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Biomedical subjects

C C Hughes

Publications and source records attributed to C C Hughes.

15 recordsLinked to original sources
PubMed1992

Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier.

Primary culture of rat brain endothelial cells is described, based on the method of C. C. W. Hughes and P. L. Lantos. The cells have been characterised using morphological and immunocytochemical techniques, and systematic studies undertaken to determine the optimal culture medium and conditions required to grow the cells at high purity on a variety of substrata. The endothelial cells have a spindle-shaped morphology, and proliferate as plaques from small clusters of cells associated with capillary fragments in the starting material. Tight junction-like cell:cell appositions are seen at the electron-microscopic level. The cells show characteristic staining for antigens recognized by antibodies against von Willebrand factor (Factor VIII-related antigen), angiotensin-converting enzyme (ACE), the transferrin receptor (Ox-26), actin and vimentin. They also show binding of the lectin from Ulex europaeus (UEA I). Potential contaminating cells include smooth muscle, fibroblasts, pericytes and meningeal cells. Contaminants can be kept to < ca. 5% by careful removal of large vessels and meninges during dissection, by brief treatment with Ca(2+)- and Mg(2+)-free saline, by growth in medium supplemented with plasma-derived serum treated for removal of platelet-derived growth factor (PDGF), and by occasional use of medium in which D-valine is substituted for L-valine. Cells attach well to collagen-coated plastic, less well to glass. Cells can be grown on transparent collagen filters (ICN, Cellagen and Costar, Transwell-Col), and on microcarrier beads (Pharmacia, Cytodex-3). The culture has proved to be a useful preparation for studies of cellular physiology, pharmacology and biochemistry of the brain endothelium, and represents a first step in producing an in vitro model of the rat blood-brain barrier.

Animals↗
PubMed1991

Endothelial cell lymphocyte function-associated antigen-3 and an unidentified ligand act in concert to provide costimulation to human peripheral blood CD4+ T cells.

Our previous studies have demonstrated that cultured human endothelial cells (EC) provide costimulation to PHA-activated CD4+ T cells, measured as augmentation of IL-2 synthesis, through a cell contact-department pathway. Here we show that fixed and living EC provide comparable degrees of costimulation to CD4+ T cell populations, indicating that EC costimulation does not depend upon active metabolism. EC achieve these effects in part by utilizing lymphocyte function-associated antigen-3 (LFA-3) to interact with T cell CD2 as shown by observations that EC augmentation of IL-2 is partially (50-70%) blocked by eight of eight mAb tested which recognize LFA-3; that purified phosphatidylinositol-linked LFA-3 (PI-LFA-3) can also provide costimulation to CD4+ T cells; and that there is a delay of the EC effect on CD4+ T cells which express low levels of CD2 compared to those which express high levels of CD2. However, three lines of evidence suggest that EC also utilize at least one additional ligand. First, there is incomplete replacement of the EC effect by PI-LFA-3 such that the costimulatory ability of EC combined with PI-LFA-3 is additive at all concentrations of PI-LFA-3 tested. Second, costimulation by PI-LFA-3, but not by EC, is fully inhibited by anti-CD2 or anti-LFA-3 mAb. Finally, costimulation by PI-LFA-3, but not by EC, is completely suppressed by cyclosporine A. We have not formally identified the second ligand but it does not appear to be intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD44, or B7/BB1.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Human coronary transplantation-associated arteriosclerosis. Evidence for a chronic immune reaction to activated graft endothelial cells.

Occlusive disease of coronary arteries of engrafted hearts is the major obstacle to long-term survival of human cardiac allografts. The pathogenesis of this process remains uncertain. The identity and localization of cells found in transplantation-associated arteriosclerosis lesions from human cardiac allografts were evaluated, and their expression of class II major histocompatibility complex (human leukocyte antigen-DR [HLA-DR]), surface molecules required for recognition of foreign cells by CD4+ T lymphocytes, was noted. Expanded intimas of transplanted coronary arteries contain T lymphocytes (both CD4+ and CD8+ in approximately equal number) and HLA-DR+ macrophages, both localized primarily in a ring immediately below the luminal endothelium, a distribution strikingly different from that in typical atherosclerosis. Coronary arterial endothelium from six of six transplanted hearts studied bore high levels of HLA-DR. Normal human arteries or usual atherosclerotic lesions have few if any HLA-DR+ endothelial cells. The significance of these findings was tested by evaluating the ability of HLA-DR+ arterial cells to interact with allogeneic T cells in vitro. Endothelial cells (but not smooth muscle cells) cultured from human arteries stimulated foreign CD4+ T cells to proliferate and augmented their secretion of interleukin-2. These findings suggest that ongoing stimulation of recipient T lymphocytes by HLA-DR+ endothelium of donor coronary arteries contributes to a sustained regional immune response. Consequent local release of cytokines may regulate smooth muscle cell proliferation and matrix accumulation within the coronary arteries of allografted hearts.

CD4 Antigens↗
PubMed1990

Antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic CD4+ T cell lines in vitro.

Experimentally induced and naturally occurring inflammatory diseases of the central nervous system (CNS) are often associated with a breakdown of the blood-brain barrier and edema within the CNS itself. CD4+ T cells are now clearly implicated in the pathogenesis of the induced CNS disease, experimental autoimmune encephalomyelitis, and previous in vivo experiments had indicated that these cells may be capable of directly damaging the CNS vasculature. To assess the capacity of CD4+ T cells to damage brain vascular endothelial cells (EC) in vitro, two lines with specificity for myelin basic protein and OVA were prepared and added to cultures of EC. We show here that both lines, when added in a resting state, severely disrupt the EC monolayers in an Ag-specific manner. The interaction is dependent on the recognition of Ag in the context of MHC class II and is blocked in the presence of mAb specific for CD4. Addition of T cell lines preactivated on irradiated thymocyte APC caused a high level of Ag nonspecific damage to the EC, which was not blocked by the addition of anti-CD4 mAb. Supernatants derived from these latter cells did not alone damage the EC monolayers despite the presence of TNF activity suggesting that T cell-EC contact may be required for these cell lines to mediate their effector function. Both resting and preactivated lines adhered strongly to the EC in the absence of Ag. The capacity of CD4+ T cells to strongly adhere to, and disrupt the integrity of, brain vascular EC may be important in the early stages of CNS disease mediated by this cell type.

Animals↗
PubMed1990

Endothelial cells augment T cell interleukin 2 production by a contact-dependent mechanism involving CD2/LFA-3 interaction.

We have demonstrated that endothelial cells (EC) augment IL-2 production by PHA-stimulated PBMC or purified CD4+ T cells and that the increase is apparent both in the amount of soluble IL-2 secreted and in the level of specific mRNA detectable by Northern blot hybridization. The ability of EC to affect levels of IL-2 cannot be reproduced by soluble factors, including the cytokines IL-1, IL-6, IFN-gamma, or TNF, conditioned medium from resting EC or IL-1, IFN-gamma- or TNF-treated EC, or from resting PBMC + EC cultures. Separation of the EC and PBMC by a Transwell membrane demonstrated that cell contact was required for augmentation of IL-2 synthesis and that this effect was unlikely to be mediated by a short-lived soluble signal. The cell-cell interaction required the ligand pair CD2/LFA-3, since augmentation could be inhibited by antibodies to these structures. Antibodies to ICAM-1, LFA-1, CD4, and MHC class II were without effect. A contact-dependent pathway involving CD2/LFA-3 interactions also may be used by EC to augment IL-2 production from T cells stimulated more specifically through the TCR/CD3 complex with antibody OKT3. This pathway provides a proliferative advantage to T cells stimulated with OKT3 in the presence of EC and may also be involved in the proliferative response of resting T cells to allogeneic class II MHC-expressing EC. We propose that EC augmentation of T cell IL-2 synthesis may be critical in the ability of EC to elicit primary T cell antigen responses and may have consequences for the development of localized cell-mediated immune reactions.

Antigens, CD↗
PubMed1990

The potential roles of vascular endothelium in immune reactions.

Cell-mediated immune reactions are initiated and regulated by antigen specific CD4+ helper T cells. However, T cells cannot function independently. In order for a CD4+ T cell to recognize antigen, it must be presented in association with a class II major histocompatibility complex molecule by another cell type and, in order to lead to functional T-cell activation, the antigen presenting cell must also provide costimulatory signals. Once activated, CD4+ T cells function in vivo by secreting cytokines that elicit an inflammatory infiltrate of other cell types that serves to eliminate the source of foreign antigen. In vivo, the development of inflammation requires vascular responses as well as contributions of blood-derived leukocytes. Although several cell types in vitro can present antigen, provide costimulation, and perform actions that contribute to inflammation, vascular endothelial cells may be uniquely important immune accessory cells because they are anatomically uniquely positioned to function in vivo during cell-mediated immune reactions. In this report, we shall review recent data from our laboratories which further characterize the immune accessory functions of endothelial cells.

Antigen-Presenting Cells↗
PubMed1990

Permeability of the blood-brain barrier to the immunosuppressive cyclic peptide cyclosporin A.

Uptake of the immunosuppressive lipophilic peptide cyclosporin A has been measured by a number of techniques. The brain uptake index (BUI) technique in the rat yields only a small BUI value that is not significantly different from that of sucrose and mannitol and is comparable to other published BUI values for this compound. Brain perfusion studies in the guinea pig produce a unidirectional cerebrovascular permeability constant (Kin) of 1.2 +/- 0.28 microliter g-1 min-1 for the hippocampus. Intravenous bolus injection techniques also in the guinea pig characteristically produce a larger Kin value of 2.53 +/- 0.38 microliter g-1 min-1 for the same brain region, even after a correction for the inulin space of the tissue has been made. Apparent penetration of cyclosporin A into the cerebrospinal fluid (CSF) determined with the intravenous bolus injection technique is small with a Kin of 0.79 +/- 0.07 microliter g-1 min-1. However it is suggested that the radioactivity present in CSF is largely tritiated water. Studies with cultured cerebral endothelial cells from the rat have also been carried out and show that the cultured cells take up and accumulate cyclosporin A in vitro, achieving a tissue-to-medium ratio of 20 after 25 min of incubation. It is suggested that cyclosporin A is primarily taken up from lipoprotein at the blood-brain interface but, because of tight junctions at the blood-brain and blood-CSF barriers, becomes effectively trapped in the cerebral endothelial cells and the choroid plexus.

Animals↗
PubMed1990

The endothelial cell as a regulator of T-cell function.

These studies have analyzed the antigen-presenting capacities of EC. EC can transcribe, translate and express MHC class II molecules in response to IFN-gamma as well as express class I molecules. The class II dimeric structure is functional, in that allospecific CTL can efficiently kill IFN-gamma-treated EC or fibroblasts, an outcome that can be blocked by antibody to non-polymorphic regions of the class II molecule. Moreover, EC can present antigen in an MHC-restricted manner to resting T cells as well as to antigen-specific cloned T-cell lines. This ability to stimulate primary as well as secondary responses has been further confirmed by experiments using purified populations of naive and memory T cells. In this regard, EC differ from fibroblasts and other non-immune cell types in that they possess costimulator activities necessary for activation of resting T cells. As the local concentration of IL-2 has been shown to be critical in determining the fate of T cells--whether they become activated or anergic--we have investigated the ability of EC to modulate T-cell IL-2 production, believing that this may underlie their ability to act as costimulatory cells. Using PHA-stimulated peripheral blood mononuclear cells or purified CD4+ T cells we have found that EC can augment IL-2 production, typically by 3- to 8-fold. This increased IL-2 production is functional as OKT3-stimulated or sub-optimally PHA-stimulated T cells proliferate more in the presence of EC than in their absence. The major pathway by which EC augment T-cell IL-2 production is cell contact-dependent and involves the CD2:LFA-3 ligand pair. However, use of blocking mAb to CD2 and LFA-3, of PI-LFA-3, and of the immunosuppressive drug CsA has allowed us to reveal the presence of a second signalling pathway. This pathway confers a certain degree of CsA resistance on T cells, but the ligands involved have not yet been identified. We do not find a role for CD28, LFA-1:ICAM-1, VLA-4:VCAM-1 or CD44 in this system. Augmentation is independent of EC metabolism or soluble factors, as fixed cells are almost as efficient as living cells. Similar mechanisms seem also to be involved in more physiological settings, such as alloresponses. Here, proliferation can be blocked by antibodies to CD2 or LFA-3, presumably by blocking of augmented IL-2 production.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Uptake of leucine and alanine by cultured cerebral capillary endothelial cells.

Pure cultures of rat cerebral capillary endothelium have been used to study the A- and L-systems of amino acid transport. Leucine is taken up by a non-concentrative mechanism that can be saturated, and competitively inhibited by phenylalanine. Uptake is rapid, with equilibration apparent after 3-5 min (all experiments performed at 37 degrees C). The Km for transport was 83 microM +/- 26 (mean +/- S.E.M., n = 3) which is in good agreement with recent in vivo reports using unanaesthetised rats. Alanine was transported by a saturable, concentrative mechanism. Dependence on Na+-ions was demonstrated by lack of specific uptake in Na+-free buffer and reduced uptake after preincubation in ouabain--a Na+,K+-ATPase inhibitor. The Km for transport was 325 microM +/- 88 (mean +/- S.E.M., n = 3). The finding of an active A-system transporter in vitro suggests that the cells may have lost the polarity they demonstrate in vivo. The relevance of these findings to transport of nutrients and drugs across the blood-brain barrier is discussed.

Alanine↗
PubMed1989

Lipid synthesis by rat brain microvessel endothelial cells in tissue culture.

Lipid synthesis and its regulation by serum lipoproteins at the microvascular blood-brain barrier were studied using primary cultures of microvascular endothelial cells from rat brain. These cells are capable of synthesizing all their lipids (neutral lipids, phospholipids, glycolipids) from the water-soluble compounds, glucose, acetate, acetoacetate and beta-hydroxybutyrate. The ketone bodies, especially acetoacetate, are the preferred substrates for lipid synthesis. The incorporation patterns of acetate, acetoacetate and beta-hydroxybutyrate are very similar, indicating that these precursors contribute to lipid synthesis via the same metabolic route. However, the metabolic pathway is different for glucose, which is preferentially incorporated into phospholipids. The existence of an inverse relationship between lipid synthesis and the serum lipoprotein concentration suggests that cultured cerebral endothelial cells are capable of taking up lipids, principally cholesterol, contained in the serum lipoproteins. Cellular lipids would thus be supplied both by intracellular lipid synthesis and by serum lipoproteins. The difference between cholesterol synthesis rates in cultured cerebral endothelial cells and in isolated brain microvessel cells could be partly explained by the fact that the lipoprotein concentration is much lower in the culture medium than in rat serum.

Acetates↗
PubMed1986

Brain capillary endothelial cells in vitro lack surface IgG Fc receptors.

A reliable technique to produce consistently pure cultures of endothelial cells prepared from isolated rat brain capillaries has been established. The cells express many of the morphological and antigenic characteristics of endothelium in vivo, including the formation of tight junctions and possession of Factor VIII/von Willebrand Factor antigen (FVIII/vWF) and angiotensin converting enzyme (ACE). Using indirect immunofluorescence, surface IgG Fc receptors (FcR) could not be demonstrated, either when the cells were incubated in aggregated IgG, or in adult rat serum. Rat peritoneal macrophages served as a positive control. However, permeabilization of the endothelial cells with glacial acetic acid/ethanol prior to incubation with IgG allowed the demonstration of internal binding sites. The lack of surface receptors in the normal state does not rule out the possibility of their induction during pathological conditions.

Animals↗
PubMed1978

Obesity and perceptual reactance.

Similarities in anomalous perception of internal gastric states and sensitivity to distraction among the obese to variations in perceptual reactance suggest that the obese tend to augment the intensity of visceral cues associated with hunger. It was hypothesized that the obese would be overrepresented at the augmenter end of the perceptual reactance continuum. Thirteen obese (six male, seven female) and 14 nonobese (eight male, six female) college students participated in a study in which perceptual reactance was assessed by degree of Kinesthetic Figural Aftereffect (KFA). A highly significant relationship in the predicted direction was observed for perceptual reactance category and mean percent weight deviation. Additionally, there was a highly significant interaction of sex by category, with the hypothesized relationship intensified for the female Ss. Results supported interpretation of obesity as a consequence of animalous perception of cues associated with consuming behavior.

Attention↗
PubMed1976

Communication patterns of doctors and their assistants.

Communication patterns between physicians and their assistants (Medex) were observed in 19 practices to describe and evaluated the nature of their relationship. Observations demonstrated that interaction around patient problems was approximately 30 minures per day and informal, with the Medex more often initiating the contact. Medex were more likely to ask for, and physicians were more likely to give, suggestions. Physicians appeared to regard the accomplishment of the immediate task as more important than maintaining good relations. In most practices, supervision was available when requested. Generally, both the physician and the Medex show a striking similarity in communication styles.

Communication↗