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Biomedical subjects

R Guy

Publications and source records attributed to R Guy.

At least 37 records · Page 2Linked to original sources
PubMed1996

Modeling acne in vitro.

To help elucidate the factors responsible for the infundibular changes seen in acne, the human sebaceous pilosebaceous infundibulum was isolated by microdissection and maintained for 7 d in keratinocyte serum-free medium supplemented with 50 micrograms/ml bovine pituitary extract, 100 units/ml penicillin and streptomycin, 2.5 micrograms/ml amphotericin B and CaCl2(10H2O) to give a final Ca2+ concentration of 2 mM. Infundibular structure was maintained over 7 d in this medium; the pattern of cell division mimicked that in vivo. The rate of cell division was significantly higher than previously described for infundibula maintained in supplemented William's E medium, and moreover did not fall over 7 d. The addition of 1 ng/ml interleukin-1 alpha (IL-1 alpha) caused hypercornification of the infundibulum similar to that seen in comedones; this could be blocked by 1000 ng/ml interleukin-1 receptor antagonist (IL-1ra). In about 20% of subjects there was spontaneous hypercornification of the infundibulum that could be blocked by 1000 ng/ml IL-1ra, suggesting that the infundibulum is capable of synthesising IL-1 alpha. The addition of 5 ng/ml epidermal growth factor or 5 ng/ml transforming growth factor-alpha to the medium caused a disorganisation of the keratinocytes of the infundibulum that resulted in rupturing similar to that seen in the more severe, purulent grades of acne. The addition of 1 microM 13-cis retinoic acid caused a significant reduction in the rate of DNA synthesis and apparent parakeratosis. We are now, therefore, able to model histologically the major infundibular changes in acne.

Acne Vulgaris↗
PubMed1996

The improved organ maintenance of the human sebaceous gland: modeling in vitro the effects of epidermal growth factor, androgens, estrogens, 13-cis retinoic acid, and phenol red.

We have previously reported that human sebaceous glands can be maintained for up to 14 d as whole organs with full retention of the physiological rate and pattern of new cell formation, but we have also reported that the newly formed cells did not differentiate normally, causing a progressive loss of lipogenesis in vitro. We now show that this abnormal sebocyte differentiation was attributable to the presence of epidermal growth factor (EGF) and phenol red in our maintenance medium. In their absence, human sebaceous glands apparently retain in vivo rates of cell division and lipogenesis over 7 d of maintenance in addition to a retention of in situ morphology. This is reversible on the re-addition of 10 ng EGF/ml and 10 mg phenol red/ml. The addition of 600 pM 17 beta-estradiol results in a significant fall in the rate of lipogenesis over 7 d of maintenance, without affecting the rate of cell division. This effect is apparently due to abnormal differentiation of newly formed sebocytes. Neither 1 nM testosterone nor 1 nM dihydrotestosterone (DHT) has any effect on rates of cell division of lipogenesis over 7 d. In the presence of phenol red, however, 1 nM testosterone or 1 nM DHT cause a significant reduction in the rate of lipogenesis over 7 d of maintenance. One micromolar 13-cis retinoic acid caused a significant reduction in the rate of lipogenesis over 7 d in both the presence and absence of phenol red. These findings show that we can model the physiological effects of steroids, EGF, and 13-cis retinoic acid in vitro.

Adult↗
PubMed1996

MHC non-restricted, CD95-independent apoptosis of immature thymocytes induced by thymic epithelial cells.

The interaction of thymocytes with thymic epithelial cells in the absence of an exogenous antigen was studied in vitro. Thymic, but not splenic epithelial cells induced apoptosis of thymocytes. A thymic epithelial cell line (TEC) induced apoptosis of thymocytes but not of splenic T-cells. The target population for TEC-induced death were immature CD4(+)8(+) (double positive), but not mature single positive thymocytes. TEC also induced DNA fragmentation in day 18 foetal thymocytes, most of which are CD4(+)8(+) cells. Radiation leukemia virus (RadLV)-transformed thymic lymphoma clones expressing various phenotypes reflected this sensitivity, in that a CD4(+)8(+)3(+) clone apoptosed by thymic epithelial cells or TEC. Other, single positive or double negative clones were resistant. Thymocytes from C3H (H-2(k)), C57BL/6 (H-2(b)) and Balb/C (H-2(d)) mice apoptosed equally in response to either C57BL/6 thymic epithelial cells or TEC (H-2(b) x H-2(d)). Likewise, thymocytes from MRLIpr((-/-)) and B6Ipr((-/-)) mice, which do not express CD95 were also apoptosed by TEC.The data suggest that thymic epithelial cells induce MHC non-restricted, Fas-independent apoptosis of immature thymocytes. This response may reflect a mechanism through which thymocytes expressing TcR with no affinity to self MHC/peptide complexes are eliminated.

Journal Article↗
PubMed1994

The collagen binding domain of fibronectin contains a high affinity binding site for Candida albicans.

A 30-kDa proteolytic fragment from the gelatin/collagen-binding domain of fibronectin is a potent inhibitor of fibronectin binding to Candida albicans, with a molar inhibition constant equal to that of intact fibronectin. Recombinant and proteolytic fragments from the cell-, the fibrin I-, and the heparin II-binding domains also inhibit fibronectin binding, but are 13-1000-fold less active. In suspension, binding of fibronectin to C. albicans is regulated by growth conditions and is specific, saturable, time-dependent, reversible, and divalent cation-independent. Scatchard plot analyses indicate the presence of high affinity (Kd = 1.3 x 10(-9) M) and low affinity (Kd = 1.2 x 10(-7) M) receptors. Recombinant or proteolytic fragments from four binding domains of fibronectin promote adhesion of C. albicans. A recombinant fragment corresponding to the cell-binding domain but with the sequence Arg-Gly-Asp-Ser deleted promotes C. albicans adhesion and inhibits fibronectin binding to C. albicans with the same activity as the natural sequence. Furthermore, four peptides containing the Arg-Gly-Asp-Val sequence and the peptides CS-1 and Arg-Glu-Asp-Val did not block the binding of fibronectin to C. albicans. Thus, in contrast to the specific binding of soluble fibronectin, recognition of immobilized fibronectin by C. albicans is mediated by several domains of the protein. Interactions with the cell-binding domain are not mediated by the Arg-Gly-Asp or other known recognition sequences as it has been suggested. Binding of fibronectin also did not correlate with C3d binding to the avirulent clones of C. albicans strain H12 or with iC3b binding to variants of the strain 4918.

Amino Acid Sequence↗
PubMed1994

Effect of advertising on awareness of symptoms of diabetes among the general public: the British Diabetic Association Study.

OBJECTIVE: To determine the impact of posters advertising symptoms of diabetes on public knowledge of these symptoms. DESIGN: Structured street interviews of members of the general public before, at the end of, and 10 weeks after a campaign advertising the main symptoms of diabetes. SETTING: Basingstoke and Wolverhampton. SUBJECTS: Three samples of 1000 members of the general public were interviewed. Samples were selected randomly but stratified to match the local population's age (20-75), sex, social class, and racial characteristics. MAIN OUTCOME MEASURES: Knowledge of symptoms of diabetes; perceived seriousness of diabetes; and induction of anxiety about symptoms in the target population. RESULTS: Advertising significantly raised knowledge (without prompting) of symptoms: thirst, 245 before v 411 at end of campaign (P < 0.0001) v 341 after (P = 0.0012 v before); polyuria, 72 v 101 (P = 0.0211) v 92 (P = 0.5169); lethargy, 180 v 373 (P < 0.0001) v 298 (P < 0.0001); knowledge of weight loss and visual disturbance was unaffected. The number of subjects lacking knowledge of any symptoms was reduced from 550 to 388 (P < 0.0001). The perceived seriousness of diabetes was unaffected (mean 7.6 in each phase on a scale of 1 (not) to 10 (very). Before advertising, 449 (45%) claimed to have one or more symptoms of diabetes, but this number fell at the end of the campaign (403; P = 0.0419) and 10 weeks afterwards (278; P < 0.0001). CONCLUSIONS: An advertising campaign raised public knowledge of diabetes symptoms without inducing fear of diabetes or anxiety about symptoms. Its potential for achieving earlier detection of non-insulin dependent diabetes should be evaluated.

Adult↗
PubMed1994

Apolipoprotein E: a potent inhibitor of endothelial and tumor cell proliferation.

Recombinant human apolipoprotein E3 (apoE), purified from E. coli, inhibited the proliferation of several cell types, including endothelial cells and tumor cells in a dose- and time-dependent manner. ApoE inhibited both de novo DNA synthesis and proliferation as assessed by an increase in cell number. Maximal inhibition of cell growth by apoE was achieved under conditions where proliferation was dependent on heparin-binding growth factors. Thus, at low serum concentrations (0-2.5%) basic fibroblast growth factor (bFGF) stimulated the proliferation of bovine aortic endothelial (BAE) cells severalfold. The bFGF-dependent proliferation was dramatically inhibited by apoE with an IC50 approximately 50 nM. Under conditions where cell proliferation was mainly serum-dependent, apoE also suppressed growth but required higher concentrations to be effective (IC50 approximately 500 nM). ApoE also inhibited growth of bovine corneal endothelial cells, human melanoma cells, and human breast carcinoma cells. The IC50 values obtained with these cells were generally 3-5 times higher than with BAE cells. Inhibition of cell proliferation by apoE was reversible and dependent on the time of apoE addition to the culture. In addition, apoE inhibited the chemotactic response of endothelial cells that were induced to migrate by a gradient of soluble bFGF. Inhibition of cell proliferation by apoE may be mediated both by competition for growth factor binding to proteoglycans and by an antiadhesive activity of apoE. The present results demonstrate that apoE is a potent inhibitor of proliferation of several cell types and suggest that apoE may be effective in modulating angiogenesis, tumor cell growth, and metastasis.

Amino Acid Sequence↗
PubMed1993

Studies on fibronectin and its domains. I. Novel recombinant cell-binding domain of fibronectin--a modulator of human platelet functions.

The DNA sequences encoding for two proteins of the cell-binding domain (CBD) of human fibronectin (FN), namely a 33-kDa protein (aa 1329-1722) and a 40-kDa (aa 1380-1851) protein, were cloned and expressed in Escherichia coli. The interactions of the resulting rCBD proteins, refolded and purified to homogeneity, with human platelets were studied in comparison with those of the pentapeptide GRGDS. The binding of both the 33-kDa and the 40-kDa proteins to washed platelets appeared to be dependent upon platelet activation. In the case of the 33-kDa protein, binding to stimulated platelets was shown to be saturable, with Kd = 2 microM (thrombin as agonist). Moreover, both the 33-kDa and the 40-kDa proteins inhibited fibrinogen binding (at 0.1 microM) to ADP- or thrombin-stimulated platelets with IC50 values in the same concentration range. Binding seemed therefore to occur mainly at the GPIIb/IIIa receptor, and accordingly monoclonal antibodies against this receptor prevented up to 85% of the binding of the 33-kDa protein to platelets. With most stimuli the 33-kDa and the 40-kDa proteins inhibited platelet aggregation at concentrations 15- to 25-fold lower than those required by GRGDS and, in the case of the 33-kDa protein, this was shown to occur in either platelet-rich plasma, washed platelets, or whole blood. The 33-kDa protein also inhibited platelet aggregation and thromboxane A2 (TXA2) generation on the subendothelial extracellular matrix, whereas the GRGDS peptide inhibited only matrix-induced platelet aggregation, but not TXA2 formation. Furthermore, the 33-kDa protein, which is derived from the human FN CBD, seemed to be highly selective, since it inhibited the aggregation of platelets from primates only, and not from other animals tested. Finally, the 33-kDa protein did not promote fibroblast cell attachment, as was observed for both whole FN and the 40-kDa protein, thus displaying a selectivity toward platelets. In conclusion, the unique properties of the 33-kDa protein, and, in particular, its special affinity directed only toward activated primate platelets, seem to hold a promising potential for the further development of an antithrombotic agent.

3T3 Cells↗
PubMed1993

Mitogenic activation of phosphoinositide turnover and DNA synthesis in murine CD4+8+ thymocytes.

Eighty percent of the lymphoid cells in the murine thymus are premature CD4+8+ (double positive) thymocytes. The vast majority of the double-positive cells do not maturate and die in the thymus. Although these cells are subjected to thymic selection processes, their activation competence has been an enigma. We have separated out CD4+8+ cells and studied their early and late responses to several mitogens. Concanavalin A, anti-CD3 (145-2C11) or anti-Thy1 (G7) monoclonal antibodies enhanced phosphoinositide turnover in double-positive thymocytes. However, DNA synthesis in the mitogen-stimulated cells was only accomplished if IL-2 or IL-4 was added. Alternatively, DNA synthesis could be induced by the calcium ionophore A23187 and phorbol myristate acetate (PMA). The latter mode of activation did not require the addition of exogenous lymphokines. CD4+8+ thymocytes did not secrete IL-2 or IL-4 following activation by either mitogens or A23187 and PMA. These findings demonstrate that CD4+8+ thymocytes resemble mature T cells in their ability to respond with DNA synthesis when activated by T-cell mitogens and IL-4 as well as IL-2. The results also delineate the difference between receptor mediated mitogenesis and pharmacological stimulation.

Animals↗
PubMed1993

Phase III trial of oral magnetic particles in MRI of abdomen and pelvis.

A prospective Phase III trial was performed to evaluate the efficacy in clinical practice of a new gastro-intestinal contrast agent, Oral Magnetic Particles (OMP), for use in abdominal and pelvic magnetic resonance imaging (MRI). This was an open study comprising 35 patients each given 800 ml of OMP at a concentration of 0.5 g/l. Distribution of the agent was most reliable in the small bowel. General contrast effect was satisfactory, and image quality and diagnostic confidence were enhanced. Administration of OMP increased diagnostic information in the magnetic resonance (MR) image in 50% of patients. No clinically significant adverse effects were observed. The results confirm in a clinical setting a potential role for this new contrast agent.

Abdominal Neoplasms↗
PubMed1993

Isolation and maintenance of the human pilosebaceous duct: 13-cis retinoic acid acts directly on the duct in vitro.

The human pilosebaceous duct was isolated and maintained for 7 days in defined medium, and defined medium supplemented with 1 microM 13-cis retinoic acid. Freshly isolated ducts retained their in vivo morphology, showing a stratified squamous keratinizing epithelium. On maintenance there was a loss of basic duct architecture, and a significant reduction in the rate of [methyl-3H] thymidine uptake. The addition of 1 microM 13-cis retinoic acid resulted in an improved duct architecture and caused a further significant reduction in the rate of [methyl-3H] thymidine uptake. [Methyl-3H] thymidine autoradiography showed that freshly isolated ducts maintained their in situ pattern of cell division. It was difficult to discern the region of cell division in ducts maintained for 7 days, but the degree of graining reflected the measured rates of [methyl-3H] thymidine uptake into PCA precipitable material. The pattern of keratin synthesis of the freshly isolated duct was similar to patterns previously described for the duct in situ. This study reports the successful isolation and maintenance of the human pilosebaceous duct, and demonstrates that 13-cis retinoic acid acts directly at the level of the duct.

Cell Division↗
PubMed1992

Haemorrhagic cerebral metastases from malignant testicular teratoma.

Cerebral metastases from malignant testicular teratoma may show complex appearances with magnetic resonance imaging (MRI), which are consistent with changes resulting from repeated haemorrhage. Haemorrhage causes enlargement and symptomatic progression of cerebral lesions, and this can occur even when extracranial disease or other cerebral lesions remain static.

Adolescent↗
PubMed1991

Subpopulations of fetal thymocytes defined by expression of T cell receptor/CD3 and IL-2 receptor. CD3 and IL-2 receptor alpha-chain are expressed on reciprocal cell populations.

The expression of the TCR/CD3 complex and the IL-2R alpha chain (p55) on fetal thymocytes has been analyzed by flow cytometry (FCM). Two-parameter immunofluorescence identified three subpopulations which were respectively IL-2R alpha-/CD3+, IL-2R alpha+/CD3-, or IL-2R alpha-/CD3-; no detectable population of IL-2R alpha+/CD3+ cells was found in unstimulated fetal thymocytes. Fractionation by "panning" and by sterile flow cytometric separation was used to characterize the functional responsiveness of these three subpopulations to a variety of stimuli. All three populations proliferated in response to PMA + ionomycin + rIL-2. In contrast, stimulation with anti-CD3 + IL-2 induced proliferation in IL-2R alpha-/CD3+ and IL-2R alpha-/CD3- but not in IL-2R alpha+/CD3- thymocytes. IL-2R alpha- cells, including sorted IL-2R alpha-/CD3- thymocytes, underwent a phenotypic change in response to in vitro stimulation with anti-CD3 + IL-2, resulting in the appearance of an IL-2R alpha+/CD3+ population that was not detected in freshly isolated thymocytes. The ability of fractionated fetal thymocytes to produce lymphokine in response to PMA + ionomycin was also evaluated. Only the IL-2R alpha-/CD3- fraction generated detectable IL-2. These findings demonstrate for the first time that CD3 and IL-2R alpha are expressed in a mutually exclusive fashion in fetal thymocytes and define three subpopulations of thymocytes that differ significantly in their proliferative and differentiative responses to TCR-mediated, IL-2R-mediated, and pharmacologic stimulation.

Animals↗
PubMed1990

Control of primary and secondary antibody responses by cytotoxic T lymphocytes specific for a soluble antigen.

BALB/c mice immunized with keyhole limpet hemocyanin (KLH) develop Thy-1+ and CD8+, KLH-specific cytotoxic T cells (Tc). Such Tc cells can lyse TNP-specific B cells activated with TNP-KLH, but not with TNP-ovalbumin. Cytotoxicity was inhibited by anti-H-2K/D antibodies, but not by anti-Ia antibodies, suggesting a major histocompatibility complex class I-restricted killing. Selective enrichment for virgin and memory TNP-specific B cells revealed that the latter cells were relatively resistant to lysis by KLH-specific Tc cells. Depletion of CD8+ T cells from cultures of TNP-specific virgin B cells activated with TNP-KLH and KLH-primed T cells, increased the titer of anti-TNP antibodies in the culture supernatants. This increase was reduced if KLH-primed CD8+ T cells were added to the culture 1 day before its termination. Anti-TNP antibody secretion by memory B cells activated in the same manner was not affected by depletion or addition of CD8+ cells. These results suggest that Tc cells are generated following immunization with a soluble antigen which may participate in the down-regulation of primary B cell responses.

Animals↗
PubMed1990

Localization and molecular modelling of the membrane-inserted domain of the ninth component of human complement and perforin.

Upon interaction with the membrane-bound C5b-8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazirine (125I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125I-TID-labeled C9, were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292-308 and 313-333) separated by a turn (309-312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perforin/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.

Affinity Labels↗