Biomedical subjects

U E Gibson

Publications and source records attributed to U E Gibson.

5 recordsLinked to original sources

PubMed1996

A novel method for real time quantitative RT-PCR.

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed1994

Interferon gamma administration increases monocyte HLA-DR antigen expression but not endogenous interferon production.

OBJECTIVE: To determine the effect of the adjuvant administration of interferon gamma on monocyte HLA-DR antigen expression and mitogen-stimulated interferon gamma production following injury. DESIGN: Double-blind, randomized, placebo-controlled trial. SETTING: University Hospital, Newark, NJ, a level I trauma center. PATIENTS: Persons older than 16 years with an Injury Severity Score greater than 20 and documented bacterial contamination at the time of injury (N = 98). INTERVENTIONS: Recombinant human interferon gamma (n = 46; 0.1 mg subcutaneously) or placebo (n = 52) was given for 10 days following injury. OUTCOMES: Incidence of major infection, monocyte and lymphocyte cell surface antigen expression, and interferon gamma production at multiple time points following injury. RESULTS: Peripheral monocyte HLA-DR was measured as percent of cells staining positive and as mean channel fluorescence. Both values were significantly increased in the interferon gamma group compared with the placebo group on days 3, 5, 8, and 11. The incidence of major infection was unaffected by interferon gamma administration. Infection decreased percent of HLA-DR-positive monocytes and mean channel fluorescence as compared with noninfected patients on postinjury days 8 and 11 in the placebo group but not in the interferon gamma group. Interferon gamma production improved from 3 +/- 3 U/mL on day 1 to 15 +/- 10 U/mL by day 30 but was always significantly lower than normal (25 +/- 3 [mean +/- SD] U/mL). Interferon gamma production was unaffected by either infection or interferon gamma administration. CONCLUSIONS: Interferon gamma administration after injury stimulated monocyte HLA-DR antigen expression and density but failed to improve interferon gamma production, a T-cell-mediated function. The incidence of infection was not decreased by the administration of interferon gamma for 10 days. Improvement in monocyte HLA-DR antigen expression did not correlate with a global restoration of immune function, and other interventions will be necessary to decrease infection after injury.

Adult↗

PubMed1992

An antibody capture bioassay (ACB) for DNase in human serum samples.

A novel assay for antibody captured bioactivity (ACB) has been developed to quantitate deoxyribonuclease I (DNase) in human serum samples. The procedure is simple, sensitive, reproducible and has a high throughput. Serum samples are diluted a minimum of 1/4 and assayed in 96-well microtiter plates coated with polyclonal antibodies specific to DNase. The serum is removed from the wells, the plates are washed and the antibody bound DNase is incubated at 37 degrees C with a DNA-methyl green substrate. The assay is sensitive to 0.8 ng/ml with a range to 10 +/- 2 ng/ml, depending upon the time of incubation (48 +/- 2 h). The recovery of rhDNase spiked into human serum samples averaged 84.4% +/- 6.7% in sera diluted 1/4 and 97.8% +/- 7.2% at a 1/8 serum dilution. Intra-assay precision ranged from 3.0 to 7.5% coefficient of variation (% CV) and interassay precision ranged from 5.0 to 10.2% CV for spiked serum controls. Endogenous DNase concentrations in 27 normal human sera were found to range from < 2.0 to 11.4 ng/ml. Endogenous DNase-like activity was found in Cynomolgus and Rhesus monkey sera; this activity diluted linearly and did not interfere with accurate quantitation of added rh DNase. No endogenous DNase-like activity could be detected in ten Sprague-Dawley rat sera. Bovine pancreatic DNase was found to have only very low cross-reactivity in this assay system. The ACB assay format can potentially be applied to the quantitation of other enzymes in serum and other biological samples.

Animals↗

PubMed1990

Increase in cyclic AMP levels by relaxin in newborn rhesus monkey uterus cell culture.

A novel relaxin sensitive cell line of apparent smooth muscle origin has been established from a newborn rhesus monkey uterus (NRMU). NRMU cells respond to relaxin, in the presence of 1 microM forskolin, by producing intracellular adenosine 3', 5'-cyclic monophosphate (cAMP). The increase in cAMP levels is dose, time and cell density dependent, reaching peak levels at 10 min when cells are seeded at 1 X 10(5) cells/well. Specificity was demonstrated by neutralization of the relaxin activity with anti-relaxin monoclonal and polyclonal antibodies, degradation of cAMP in the presence of phosphodiesterase, and confirmation of the absence of cGMP. Three synthetic analogs of human relaxin generated a dose-related cAMP response as did synthetic native human relaxin. Natural relaxin purified from human corpora lutea tissue also generated a response similar to synthetic human relaxin. Porcine and rat relaxins also increased levels of cAMP. Insulin, but not IGF I or IGF II, was capable of increasing cAMP levels in NRMU cells, however, 200 ng/mL were required to achieve cAMP levels comparable to 6.25 ng/ml relaxin. Combinations of relaxin with insulin, IGF I or IGF II did not increase cAMP levels above levels obtained with relaxin alone. The effect on NRMU cells of other hormones, growth factors and drugs potentially present in cell culture systems or serum samples was evaluated. In combination with relaxin, oxytocin significantly decreased the cAMP production below the levels induced by relaxin alone, whereas progesterone and prostaglandin E2 resulted in additive increases in cAMP. These data suggest that the NRMU cell line is an appropriate target tissue for studying relaxin-mediated biological responses in vitro as well as functioning as the primary component of a relaxin in vitro bioassay.

Actins↗

PubMed1989

Enzyme-linked bio-immunoassay for IFN-gamma by HLA-DR induction.

A novel enzyme-linked bio-immunoassay (bio-ELISA) has been developed to detect interferon-gamma (IFN-gamma) induced HLA-DR antigen on the surface of human tumor cells. Cells are cultured at 37 degrees C in 96-well microtiter plates in the presence of IFN-gamma for 2 days. After fixation with reagent alcohol the HLA-DR antigen is detected using a monoclonal antibody, followed by goat anti-mouse IgG-HRP conjugate. Four human cell lines were evaluated and all expressed HLA-DR in response to IFN-gamma in a dose-related fashion. Based on sensitivity, reproducibility and absence of antiproliferative effect by IFN-gamma, the COLO 205 cells (colon adenocarcinoma) were determined to be optimal. The bioassay is sensitive to 0.3 ng/ml IFN-gamma with a range to 10 ng/ml. The specificity of HLA-DR induction by IFN-gamma was demonstrated using an isotype specific monoclonal antibody as well as IFN-gamma neutralizing monoclonal and polyclonal antibodies. The effect of other cytokines on HLA-DR induction with COLO 205 cells was also investigated in this bioassay and only IFN-beta and interleukin-1 (IL-1) showed slight induction of HLA-DR. IFN-alpha had no effect at the concentration tested. Evaluation of assay parameters including reproducibility, sensitivity, simplicity, speed, cost and ability to standardize support the conclusion that this bioassay is a substantial improvement over the routinely used viral inhibition assay as a measure of IFN-gamma biological activity. The bio-ELISA technique also has potential applications for the quantitation of other cellular surface antigens induced by cytokines.

Biological Assay↗