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Biomedical subjects

D T Nguyen

Publications and source records attributed to D T Nguyen.

At least 19 recordsLinked to original sources
PubMed1992

Prognostic implications of evaluation for lymph node involvement by T-cell antigen receptor gene rearrangement in mycosis fungoides.

We investigated the correlation between the detection of clonal rearrangement of the T-cell antigen receptor gene (TCRR) in lymph node tissue with histopathologic lymph node classification in 33 patients with mycosis fungoides with and without the Sezary Syndrome. We analyzed DNA extracted from lymph nodes that were histologically uninvolved (LN1-2), dermatopathic nodes with clusters of atypical cells (LN3), and nodes effaced with lymphoma (LN4) and found TCRR in none of five LN1-2 nodes, 8 of 17 LN3 nodes, and 10 of 11 LN4 nodes. Further, the detection of TCRR correlated with presence of palpable adenopathy (P2 less than .0001) and was associated with a worse survival (P2 = .0024). Within the subgroup of patients with LN3 nodes, there was a trend (P2 = .14) toward inferior survival if nodes were involved by TCRR, irrespective of extent of skin disease. We conclude that detection of TCRR in nodes from mycosis fungoides patients is an objective and reliable means of assessing tumor infiltration of lymph node and is associated with an inferior survival.

Blotting, Southern↗
PubMed1992

Anti-tumor necrosis factor antibody therapy fails to prevent lethality after cecal ligation and puncture or endotoxemia.

Cytokines have been studied intensively to delineate their role in the altered pathophysiology observed in septic shock. We studied the role of TNF in the lethality of two well characterized models of septic shock by inhibiting TNF's activity with a specific antibody. In the first model, sepsis was induced by cecal ligation and puncture (CLP), and in the second model sepsis was induced by either an i.p. or i.v. injection of LPS. After CLP, plasma endotoxin was detectable within 4 h and reached a peak at 8 h (136 +/- 109 ng/ml). TNF bioactivity peaked at 12 h (528 +/- 267 pg/ml) at a significantly higher level than sham-operated control mice (64 +/- 31 pg/ml). After i.p. LPS, TNF peaked much more quickly (90 min) compared with CLP and at a significantly higher level (107,900 +/- 25,000 pg/ml). Another cytokine studied in septic shock, IL-6, peaked at 12 h after CLP at 1011 +/- 431 pg/ml, and at 90 min after lethal LPS at 16,300 +/- 3,700 pg/ml. Mice were treated with an anti-TNF antibody that has been shown previously to inhibit in vivo TNF activity. Antibody treatment of mice subjected to CLP significantly reduced TNF bioactivity but did not reduce mortality or pulmonary neutrophilic infiltration. In the i.v. LPS model, anti-TNF antibody treatment concomitant with LPS injection reduced plasma TNF activity from 80,000 +/- 20,000 pg/ml to undetectable levels. However, anti-TNF treatment immediately before either i.v. or i.p. LPS did not reduce mortality. Additionally, when the antibody was administered 4 h before the lethal i.v. LPS, there was no reduction in lethality. These data show that in two separate models of septic shock blockade of TNF biologic activity will not prevent lethality.

Animals↗
PubMed1992

Expert system design in hematology diagnosis.

A two-part study was designed to test the hypothesis that sufficient information is available from a modern hematology analyzer (the Coulter STKS) to reach a reliable intermediate conclusion which can be used as input to the next decision-making level in the design of a high-performance expert system for hematology diagnosis. In phase one, we analyzed the performance of three probabilistic systems (using Bayes' rule) which interpret STKS data: a control system which took the traditional approach of classifying cases into specific diagnoses, and two test systems which were designed to reach only an intermediate conclusion but not a final diagnosis. One of the test systems classified cases into "textbook categories" of disease and the other utilized defined diagnostic patterns. The systems were tested with 150 cases. The pattern approach ranked the correct choice first in 141 of 150 cases (94%). In phase two, we abandoned Bayes' rule, reformulated the pattern approach into a heuristic classification system, and tested its reliability on 820 cases. The algorithm of the reformulated system was able to classify all 820 cases into the same predominant pattern as a panel of three experienced laboratory hematologists.

Algorithms↗
PubMed1991

Alternating zinc fingers in the human male associated protein ZFY: 2D NMR structure of an even finger and implications for "jumping-linker" DNA recognition.

ZFY, a sex-related Zn-finger protein encoded by the human Y chromosome, is distinguished from the general class of Zn-finger proteins by the presence of a two-finger repeat. Whereas odd-numbered domains and linkers fit a general consensus, even-numbered domains and linkers exhibit systematic differences. Because this alternation may have fundamental implications for the mechanism of protein-DNA recognition, we have undertaken biochemical and structural studies of fragments of ZFY. We describe here the solution structure of a representative nonconsensus (even-numbered) Zn finger based on 2D NMR studies of a 30-residue peptide. Structural modeling by distance geometry and simulated annealing (DG/SA) demonstrates that this peptide folds as a miniglobular domain containing a C-terminal beta--hairpin and N-terminal alpha-helix (beta beta alpha motif). These features are similar to (but not identical with) those previously described in consensus-type Zn fingers (derived from ADR1 and Xfin); the similarities suggest that even and odd ZFY domains bind DNA by a common mechanism. A model of the protein-DNA complex (designated the "jumping-linker" model) is presented and discussed in terms of the ZFY two-finger repeat. In this model every other linker is proposed to cross the minor groove by means of a putative finger/linker submotif HX4HX3-hydrophobic residue-X3. Analogous use of a hydrophobic residue in a linker that spans the minor groove has recently been described in crystallographic and 3D NMR studies of homeodomain-DNA complexes. The proposed model of ZFY is supported in part by the hydroxyl radical footprint of the TFIIIA-DNA complex [Churchill, M.E.A., Tullius, T.D., & Klug, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5528-5532].

Amino Acid Sequence↗
PubMed1991

Maximal activities of glutaminase, citrate synthase, hexokinase, 6-phosphofructokinase and lactate dehydrogenase in skin of immune-competent Balb/c and immune-deficient Balb/c (nu/nu) mice during wound healing.

1. The maximal activities of hexokinase (HK), 6-phosphofructokinase (PFK), lactate dehydrogenase, citrate synthase (CS) and glutaminase (GLU) which provide quantitative and qualitative indices of flux through several important metabolic pathways have been examined in the wounded skin of haired immune competent Balb/c mice and hairless immune deficient Balb/c (nu/nu) mice of various ages during the first ten days of wound healing. 2. The potential for glucose utilization and for aerobic metabolism as suggested by the maximal activities of HK, PFK, CS, were raised in the skin of Balb/c mice of various ages on all post wounding days. Increases in the maximal activity of GLU was observed only in the skin of 6 and 10 weeks old Balb/c mice during wound healing. 3. There was no evidence of a contribution to the maximal activity of GLU by infiltrating cells of the immune system to the wound site in the skin of either haired or hairless mice.

Aging↗
PubMed1991

Interleukin 2 induces tumor necrosis factor gene expression in vivo.

Interleukin 2 (IL-2) treatment of malignancies is often associated with severe toxicity, and the alterations observed after high dose administration of IL-2 are similar to those induced by recombinant tumor necrosis factor (TNF). We therefore examined the hypothesis that IL-2 induces TNF gene expression in vivo. Purified, recombinant human IL-2 was injected intraperitoneally into mice which had been previously primed with complete Freund's adjuvant (CFA). Biologically-active TNF was detected in the ascites fluid of CD-1 mice; it was detectable 30 minutes after IL-2 and peaked at 1 hour (500 +/- 158 units/ml). Plasma levels of TNF also peaked at 1 hour at 32 +/- 4 units/ml. Similar kinetics were observed in CBA/J mice. TNF specific mRNA was also present in the ascites cells, and peaked 30 minutes after IL-2 injection into CBA/J mice. Injection of vehicle containing 10 times the maximum contaminating dose of endotoxin did not induce TNF above background levels. As a further control for potential endotoxin contamination, IL-2 was injected into endotoxin hyporesponsive C3H/HeJ mice. These mice also demonstrated the rapid upregulation of biologically-active TNF in the ascites, with peak production occurring at 1 hour (125 +/- 47 units/ml). The induction of biologically-active TNF in the C3H/HeJ mice was associated with a peripheral blood neutrophilia and lymphopenia, pathophysiologic alterations that have been attributed to TNF. These data show that a single injection of purified, recombinant IL-2 induces TNF gene expression in vivo.

Animals↗
PubMed1990

Cyclosporin a modulation of tumor necrosis factor gene expression and effects in vitro and in vivo.

We investigated, in vitro and in vivo, the cyclosporin A (CsA) regulation of LPS-induced TNF gene expression and subsequent pathophysiologic changes. In vitro dose-response kinetics data showed that CsA inhibited TNF bioactivity in the supernatant without delaying its production, whereas Northern blot and in situ hybridization analysis demonstrated that CsA did not inhibit TNF mRNA expression. We then sought to examine the in vivo effects of CsA (75 mg/kg) in CBA/J mice that were primed with CFA, and injected 2 wk later with LPS. CsA demonstrated suppression of local levels (ascites) of TNF as measured by either bioactivity or an anti-murine TNF ELISA. However, CsA did not decrease mRNA for TNF, or cell-associated TNF. In vivo kinetics studies were performed to show that CsA blocked both local (ascites) and systemic (plasma) LPS-induced TNF production without delaying these effects. CsA inhibited the neutrophilia and lymphopenia that developed after the LPS challenge, but did not block the lung neutrophilic infiltrate. These observations are helpful in understanding the role of the macrophage in CsA immunosuppression, particularly with regard to the ability of CsA to block LPS-induced TNF secretion.

Animals↗
PubMed1990

WEHI 164 subclone 13 assay for TNF: sensitivity, specificity, and reliability.

Tumor necrosis factor alpha (TNF) is a peptide monokine involved in a number of immune reactions. To further understand the role of TNF in disease states it is critical to have an inexpensive, yet sensitive and specific assay. Additionally, the effects of prostaglandin E2 (PGE2), dexamethasone (dex), and cyclosporine A (CsA) on TNF gene expression have been studied, although little is known of the effects these compounds have on TNF containing samples. The aim of this study is to determine the sensitivity and specificity of a highly sensitive cell line to the actions of TNF, and to elucidate parameters which affect the stability of TNF in biological fluids. Dex and PGE2 at concentrations of 10(-5), 10(-7), and 10(-9) M, were shown not to effect the WEHI assay, and neither did CsA (10 ng/ml-1 ug/ml). The cells were not lysed by recombinant murine IL-1 alpha or beta, human recombinant IL-1 alpha or beta, human recombinant IL-2 or human recombinant IL-6 at concentrations ranging from 0.02 pg/ml to 1.0 ug/ml, or murine gamma-IFN from 100 pg/ml to 10 ng/ml. TNF containing samples with 1%-10% fetal calf serum maintained their cytolytic activity even after three freeze-thaw cycles. Serum samples did not lose any cytolytic activity with up to 11 cycles of freezing and thawing whereas, tissue culture media, containing TNF, lost significant activity with freeze-thawing. The WEHI assay has successfully detected cytolytic activity from lipopolysaccharide stimulated specimens from a number of different species. These data show the utility of this highly sensitive and specific assay. Furthermore, the WEHI assay showed a high degree of reproducibility in repeated assays.

Animals↗
PubMed1990

Role of tumor necrosis factor-alpha in lipopolysaccharide-induced pathologic alterations.

Tumor necrosis factor-alpha (TNF) has been implicated strongly as a principal mediator in the pathogenesis of septic shock. The authors investigated the in vivo production of TNF in CBA/J and CD-1 mice that had been primed by an intraperitoneal injection of complete Freund's adjuvant followed 2 weeks later by an intraperitoneal injection of lipopolysaccharide (LPS). TNF bioactivity peaked in both the ascites and plasma one hour after challenge, and TNF mRNA expression in the ascites cells peaked 30 minutes after LPS. After the induction of bioactivity, an interstitial pulmonary neutrophilic infiltrate occurred that was quantitated both morphometrically and by a myeloperoxidase (MPO) assay. Peripheral blood neutrophilia and lymphopenia developed after the LPS injection (PMNs: control, 46 +/- 2%; LPS, 65 +/- 3%; Lymphs control, 53 +/- 2%; LPS, 37 +/- 3%). Treatment with dexamethasone (Dex) completely inhibited the pulmonary neutrophilic infiltrate as measured by the (MPO) assay. Because Dex will inhibit the production of several cytokines, anti-TNF antiserum was given to mice at the same time as the LPS challenge to assess specifically the role of TNF in inducing these changes. This antiserum partially blocked the pulmonary neutrophil infiltrate, and completely blocked the peripheral blood changes at one hour after LPS. These data demonstrate that TNF plays an important role in the early pathophysiologic alterations that occur after systemic exposure to LPS.

Animals↗
PubMed1989

Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: assignment of aromatic resonances with application to protein folding, structure, and dynamics.

The aromatic 1H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25----Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constraints in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. In the monomer large variations are observed in the line widths of amide resonances, suggesting intermediate exchange among conformational substates; such substates may relate to conformational changes observed in different crystal states and proposed to occur in the hormone-receptor complex. Additional evidence for multiple conformations in solution is provided by comparative studies of an insulin analogue containing a peptide bond between residues B29 and A1 (mini-proinsulin). This analogue forms dimers and higher-order oligomers under conditions in which native insulin is monomeric, suggesting that the B29-A1 peptide bond stabilizes a conformational substate favorable for dimerization. Such stabilization is not observed in corresponding studies of native proinsulin, in which a 35-residue connecting peptide joins residues B30 and A1; this extended tether is presumably too flexible to constrain the conformation of the B-chain. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures.

Circular Dichroism↗
PubMed1989

Cyclosporine A inhibits TNF production without decreasing TNF mRNA levels.

The role of cytokines in health and disease has received increasing attention and numerous investigations have explored the regulation of cytokine gene expression. Tumor necrosis factor-alpha (TNF) has received particular attention because of its central role in septic shock and more recent work has shown its participation in transplant immunology. We explored the mechanism of cyclosporine A (CsA) modulation of complete Freunds adjuvant macrophage (CFA-MO) TNF gene expression. From 0.001 to 1 microgram/ml, CsA dose-dependently inhibited lipopolysaccharide (LPS) induced secreted bioactivity; at doses above 10 micrograms/ml CSA was directly toxic to CFA-MO. However, there was no suppression of TNF mRNA levels, and CsA also did not inhibit the accumulation of cell-associated TNF. Thus, CsA modulates TNF gene expression in a previously undescribed manner.

Animals↗
PubMed1989

Adjuvant activity of respirable iron ore dust.

The influence of the respirable fraction of an iron ore dust on immunity has been studied. The iron ore dust enhanced the specific immune responses to both sheep erythrocytes and a bacterial lipopolysaccharide. The IgG class of antibody was enhanced significantly and its pattern of enhancement suggested that the iron ore dust was functioning as an adjuvant. In studies to test this possibility further, the pattern of antibody development over both the primary and the secondary immune response to sheep erythrocytes closely matched that of Freund's incomplete adjuvant and low levels of silica. The splenocytes, from animals that had received iron ore dust for various lengths of time, exhibited enhanced polyclonal mitogenic activity for phytohaemagglutinin, in vitro. These properties were seen when the dust had been inhaled or injected into the pleural cavity and persisted for over 1 year following the injection or inhalation of the dust. Attempts to show the induction of interleukin-1 by the macrophages from mice implanted intraperitoneally with the iron ore dust were unsuccessful.

Adjuvants, Immunologic↗
PubMed1987

Stimulation of prostaglandin E2 and thromboxane B2 production by human monocytes in response to interleukin-2.

Interleukin 2 (IL-2) is a potent lymphokine involved in the regulation of immune responses and is classically regarded as a stimulus for the activation and growth of T-cells. Recent reports have demonstrated the IL-2 dependent activation of human peripheral blood lymphocytes into lymphokine activated killer cells capable of lysing tumor cells both in vitro and in vivo. In this study we report data which clearly show IL-2 may also act to down-regulate the immune response by inducing the synthesis of arachidonic acid metabolites with known immunosuppressive actions. Stimulation of peripheral human blood monocytes with IL-2 caused an increased production of prostaglandin E2 (PGE2) and thromboxane (TXB2) in a dose-dependent manner. Kinetic analysis showed no increase above controls after 6 hours and maximal levels by 10 hours; elevated levels were maintained after 45 hours of incubation. After 20 hours of stimulation with 2000 U/ml IL-2, the level of PGE2 and TXB2 were greater than three-fold above controls, 0.7 and 19 ng/10(6) cells, respectively. The stimulation was relatively specific in that neither prostacyclin nor leukotrienes were produced in response to IL-2. These data demonstrate that IL-2 acts on human monocytes to induce the secretion of PGE2 and TXB2.

6-Ketoprostaglandin F1 alpha↗