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Biomedical subjects

J G Lewis

Publications and source records attributed to J G Lewis.

At least 73 records · Page 4Linked to original sources
PubMed1987

An enzyme-linked immunosorbent assay (ELISA) for plasma progesterone: immobilised antigen approach.

A single extraction ELISA for plasma progesterone is described using the fixed antigen approach. Progesterone is covalently coupled to bovine thyroglobulin and adsorbed onto a 96-well microtitre plate in guanidine hydrochloride. The assay, performed on an automatic ELISA processor, follows an established methodology used for other steroid hormones analysed in this laboratory with concomitant advantages in assay standardisation, cost structure and result throughput. A comparison with an established RIA shows the assay to be rapid, of similar specificity and accuracy with a sensitivity of less than 0.5 nmol/l and is suitable for use in a routine endocrine laboratory for determination of luteal function.

Animals↗
PubMed1987

Early inflammatory changes in the skin of SENCAR and C57BL/6 mice following exposure to 12-O-tetradecanoylphorbol-13-acetate.

Tumor-promoting phorbol esters are potent inflammatory agents. Inflammation has long been associated with carcinogenesis, particularly the promotion phase. SENCAR mice have been bred for their sensitivity to the promotion of skin tumors by phorbol esters whereas C57BL/6 mice have been shown to be resistant. When 12-O-tetradecanoylphorbol-13 acetate (TPA) was applied to the skin of SENCAR and C57BL/6 mice, SENCAR mice had a much more intense inflammatory response than the C57BL/6 animals. There was more edema formation at the site of application and vascular permeability was also greatly enhanced in the SENCAR strain. When samples of skin were examined microscopically, a fulminant, acute inflammatory cellular infiltrate was observed in SENCAR mice. Very few inflammatory cells migrated into the skin of C57BL/6 mice. At higher doses and multiple exposures to TPA there was a hyperplastic response of the keratinocytes in C57BL/6 mice, but it was less intense than in SENCAR mice. These data demonstrate that enhanced inflammatory responses in TPA in the skin correlate with the sensitivity to the promotion of skin tumors by TPA.

Animals↗
PubMed1987

Inflammation, oxidative DNA damage, and carcinogenesis.

Inflammation has long been associated with carcinogenesis, especially in the promotion phase. The mechanism of action of the potent inflammatory agent and skin promoter 12-tetradecanoyl phorbol-13-acetate (TPA) is unknown. It is thought that TPA selectively enhances the growth of initiated cells, and during this process, initiated cells progress to the preneoplastic state and eventually to the malignant phenotype. Many studies support the multistep nature of carcinogenesis, and a significant amount of evidence indicates that more than one genetic event is necessary for neoplastic transformation. Selective growth stimulation of initiated cells by TPA does not explain how further genetic events may occur by chronic exposure to this nongenotoxic agent. We and others have proposed that TPA may work, in part, by inciting inflammation and stimulating inflammatory cells to release powerful oxidants which then induce DNA damage in epidermal cells. Macrophages cocultured with target cells and TPA induce oxidized thymine bases in the target cells. This process is inhibited by both catalase and inhibitors of lipoxygenases, suggesting the involvement of both H2O2 and oxidized lipid products. Furthermore, macrophage populations that release both H2O2 and metabolites of arachidonic acid (AA) are more efficient at inducing oxidative DNA damage in surrounding cells than populations which only release H2O2 or metabolites of AA. In vivo studies demonstrated that SENCAR mice, which are sensitive to promotion by TPA, have a more intense inflammatory reaction in skin than C57LB/6 mice, which are resistant to promotion by TPA. In addition, macrophages from SENCAR mice release more H2O2 and metabolites of AA, and induce more oxidative DNA damage in cocultured cells than macrophages from C57LB/6 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

A monoclonal antibody to prednisone: use of enzyme-linked immunosorbent assay (ELISA) for screening and characterization of antigenic determinants.

ELISA technology using immobilized steroid conjugates has allowed the rapid screening and characterization of hybridoma supernatants. Although cortisol-3CMO-BSA was the immunogen, a monoclonal antibody with exceptionally high cross-reactivity (greater than 1000%) to prednisone was obtained. Characterization of the antigenic determinants shows a requirement for overall glucocorticoid 21-hydroxyl and 17 alpha-hydroxyl groups with additional high specificity for the 1,2-double bond and 11-position. There is potential use for this antibody in the assay of prednisone.

Animals↗
PubMed1986

A competitive enzyme-linked immunosorbent assay for plasma 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione).

An enzyme-linked immunosorbent assay (ELISA) for 11-deoxycortisol is described for the first time. 11-Deoxycortisol-thyroglobulin conjugate is adsorbed onto the wells of a 96-well ELISA plate and competes with 11-deoxycortisol in the standards or plasma extract for antibody binding sites. After washing, immobilized primary antibody is probed with peroxidase-labeled goat anti-rabbit IgG. The ELISA plate is further washed and o-phenylenediamine added, color developed and the absorbance read at 492 nm. The ELISA shows good agreement with our existing 11-deoxycortisol radioimmunoassay (RIA) and has similar specificity and performance which allow its use in the routine steroid laboratory for assessing pituitary adrenal function by the metyrapone test.

17-Hydroxycorticosteroids↗
PubMed1986

The effect of macrophage development on the release of reactive oxygen intermediates and lipid oxidation products, and their ability to induce oxidative DNA damage in mammalian cells.

Inflammation and the release of potentially damaging substances, such as reactive oxygen intermediates (ROI) and lipid oxidation products from inflammatory cells, have been linked to the potentiation of carcinogenesis. Murine macrophages when stimulated with phorbol esters induce 5,6 ring saturated thymine residues (T'), a lesion of known oxidative origin, in co-cultivated mammalian cells. Induction of this damage was inhibited by catalase and induced in target cells by reagent H2O2 alone. In the present studies, we used defined populations of macrophages with high, low and intermediate capacities for the release of H2O2 or metabolites of arachidonic acid (AA) to assess the relative contribution of these classes of compounds to the induction of saturated thymines. Macrophages activated with Bacillus Calmette-Guerin (BCG), which have the highest capacity for the release of H2O2 and the lowest for the release of metabolites of AA, induced the lowest levels of saturated thymines. Resident macrophages from the unmanipulated peritoneum, which have the lowest capacity for the production of H2O2 and the highest capacity for release of AA metabolites, induced more saturated thymines than did the BCG macrophages. Inflammatory macrophages elicited by casein, which have an intermediate capacity for release of H2O2 and AA metabolites, induced the highest level of saturated thymines. Zymosan, which induced more release of AA metabolites than release of H2O2, was a better stimulant for the induction of T' than TPA, which is a better stimulant for secretion of H2O2. Nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase and cyclooxygenase pathways for metabolism of AA, inhibited the induction of T' by resident macrophages. Indomethacin, an inhibitor of the cyclo-oxygenase path, enhanced induction of T'. Taken together, the data suggest that while H2O2 has the capacity to induce T' in 3T3 cells, it may not be the only mediator of DNA damage and that lipoxygenase generated metabolites of AA may (alone or in concert with ROI) play an important role in the induction of oxidative DNA damage by macrophages.

Animals↗
PubMed1986

Enhanced release of hydrogen peroxide and metabolites of arachidonic acid by macrophages from SENCAR mice following stimulation with phorbol esters.

Chronic inflammation has long been associated with carcinogenesis. Phorbol esters which are potent promoters of tumors in mouse skin are also potent inflammatory agents in skin and cause inflammatory cells to release large quantities of reactive oxygen intermediates and oxidized lipid products. SENCAR mice have been bred for their sensitivity to the promotion of tumors by phorbol esters and C57BL/6 mice have been shown to be resistant. We quantified the release of H2O2 and metabolites of arachidonic acid by macrophages obtained from SENCAR and C57BL/6 mice, following exposure to phorbol esters and other stimulants. The basal level for secretion of H2O2 in resident peritoneal macrophages was negligible in cells from both strains. Conversely, inflammatory macrophages from SENCAR mice, elicited by the injection of sterile casein, secreted 4 times more H2O2 than the corresponding cells from C57BL/6 mice. Furthermore, cells from SENCAR mice required less than one-third the amount of phorbol ester to obtain 50% of the maximal response than that required by cells from C57BL/6 mice. This difference was less when zymosan was used as a stimulant. Both resident and inflammatory macrophages from SENCAR mice released more metabolites of arachidonic acid than cells from C57BL/6 mice when exposed to phorbol esters, but macrophages from C57BL/6 mice released more metabolites when stimulated with zymosan. Few differences in the pattern of released metabolites were noted between the strains of mice. There were large differences in the relative amounts of individual metabolites released when different stimulants were used. The enhanced response to phorbol esters of chronic inflammatory cells from SENCAR mice correlates with the enhanced sensitivity to the promotion of tumors by phorbol esters in these animals.

Animals↗
PubMed1985

An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone.

A rapid, single extraction ELISA for testosterone in plasma is described, using a standard 96 well microtitre plate. Testosterone is covalently bonded to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to the ELISA plate, giving an immobilised antigen approach which simplifies subsequent assay standardisation for steroid hormone assays. The addition of standard, sample and first antibody (rabbit anti-testosterone), which is unique for each different assay, is followed by a general procedure which includes washing, addition of peroxidase labelled goat antirabbit IgG, further washing and finally, addition of o-phenylenediamine substrate with colour development and reading of the plate at 492 nm on an automatic ELISA processor. The ELISA assay is compared to a testosterone RIA with 125I-label and has similar specificity and precision to the latter with a quicker processing time, and is more cost effective. The added advantages that ELISA assays confer over RIAs in terms of isotope purchase and disposal make this an ideal procedure for use in a routine steroid laboratory.

Antibody Specificity↗
PubMed1985

An enzyme-linked immunosorbent assay (ELISA) for plasma cortisol.

A direct ELISA for plasma cortisol is described which is carried out in a standard 96 well microtitre plate. In this ELISA cortisol-thyroglobulin conjugate is immobilised to the microtitre plate and competes with cortisol in the standard or plasma sample for antibody binding sites. Following washing the rabbit cortisol antibody bound to immobilised cortisol is incubated with peroxidase labelled goat antirabbit IgG. Following further washing o-phenylenediamine is added, colour developed, and the plate read at 492 nm on a standard ELISA plate reader. This ELISA shows good agreement with RIA and its sensitivity, specificity and precision allow its use in the routine steroid laboratory.

Adult↗
PubMed1985

Fractionation of cortisol antisera by immunoadsorption chromatography: characterisation and use in an enzyme-linked immunosorbent assay (ELISA).

The use of affinity chromatography in the presence of 20% acetonitrile combined with a decreasing pH gradient has allowed the fractionation of two cortisol antisera into components of varying affinity. The high affinity fractions generate considerably improved ELISA standard curves compared to the intact sera but do not grossly alter the specificity of the antisera. Accordingly, the high affinity fraction of the least cross-reactive antiserum was used for a plasma cortisol ELISA. The cortisol ELISA, although returning slightly higher values than RIA, was comparable in the ability to distinguish dexamethasone suppression and cortisol response to synacthen and should thus prove of value in the assay of plasma cortisol.

Animals↗
PubMed1985

Induction of 5,6-ring-saturated thymine bases in NIH-3T3 cells by phorbol ester-stimulated macrophages: role of reactive oxygen intermediates.

Because oxygen intermediates secreted by inflammatory leukocytes are postulated to play a role in potentiating carcinogenesis, we investigated the ability of macrophages to induce oxidative DNA damage in eukaryotic cells. Murine macrophages, obtained from sites of inflammation and stimulated with 12-O-tetradecanoylphorbol-15-acetate, induced the formation of 5,6-ring-saturated thymine bases in the DNA of cocultured NIH-3T3 cells; macrophages or 12-O-tetradecanoylphorbol-15-acetate alone did not induce such alterations. Reagent H2O2, at concentrations produced by macrophages in the ambient medium (i.e., approximately 10(-5) M), induced saturated thymines in the target cells in a dose-dependent manner. The reaction between reagent H2O2 and cellular DNA was rapid, reaching maximum levels in 30 min, and similar amounts of saturated thymines were induced at 4 degrees or 37 degrees. The 3T3 targets were able to repair the saturated thymines rapidly (i.e., over 70% of the lesion was removed in 2 hr). Catalase completely inhibited macrophage-mediated induction of saturated thymines, although superoxide dismutase enhanced induction. Taken together, the data indicate that macrophages exposed to phorbol diesters can induce a specific, quantifiable lesion in the DNA of bystander eukaryotic cells and that reactive oxygen species from the macrophages participate in producing the lesion.

Animals↗
PubMed1984

The effect of alpha 2HS glycoprotein on lymphocyte reactivity to phytohemagglutinin.

The effect of alpha 2HS glycoprotein on lymphocyte reactivity to phytohemagglutinin (PHA) was investigated. The results show that alpha 2HS glycoprotein, rather than enhancing lymphocyte reactivity to PHA as suggested by earlier correlation studies, inhibits such reactivity. Experiments suggest that this inhibition is unlikely to be the result of alpha 2HS glycoprotein binding either PHA or 3H-thymidine but is more likely to act at the cellular level.

Blood Proteins↗
PubMed1983

Evidence that the alpha 2HS glycoprotein receptor and the Epstein-Barr virus nuclear antigen (EBNA) are identical.

Evidence is presented which suggests that the alpha 2HS glycoprotein (alpha 2HS) receptor on lymphocytes transformed by Epstein-Barr virus (EBV) and the EBV-determined nuclear antigen (EBNA) are immunologically related, and may be identical. Western blotting, using both anti-EBNA and anti-alpha 2HS receptor serum identified a similar Mr 48 000 component in labelled detergent-solubilized transformed cell supernatants. The Mr 48 000 component recognized by anti-EBNA serum had a pI between 7-8, similar to that reported for the alpha 2HS receptor.

Antigens, Viral↗