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Biomedical subjects

D M Knight

Publications and source records attributed to D M Knight.

At least 19 recordsLinked to original sources
PubMed2002

Matrix effects in applying mono- and polyclonal ELISA systems to the analysis of weathered oils in contaminated soil.

Commercial mono- and polyclonal enzyme-linked immunosorbent assay (ELISA) systems were applied to the on-site analysis of weathered hydrocarbon-contaminated soils at a former integrated steelworks. Comparisons were made between concentrations of solvent extractable matter (SEM) determined gravimetrically by Soxhlet (dichloromethane) extraction and those estimated immunologically by ELISA determination over a concentration range of 2000-330,000 mg SEM/kg soil dry weight. Both ELISA systems tinder-reported for the more weathered soil samples. Results suggest this is due to matrix effects in the sample rather than any inherent bias in the ELISA systems and it is concluded that, for weathered hydrocarbons typical of steelworks and coke production sites, the use of ELISA requires careful consideration as a field technique. Consideration of the target analyte relative to the composition of the hydrocarbon waste encountered appears critical.

Enzyme-Linked Immunosorbent Assay↗
PubMed2000

Humphry Davy: science and social mobility.

Humphry Davy (1778-1829) was one of the first professional scientists, earning his living and rising spectacularly from an impoverished upbringing in Cornwall to be President of the Royal Society and a baronet. He owed his rise to patronage as well as to his range of abilities: as a lecturer, as a chemical theorist and as a very early applied scientist. But his exalted position brought him little happiness, for he could not satisfy all the hopes put upon him as the successor to Sir Joseph Banks. Admired rather than loved, he became unpopular and was seen as haughty. In his last two years, spent wandering lonely and sickly in Italy and the Alps, he sought to make sense of his life, writing dialogues as his bequest to the new generation.

England↗
PubMed1998

Sudden death due to intravenous infusion of hair conditioner.

A case of sudden death in a 14-year-old girl due to self administration of hair conditioner through an intravenous infusion pump is described. This report demonstrates difficulties that may occur in determining the manner of death in such cases and outlines a specific danger that may occur when adolescents have unsupervised access to intravenous infusion equipment.

Adolescent↗
PubMed1996

Reduced prostacyclin and increased leukotriene B4 synthesis in porcine venous-arterial grafts.

BACKGROUND: Migration and proliferation of vascular smooth muscle cells in the intima and superimposed atheroma are the main changes underlying late failure of saphenous vein bypass grafts. There is evidence that these events are partly modulated by complex interactions between inhibitors of vascular smooth muscle cell proliferation, such as prostacyclin (PGI2), and mitogens, such as leukotriene B4 (LTB4). Because the relative balance between these eicosanoids may play a role in vein graft failure, the synthesis of PGI2 and LTB4 was measured in porcine saphenous vein-carotid artery grafts 4 weeks after implantation and compared with ungrafted vein and common carotid artery from the same animal. METHODS: Vessels were cut into 2-mm squares and preincubated in Dulbecco's minimum essential medium for 4 hours at 37 degrees C. Tissues were then further incubated with Dulbecco's minimum essential medium containing a range of concentrations of noradrenaline, arachidonate, and calcium ionophore A23187. Release of PGI2 and LTB4 into the supernatant was then assessed by radioimmunoassay. RESULTS: In response to all stimulators, PGI2 release was markedly diminished in vein grafts compared with ungrafted saphenous veins and carotid arteries. The patterns of responses were similar in each vessel type. In contrast, LTB4 release was significantly enhanced in vein grafts compared to ungrafted saphenous veins and carotid arteries. CONCLUSIONS: These data indicate that there is a down-regulation of cyclooxygenase or PGI2 synthase in porcine vein grafts, which may constitute a further phenotypic change that would augment the hyperplastic process. Local increases in LTB4 synthesis in the vein graft, which indicates an induction of lipoxygenase and LTB4 synthase enzymes (and possibly reflects release from leukocytes which have infiltrated the graft), may contribute to increased intimal proliferation by direct promitogenic effects on smooth muscle cells.

Animals↗
PubMed1995

Chimeric anti-TNF-alpha monoclonal antibody cA2 binds recombinant transmembrane TNF-alpha and activates immune effector functions.

Results of clinical trials have indicated that cA2, a neutralizing mouse/human IgG1 chimeric anti-human TNF-alpha monoclonal antibody, may have therapeutic benefit for rheumatoid arthritis patients. Arthritic joints contain, in addition to elevated levels of soluble TNF-alpha, high numbers of CD4+ T cells and macrophages, cells known to express transmembrane TNF-alpha upon activation. For that reason, we sought to determine if cA2 binds to transmembrane TNF-alpha and what effects such binding may have on TNF-alpha-expressing cells. A cell line expressing a cell-surface, mutant form of transmembrane TNF-alpha was prepared for these studies. Analysis of these TNF+ cells by flow cytometry, direct binding, and competitive binding assays showed that cA2 binds to the transmembrane form of TNF-alpha with high avidity. Binding of the IgG1 isotype of cA2, but not an IgG4 version of cA2, resulted in efficient killing of the TNF+ cells by both antibody-dependent cellular toxicity and complement-dependent cytotoxicity effector mechanisms. These findings indicate that, in addition to blocking soluble TNF-alpha activity, cA2 can bind to transmembrane TNF-alpha in vitro and suggest that cA2 binding may lead to lysis of TNF-alpha-expressing cells in vivo.

Animals↗
PubMed1995

The immunogenicity of the 7E3 murine monoclonal Fab antibody fragment variable region is dramatically reduced in humans by substitution of human for murine constant regions.

A murine monoclonal antibody (7E3) directed against the platelet glycoprotein IIb/IIIa was engineered to reduce immunogenicity by substituting human for murine constant regions. The chimeric antibody is functionally identical to the murine antibody in vitro. Results from clinical trials with 7E3 Fab antibody fragments, however, show that the 7E3 variable region, which elicits the vast majority of the immune response to murine 7E3 Fab, is rendered dramatically less immunogenic (incidence reduced from 17% to 1%) when the identical variable region is linked to human rather than murine constant regions. Neither murine nor human constant regions were highly immunogenic themselves. We conclude that the constant regions of the Fab fragments are critical in modulating the immune response elicited by the linked 7E3 variable region. Because naturally occurring anti-human Fab fragment antibodies are prevalent both in the normal human population and in the patient population studied here, murine 7E3 Fab and chimeric 7E3 Fab may be fundamentally different in their interactions with the human immune system. This difference may be related to the dramatic difference in immunogenicity observed between murine 7E3 Fab and chimeric 7E3 Fab.

Animals↗
PubMed1993

Construction and initial characterization of a mouse-human chimeric anti-TNF antibody.

Tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of a variety of human diseases including septic shock, cachexia, graft-versus-host disease and several autoimmune diseases. Monoclonal antibodies directed against TNF provide an attractive mode of therapeutic intervention in these diseases. We have generated a murine monoclonal antibody (A2) with high affinity and specificity for recombinant and natural human TNF. To increase its therapeutic usefulness, we used genetic engineering techniques to replace the murine constant regions with human counterparts while retaining the murine antigen binding regions. The resulting mouse-human chimeric antibody should have reduced immunogenicity and improved pharmacokinetics in humans. Molecular analysis of light chain genomic clones derived from the murine hybridoma suggests that two different alleles of the same variable region gene have rearranged independently and coexist in the same hybridoma cell. The chimeric A2 antibody (cA2) exhibits better binding and neutralizing characteristics than the murine A2 which was shown to contain a mixture of two kappa light chains. The properties of cA2 suggest that it will have advantages over existing murine anti-TNF antibodies for clinical use.

Animals↗
PubMed1993

Effects of isotype and Fc region on in vitro function of a mouse/human chimeric CD4 antibody.

Murine CD4 mAbs have shown potential for the treatment of allograft rejection and autoimmune disorders including rheumatoid arthritis. Clinical usefulness of the murine mAbs has been limited by immunogenicity and a short circulating half-life. Mouse/human chimeric antibodies have been constructed, composed of the variable region of M-T412 (a murine G2a mAb specific for the human CD4 molecule) and human G1 (cM-T412 G1) or G4 (cM-T412 G4) Fc regions. F(ab')2 and F(ab) fragments of the murine G2a and chimeric G1 mAbs were generated by enzymatic digestion. The chimeric mAbs and all fragments retained the avidity and specificity of the murine M-T412 and were evaluated in in vitro assays measuring Ig production by pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC), sIL-2R produced by phytohemagglutinin-stimulated PBMC, and proliferation in response to tetanus toxoid, CD3 mAb plus IL-2, and mixed lymphocyte response (MLR). When PBMC were stimulated with tetanus toxoid, 10 ng/ml of cM-T412 G1 inhibited proliferation by 90%, while neither the cM-T412 G4, M-T412 G2a, nor any mAb fragment produced > 65% inhibition, even at 1000-fold higher concentrations. A similar pattern of inhibition was observed in MLR assays. In contrast, the F(ab')2 fragment of the cM-T412 G1 was as effective as the whole antibody in inhibiting PWM-stimulated IgM synthesis and PBMC proliferation in response to stimulation by a CD3 mAb plus IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Stable expression of cloned human antibody genes in murine myeloma cells.

Human monoclonal antibodies (MAbs) offer potential advantages over murine MAbs for therapy because they are not likely to elicit immune responses and are expected to interact more efficiently with the human immune system to activate therapeutically useful functions. Traditional methods for obtaining human MAbs (i.e., immortalization of B cells by cell fusion or transformation) can result in low and unstable antibody secretion. Recently, methods have been devised for direct cloning of human variable region genes via polymerase chain reaction and phage combinatorial libraries. Both types of human MAb production can benefit from expression systems that support the stable, high-level antibody secretion required for therapeutic use. Using an existing human-derived hybridoma that secretes a human IgM antibody as a convenient source of antibody genes, we have demonstrated that cloned human antibody genes can be efficiently expressed in murine myeloma cells and that cell lines with properties suitable for large-scale economical production can be obtained. We were unable to detect any differences between the antibodies produced by the original hybridoma and the engineered cell line. In addition, we were able to express an IgG form of the antibody, showing that expression of a recombinant human antibody need not be limited to the original antibody class.

Animals↗
PubMed1992

High-level expression and characterization of a mouse-human chimeric CD4 antibody with therapeutic potential.

The use of murine anti-CD4 monoclonal antibodies (MAbs) has shown considerable promise for the treatment of allograft rejection and rheumatoid arthritis. We have constructed mouse-human anti-CD4 antibodies with the goal of increasing their clinical potential by decreasing immunogenicity and improving effector functions. The chimeric antibodies were constructed by cloning the heavy and light chain variable regions of M-T412, a murine antibody raised against the human CD4 antigen, and joining them to the human G1, G4, or kappa constant regions in mammalian expression vectors. After transfection into mouse myeloma cells, stable cell lines were isolated that secrete up to 140 micrograms/ml chimeric antibody in static culture. The chimeric antibodies were equivalent to the murine antibody in their binding characteristics and relative affinities. However, the chimeric M-T412 MAbs have enhanced activity when compared to the murine G2a MAb in mediating antibody-dependent cell-mediated cytotoxicity using human CD4+ target and effector cells.

Animals↗
PubMed1987

Expression of the art/trs protein of HIV and study of its role in viral envelope synthesis.

The art/trs transactivator protein of human immunodeficiency virus (HIV) was expressed in mammalian cells as a 19-kilodalton protein that was immunoreactive with sera from HIV-infected patients. Separate plasmids encoding the art/trs protein, the tat protein, or the envelope glycoprotein gp120 were used to demonstrate that both art/trs and tat are absolutely required for the synthesis of gp120 from its cognate messenger RNA. In addition, both the tat and art/trs proteins influence the level of envelope RNA. The results suggest that art/trs and tat may be ideal targets for potential anti-HIV agents in AIDS therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Requirements for triggering of adipocyte differentiation by glucocorticoids and indomethacin.

The stable adipogenic cell line TA1, spontaneously differentiates into mature adipocytes after several days at confluence. Glucocorticoids (e.g. dexamethasone) accelerate the onset of differentiation by precociously activating the transcription of genes that are expressed in the mature adipocyte but not in the preadipocyte. Thus the hormone may induce a critical regulatory factor required for activating the entire set of differentiation-dependent genes. We have found that the nonsteroidal antiinflammatory drug indomethacin also stimulates differentiation of TA1 cells but even more rapidly and completely than does dexamethasone. Contrary to previous suggestions we find that this activity of indomethacin's cannot be ascribed to inhibition of cyclo-oxygenase, the critical enzyme in prostaglandin biosynthesis. Finally, indomethacin's ability to stimulate TA1 cell differentiation synchronously and rapidly has allowed us to document that cell confluence is required for efficient differentiation and that the drug needs only to trigger rather than maintain the differentiation process.

Adipose Tissue↗
PubMed1986

Glucocorticoid control of developmentally regulated adipose genes.

We have analyzed the hormonal basis for the acceleration of differentiation by dexamethasone in the stable adipogenic cell line TA1. These cells, which were derived from 5-azacytidine-treated 10T1/2 mouse embryo fibroblasts, undergo differentiation in culture after reaching confluence. Using cDNA clones corresponding to mRNAs that are induced during adipogenesis, we find that dexamethasone elicits the precocious accumulation of differentiation-specific gene products. This effect appears to be mediated by the glucocorticoid receptor, yet unlike standard steroid inductions, most of the RNAs reach the same maximal levels in the absence of dexamethasone. Glucocorticoids thus may increase the expression of a regulatory factor required for activating the entire set of differentiation-dependent genes. We also describe a gene whose transcription is not only activated during adipogenesis, but is also specifically inducible by dexamethasone in the mature adipocyte. Moreover, the glucocorticoid responsiveness of this gene in differentiated cells appears to be dependent on its prior developmental activation.

Adipose Tissue↗
PubMed1986

Hormonal control of adipogenesis.

The concept that hormonal substances can alter the expression of entire developmental programs is in itself not particularly new. The ability to define conditions under which a specific hormone can precociously activate the differentiation of a well-defined population of cells and under which another hormone can both block and reverse such a developmental progression, however, provides a major step forward toward unraveling the biochemical events that define the transition from a committed precursor to a fully differentiated cell. Further analysis of the molecular events initiated by glucocorticoids and TNF should provide insights into the control of adipogenesis and may generate a foundation for understanding the mechanisms by which other cells enter a particular differentiative lineage. In a more applied sense, such knowledge may also provide a rational approach to controlling metabolic disease syndromes related to adipogenesis gone awry such as obesity-associated diabetes and cachexia.

Adipose Tissue↗
PubMed1985

Glucocorticoid regulation of adipocyte differentiation: hormonal triggering of the developmental program and induction of a differentiation-dependent gene.

We have analyzed the hormonal basis for the acceleration of differentiation by dexamethasone and insulin in the stable adipogenic cell line TA1. These cells, which were derived from 5-azacytidine-treated 10T1/2 mouse embryo fibroblasts, undergo differentiation in culture after reaching confluence. The ensuing morphological changes are accompanied by widespread alterations in the pattern of protein synthesis and the increased accumulation of specific mRNAs. Using cDNA clones corresponding to mRNAs that are induced during adipogenesis, we find that dexamethasone elicits the precocious accumulation of differentiation-specific gene products. This effect appears to be mediated by the glucocorticoid receptor, yet unlike standard steroid inductions, most of the RNAs reach the same maximal levels in the absence of dexamethasone. Glucocorticoids thus may increase the expression of a regulatory factor required for activating the entire set of differentiation-dependent genes. We also describe a gene whose transcription is not only activated during adipogenesis but is also specifically inducible by dexamethasone in the mature adipocyte. Moreover, the glucocorticoid responsiveness of this gene in differentiated cells appears to be dependent on its prior developmental activation.

Adipose Tissue↗