigens with molecular masses 18,000 (CENP-A), 50,000 (CENP-D), and 80,000 (CENP-B). For CENP-A, IgM was present before the appearance of the IgG response. Anti-CENP-D revealed an IgM response that decreased over time but no IgG, while CENP-B showed an IgG response that strengthened and then weakened over time. The appearance of an anti-centromere nuclear fluorescence pattern correlated with the appearance of IgG anti-CENP-A. Signs and symptoms typical of CREST began about 4 years after antibodies to centromere antigens were found. The development of the CREST syndrome in our patient was preceded by the appearance of anti-centromere autoantibodies. For at least one of the antigens (CENP-A), there was an immunoglobulin class switch from IgM to IgG.

Adult↗
PubMed1996

Microglia induce CD4 T lymphocyte final effector function and death.

Microglia, a type of tissue macrophage, are the only cells in the central nervous system (CNS) parenchyma to express some major histocompatibility complex (MHC) class II constitutively or to upregulate expression readily. They are thought to play a role in CD4 T cell activation in autoimmune diseases such as multiple sclerosis, as well as in neurodegenerative conditions, Alzheimer's disease in particular. We show here that highly purified MHC class II+ microglia when tested directly ex vivo do indeed support an effector response by an encephalitogenic myelin basic protein-reactive CD4 T cell line from which production of the proinflammatory cytokines, interferon gamma and tumor necrosis factor, is elicited, but not interleukin (IL)-2 secretion or proliferation. After this interaction, the T cells die by apoptosis. Other nonmicroglial but CNS-associated macrophages isolated in parallel stimulate full T cell activation, including IL-2 production, proliferation, and support T cell survival. Neither CNS-derived population expresses B7.1/B7.2. Resident macrophages that terminate effector T cells in tissues constitute a novel and broadly applicable regulatory measure of particular relevance to processes of self-tolerance against sequestered antigens.

Animals↗
PubMed1996

Tissue digestion with dispase substantially reduces lymphocyte and macrophage cell-surface antigen expression.

Dispase was used to digest central nervous system (CNS) tissue for isolation of the fixed tissue macrophage population (CNS microglia) and other leucocytes present. An apparent reduction in expression of some leucocyte cell surface markers was investigated further by addition of CD45b allotype-marked post-activated CD4+ T cells of known phenotype to CNS tissue preparations derived from a CD45a-expressing rat strain, before enzymatic treatment. Assessment of these T cells for expression of a range of surface molecules after completion of the isolation procedure revealed almost complete loss of expression of CD4 and CD25 and reduced expression of a number of other surface antigens.

Animals↗
PubMed1995

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.

Animals↗
PubMed1995

Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.

Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4+ T cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45lowCD11b/c+ is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45highCD11b/c+) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45highCD11b/c+ transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4+ myelin basic protein (MBP)-reactive T cells. CD45highCD11b/c+ CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture, suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4+ T cells to proliferate or secrete IL-2.

Animals↗
PubMed1995

Developmental progression of Gpd expression from the inactive X chromosome of the Virginia opossum.

Metatherian (marsupial) mammals possess a non-random form of X-chromosome inactivation in which the paternally-derived X is always the one inactivated. To examine the progression of X-linked gene expression during metatherian development, we compared relative levels of the maternally and paternally encoded Gpd gene products in heterozygous female Virginia opossums (Didelphis virginiana) across a major portion of the developmental period. Panels of tissues obtained from fetuses, newborns, and pouch young were examined via polyacrylamide gel electrophoresis of the G6PD protein. As in adults, G6PD phenotypes in these developmental stages were highly skewed in favor of the maternal allele product, but in some tissues there was a marked increase in paternal allele expression with advancing developmental age. However, even by 42 days of post-partum development, expression of the paternal Gpd allele had not attained the adult, tissue-specific activity pattern. Our findings indicate remarkable developmental changes in the activity of the paternal allele in several tissues/organs continuing well into mid pouch-life stages and beyond. Specifically we found that 1) a substantially repressed paternal Gpd gene is present in the cells of female stage 29 fetuses and later developmental stages, 2) the activity state of the paternal Gpd gene is not fixed during early embryonic development in this species, 3) major changes in paternal Gpd expression occur in advanced developmental stages and comprise a maturation of the gene expression pattern during ontogeny, and 4) alterations of paternal Gpd allele activity during development occur in a tissue-specific manner.

Alleles↗
PubMed1995

Flow cytometric identification of a minority population of MHC class II positive cells in the normal rat retina distinct from CD45lowCD11b/c+CD4low parenchymal microglia.

AIMS: This study aimed to isolate and classify by flow cytometry, the cell surface phenotype of microglia in the normal rat retina with a view to identifying putative antigen presenting cells (APC) within the retina, which has to date not been possible by immunohistochemistry. METHODS: Normal rat retinal microglia were isolated and classified using a modification of an isolation technique employing graduated Percoll density gradient cell separation and flow cytometric phenotypic criteria used for CNS microglia. RESULTS: Retinal microglia can be defined by flow cytometry on the basis of their CD45lowCD11b/c+CD4low cell surface expression. Constitutive MHC class II expression in the normal rat retina was confined almost exclusively to a very minor population of cells expressing neither low (microglia) nor high levels of CD45. Three colour flow cytometric analysis confirmed that these MHC class II positive cells were ED2+. CONCLUSIONS: Using this sensitive isolation technique we have identified the cell surface characteristics of ramified, resident microglia, and found that they do not constitutively express MHC class II. There is, however, constitutive MHC class II expression on a phenotypically distinct population of cells (CD45low/highED2+). We propose these cells are the counterpart of the perivascular macrophages found in the CNS which present antigen to extravasating T cells, although their exact retinal location can only be confirmed by immunohistochemical analysis. The role of parenchymal microglia as APC remains undefined. Future isolation of microglia and putative perivascular cells using this technique will help identify the role these cells play in the initiation and perpetuation of immune responses within the retina.

Animals↗
PubMed1993

Schwann cells are able to present exogenous mycobacterial hsp70 to antigen-specific T lymphocytes.

Peripheral nerves are frequently damaged during infection with Mycobacterium leprae. Although Schwann cells are host for this obligate intracellular parasite, the mechanisms of immunopathology are unresolved. This study examines the ability of Lewis rat Schwann cells to present an exogenous Mycobacterium leprae protein, the heat shock protein 70 (hsp70), to antigen-specific T lymphocytes isolated from the lymph nodes of immunised rats. Secondary reactivation of hsp70-specific T lymphocytes occurred producing an antigen-specific lymphoproliferative response. This was inhibited by monoclonal antibodies against rat major histocompatibility complex (MHC) class II molecules, but not antibodies against MHC class I molecules. Coculture of Schwann cells with the M.leprae hsp70-specific T lymphocytes and antigen (MLrp70) induced the expression of MHC class II molecules on the Schwann cell's surface. Although M.leprae hsp70 is immunodominant in the host response to the bacillus, there is a high degree of homology between human and M.leprae hsp70. The M.leprae hsp70-specific T lymphocytes also recognised human hsp70 presented by Schwann cells confirming that antigenic determinants are conserved between the proteins. The ability of Schwann cells to present protein antigens in an MHC class II-restricted manner, to antigen-specific T lymphocytes involved in surveillance of the peripheral nervous system, may play an important role in the activation of an immunological reaction associated with nerve damage seen in tuberculoid leprosy.

Animals↗
PubMed1992

Production of polyacrylamide gradient gels for the electrophoretic resolution of lipoproteins.

We describe a protocol to cast nondenaturing polyacrylamide gradient gels (SFBR3/31) for the size resolution of lipoproteins. The protocol yields gels with minimal lot-to-lot variation in length and electrophoretic properties. Absorbance profiles of cholesterol-stained lipoproteins in baboon sera were used to estimate the relative amounts of stain in four lipoprotein size classes (VLDL+LDL, HDL1, HDL2, and HDL3). When compared with gels from a commercial source, the SFBR3/31 gels gave very similar results in terms of precision (coefficients of variation) and of estimated amounts of lipoproteins in the four size classes. In other studies, we estimated peak diameters of protein-stained human lipoproteins after calibrating the gels with size standards. Peak diameters estimated using SFBR3/31 gels were highly correlated (r2 = 0.99, n = 33) with those estimated using gels from a commercial source. We conclude that the protocol reliably produces gradient gels that are suitable for the analysis of lipoprotein phenotypes.

Acrylic Resins↗
PubMed1990

Biochemical characteristics and subcellular localizations of rat liver neuraminidase isozymes: a paradox resolved.

A striking discrepancy in the abilities of two analytical approaches (fluorometric and electrophoretic) to detect the effect of a gene, Neu-2, on rat liver neuraminidase phenotypes led us to examine the biochemical and physical properties of the liver isozymes NEU-1 and NEU-2 that might be responsible for this difference. Cell fractionation via Percoll gradient centrifugation revealed NEU-1 activity almost exclusively in the lysosomal cell fraction, while NEU-2 was strictly cytosolic in distribution. The two isozymes were also found to differ in pH activity curves and optima (optima: 4.6-4.8 and 5.4-5.8 for NEU-1 and NEU-2, respectively) and in solubility characteristics (NEU-2 highly soluble; NEU-1 relatively insoluble but solubilized by freezing/thawing). Both isozymes were found to be freeze-thaw stable in crude, whole-cell extracts, but NEU-1 was destabilized in the enriched (partially purified) lysosomal subcellular fraction. Consideration of these properties relative to those described previously for unidentified cytosolic and membrane bound (lysosomal) rat liver neuraminidases (Tulsiani, D. R. P., and Carubelli, R., J. Biol. Chem. 245:1821, 1970) leads us to believe that NEU-2 also is destabilized by partial purification and that NEU-1 and NEU-2 have very different relative abundances within the cell. The biochemical and physical differences between NEU-1 and NEU-2 can account for the discrepant abilities of the fluorometric and electrophoretic approaches to detect the effects of Neu-2. Ways to increase the sensitivity of the fluorometric approach for quantitative assays of specific NEU-1 and NEU-2 activity are discussed.

Animals↗
PubMed1989

X-linked gene expression in metatherian fibroblasts: evidence from the Gpd and Pgk-A loci of the Virginia opossum and the red-necked wallaby.

Fibroblasts cultured from ear pinna biopsies of Virginia opossums (Didelphis virginiana) and red-necked wallabies (Macropus rufogriseus) were examined electrophoretically to determine the relative expression levels of the maternally and paternally derived alleles at X-linked, enzyme-coding loci. Only the maternally derived allele was expressed at the Pgk-A locus in fibroblasts of heterozygous D. virginiana (M. rufogriseus not examined), but fibroblasts of both species exhibited evidence of paternal allele expression at the Gpd locus. Furthermore, the heterozygous G6PD phenotypes in both species were skewed in favor of the maternal gene product, as expected if the paternal allele is only partially (incompletely) expressed. For M. rufogriseus this result is contrary to a previous finding which suggested equal expression of both Gpd alleles in cultured fibroblasts of this species. The present results suggest that X-linked genes in metatherian fibroblasts are subject to the same kind of determinate, paternal allele inactivation, incomplete at some loci, described previously for X-linked genes in adult tissues and that the pattern of paternal X-linked gene expression in these cells is independent of the patterns in the tissues from which the fibroblasts are derived.

Alleles↗
PubMed1987

X-linked gene expression in the Virginia opossum: differences between the paternally derived Gpd and Pgk-A loci.

Expression of X-linked glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase-A (PGK-A) in the Virginia opossum (Didelphis virginiana) was studied electrophoretically in animals from natural populations and those produced through controlled laboratory crosses. Blood from most of the wild animals exhibited a common single-banded phenotype for both enzymes. Rare variant animals, regardless of sex, exhibited single-banded phenotypes different in mobility from the common mobility class of the respective enzyme. The laboratory crosses confirmed the allelic basis for the common and rare phenotypes. Transmission of PGK-A phenotypes followed the pattern of determinate (nonrandom) inactivation of the paternally derived Pgk-A allele, and transmission of G6PD also was consistent with this pattern. A survey of tissue-specific expression of G6PD phenotypes of heterozygous females revealed, in almost all tissues, three-banded patterns skewed in favor of the allele that was expressed in blood cells. Three-banded patterns were never observed in males or in putatively homozygous females. These patterns suggest simultaneous, but unequal, expression of the maternally and paternally derived Gpd alleles within individual cells (i.e., partial paternal allele expression). The absence of such partial expression was noted in a parallel survey of females heterozygous at the Pgk-A locus. Thus, it appears that Gpd and Pgk-A are X-linked in D. virginiana and subject to preferential paternal allele inactivation, but that dosage compensation may not be complete for all paternally derived X-linked genes. The data establish the similarity between the American and Australian marsupial patterns of X-linked gene regulation and, thus, support the hypothesis that this form of dosage compensation was present in the early marsupial lineage that gave rise to these modern marsupial divisions. In addition, the data provide the first documentation of the differential expression of two X-linked genes in a single marsupial species. Because of its combination of X-linked variation, high fecundity, and short generation time, D. virginiana is a unique model for pursuing questions about marsupial gene regulation that have been difficult to approach through studies of Australian species.

Alleles↗
PubMed1986

Genetic analysis of liver neuraminidase isozymes in Rattus norvegicus: independent control of NEU-1 and NEU-2 phenotypes.

Two recently identified isozymes of neuraminidase in rat liver were examined for transmission patterns and linkage relationships, and for variation among inbred strains. The isozymes, designated neuraminidase-1 (NEU-1) and neuraminidase-2 (NEU-2), exhibited no electrophoretic mobility variants among the 22 inbred strains examined, but did possess striking interstrain variation in activity phenotypes on electrophoretic gels. The results of a backcross analysis involving the KGH and ACP strains revealed that NEU-1 and NEU-2 phenotypes are independently controlled, each by a single autosomal locus with additively acting alleles. The two loci are unlinked to one another, but the gene controlling NEU-1 is tightly linked to RT1, the rat major histocompatibility complex. This gene is almost certainly identical to Neu-1, a gene identified previously through its effect on "total" activity levels of liver neuraminidase as determined by fluorometric assay of tissue homogenates. NEU-2 and the gene controlling its phenotype were not detected by the fluorometric technique. We designate the genes controlling the NEU-1 and NEU-2 phenotypes as Neu-1 and Neu-2, respectively. Data from this and other studies place Neu-1 between Glo-1 and dw-3. The location of Neu-2 is unknown.

Animals↗
PubMed1986

Electrophoretic analysis of liver neuraminidase-1 variation in mice and additional evidence concerning the location of NEU-1.

Neuraminidase-1 (NEU-1) is one of two neuraminidase isozymes which can be detected electrophoretically in mouse liver extracts. The inheritance of variation in NEU-1 and the linkage relationships of the gene controlling this variation were studied through a backcross analysis involving the SM/J and MA/MyJ inbred strains, and by examination of NEU-1 phenotypes in three congenic strains: B10.SM, B10.SM(22R) and B10.RVB. The data indicate that NEU-1 is controlled by Neu-1, a gene previously identified by its effect on total liver neuraminidase activity in whole tissue homogenates. Analysis of the congenic strains revealed identical low activity (SM/J-type: Neu-1a/Neu-1a) NEU-1 phenotypes in all three strains. This indicates that Neu-1 lies in the segment of the SM/J-derived H-2 region that is common to all three strains: H-2E alpha to H-2D. In addition, we examined the relationship between NEU-1 and phenotypic variation in liver acid phosphatase (AP; for which a new typing method is described) and linkage order among several other enzyme-coding genes linked to H-2. In all animals that could be scored confidently for AP, the NEU-1 and AP phenotypes were concordant, adding support to the hypothesis that both phenotypes are controlled by Neu-1. Recombination rates among six H-2-linked marker loci were unexpectedly low, but were sufficient to verify the position of Upg-1 as the telomeric flanking marker relative to Glo-1, H-2 (C4), Neu-1 (Apl), Ce-2 and Pgk-2.

Acid Phosphatase↗
PubMed1979

A prediction of internship performance.

Previous studies of predictors for occupational therapy internship performance were based on the Report of Student Performance, the tool used before 1974 to evaluate students' clinical performance. The instrument was never tested for validity or reliability. No studies for prediction of clinical performance used the present Field Work Performance Report (FWPR), which was tested for validity and reliability before its adoption late in 1973. Thus, new studies using the FWPR were indicated. Grades from 16 selected college courses and the pre-occupational therapy grade-point average were studied to determine significant relationships with the physical disability intership grades of 72 students from Colorado State University. Neurology was significant at the .0004 level. Further study is necessary to confirm the present findings and to determine additional predictors for clinical performance of Colorado State University students.

Adult↗