Biomedical subjects

D Xu

Publications and source records attributed to D Xu.

At least 343 records · Page 19Linked to original sources

PubMed1995

Protection against Leishmania major infection in genetically susceptible BALB/c mice by gp63 delivered orally in attenuated Salmonella typhimurium (AroA- AroD-).

The gene encoding the Leishmania major (L. major) promastigote surface glycoprotein, gp63, was introduced into the Salmonella typhimurium (S. typhimurium) aroA- aroD- live oral vaccine strain BRD509 and expressed under the control of a constitutive tac promoter in plasmid pKK233-2. This construct (GID101) expressed gp63 in vitro and was used to immunize highly susceptible BALB/c mice by the oral route. The plasmid was relatively stably inherited by bacteria growing or persisting in the mesenteric lymph nodes of immunized mice. Mice immunized with GID101 developed significant resistance against a challenge infection with L. major compared to controls immunized with BRD509 alone. Spleen and lymph node cells from immunized mice developed a strong in vitro proliferative T-cell response to killed or live L. major. The activated T cells secreted interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) which was abrogated by treatment with anti-CD4 but not with anti-CD8 antibody. The cells did not produce detectable levels of interleukin-4 (IL-4). The immunized mice also produced significant amounts of leishmanial specific IgG2a antibody but did not develop delayed-type hypersensitivity (DTH) to live parasites. No IgG1 antibody was detected. These data therefore demonstrate that gp63 gene delivered orally by a vaccine strain of S. typhimurium can preferentially induce the development of Th-1 subset of CD4+ T cells and protective immunity in the highly susceptible BALB/c mice.

Animals↗

PubMed1995

[Influence of PGE2 on the oxidative modification of LDL and the scavenger receptor activities of macrophages].

The influence of oxidized LDL (OX-LDL) on scavenger receptor of macrophages and the protective effects of PGE2 and selenium was studied by means of BA-ELISA technique, immunohistochemical methods and oil red O histochemistry. The results indicated that the scavenger receptor activities of macrophages in two treated groups were much lower than that of OX-LDL group. Oil red O staining showed that the macrophages in OX-LDL group were full of fatty drops and became foam cells while little lipid could be found in the treated groups. Immunohistochemical study revealed that the entry of OX-LDL into the macrophages was by way of scavenger receptors. The results confirmed that both PGE2 and selenium had marked antioxidizing effects and that PGE2 was slightly more effective than selenium.

Animals↗

PubMed1995

Protection against leishmaniasis by injection of DNA encoding a major surface glycoprotein, gp63, of L. major.

cDNA encoding the highly conserved major surface glycoprotein, gp63, of Leishmania major was cloned, together with a signal sequence, into an eukaryotic expression vector, pCDNAI, which carries the human cytomegalovirus (CMV) promoter. This construct, pCMV/glycoprotein 63 (gp63), when injected into the skeletal muscle of BALB/c mice expressed sustained levels of gp63 in the muscle tissue for at least 40 days. Spleen and lymph node cells from the immunized mice produced significant amounts of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) but no detectable IL-4 when cultured with L. major antigens in vitro. The immunized mice also developed significant resistance against L. major infection compared to control mice injected with the empty plasmid. These results suggest that nucleic acid vaccine is effective against parasite infections.

Animals↗

PubMed1995

Expression of TGF-beta in attenuated Salmonella typhimurium: oral administration leads to the reduction of inflammation, IL-2 and IFN-gamma, but enhancement of IL-10, in carrageenin-induced oedema in mice.

Mice injected with carrageenin in the footpad developed local inflammation which peaked at 48 hr. This was significantly reduced in mice inoculated orally with an attenuated Salmonella construct expressing transforming growth factor-beta (TGF-beta). Administration of the Salmonella construct alone had no effect on inflammation. High levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were secreted by draining lymph node cells from mice injected with carrageenin following stimulation in vitro. Prior inoculation with Salmonella enhanced the production of IL-2 and IFN-gamma from the draining lymph node cells. Administration of the Salmonella-TGF-beta construct significantly inhibited the production of these cytokines. In contrast, IL-10 only was secreted from draining lymph node cells of animals inoculated with the Salmonella-TGF-beta construct. Thus, oral administration of TGF-beta can significantly inhibit local inflammation and alter the cytokine secretion pattern of cells from lymph nodes draining the site of inflammation.

Administration, Oral↗

PubMed1995

Pharmacological evaluation of selected, orally active, peptidyl inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) is a serine proteinase capable of degrading a number of connective tissue macromolecules and has been implicated in the destructive processes associated with several chronic inflammatory diseases. A large series of peptidyl electrophilic ketones have been shown to be potent inhibitors of HNE in vitro and in vivo. We report the pharmacology and pharmacokinetics of selected inhibitors from this series. MDL 101, 146, MDL 102, 111, MDL 102,823 and MDL 100,948A are -Val-Pro-Val-pentafluoroethylketones with various amino-terminal protecting groups. Although their Ki values varied considerably, (25-170 nM), these compounds demonstrated similar ED50 values after oral administration in the HNE-induced hemorrhage model in hamsters and rats. The duration of action of MDL 102,111 was shorter than that of the other analogs in the HNE-induced pulmonary hemorrhage model in both species. The duration of action of all of the compounds was longer in the rat than in the hamster. Isolated sections of rat jejunum were used to determine the in situ absorption of these compounds. MDL 102,111 showed the greatest extent of absorption, with MDL 102,823, MDL 100,948A and MDL 101,146 following in descending rank order. The comparative metabolic stability of these analogs was measured over a 2-hr incubation period using rat liver homogenates. MDL 101,146 was the most stable, followed by MDL 102,823, MDL 102,111 and MDL 100,948A. MDL 101,146 was more stable in a liver homogenate from rats compared with a liver homogenate from hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

PubMed1995

Studies of protective properties of pluronic and other agents on the hybridoma cell culture.

The potential toxicity and optimal concentrations of different protective agents such as pluronic F-68, methylcellulose (MC), CMC, and GPE on the in vitro growth of murine hybridoma 2F7 cells that secrete monoclonal antibody against small-cell lung cancer were studied. The effect on the rate of glucose utilization of adding protective agents was investigated. The protective effects of different concentrations of the protective agents at high agitation speed were also observed. It showed that 0.05% to 0.10% (w/v) of pluronic F-68 and 0.10% to 0.20% (w/v) of MC could protect hybridoma cells from shear stress at high agitation speed. Adding pluronic F-68 could increase glucose utilization rate, but increased the ammonia production rate. Although CMC did not affect 2F7 cell growth at a concentration less than 0.10% (w/v), it exhibited no protective property. GPE could lyze hybridoma cells. In a 1.5-L CelliGen bioreactor, when the pluronic F-68 concentration was 0.10% (w/v) in the medium and agitation speed was 70 r/min, the hybridoma cells could grow normally.

Ammonia↗

PubMed1995

Human immunodeficiency virus load. Quantitative assessment in semen from seropositive individuals and in spiked seminal plasma.

OBJECTIVE: To establish criteria for the quantitation of the human immunodeficiency virus (HIV) in seminal plasma, seminal cells and the whole semen of HIV-infected individuals. The reverse transcription polymerase chain reaction (RT-PCR), DNA-PCR and semen HIV culture assays were standardized by testing seminal plasma spiked separately with serial dilutions of cell-free and cell-associated HIV stocks of known titers. The standardized assays were then used to assess the quantity of virus in the freshly collected seminal cells and seminal plasma. RESULTS: Analysis of freshly collected peripheral blood mononuclear cells (PBMCs) and paired semen from HIV-seropositive men who had received antiviral drugs and/or immunemodulators indicated that HIV could be isolated from 42 of 55 (76%) samples of peripheral blood mononuclear cells (PBMCs) and 13 of 55 (24%) samples of ejaculates. Since no semen sample was culture positive in the absence of culturable HIV in PBMCs of the same individual, RT-PCR was 5-125 times more sensitive than cell cultures for the quantitation of HIV spiked in seminal plasma, freshly collected seminal fluid and whole semen. Further, HIV-RNA was detected in samples containing higher dilutions of virus from which HIV was not isolated by culture. CONCLUSION: We conclude that cell-free HIV is present in excess of the culturable virus in all specimens tested and that the high sensitivity of HIV-RNA detection is useful for quantitation of the virus directly in seminal fluid, seminal cells and whole semen.

Cell Survival↗

PubMed1994

Inhibition of adrenal cytochromes P450 by 1-aminobenzotriazole in vitro. Selectivity for xenobiotic metabolism.

Studies were done to determine the effects of a P450 suicide inhibitor, 1-aminobenzotriazole (ABT), on adrenal steroid and xenobiotic metabolism. Incubation of guinea pig adrenal microsomes with ABT plus an NADPH-generating system caused a time-dependent decline in total P450 concentrations. The maximal decrease in P450 levels was approximately 35% and was accompanied by an equimolar decrease in heme content. Western blot analyses indicated that ABT had no effect on P450 apoprotein levels. Benzphetamine (BZ) N-demethylase and benzo[a]pyrene (BP) hydroxylase activities were inhibited almost completely by microsomal incubations with ABT. In contrast, neither steroid 17 alpha-hydroxylase nor 21-hydroxylase activity was affected by ABT. The steroid-induced type I spectral change in adrenal microsomes also was not affected by ABT, whereas that induced by BZ was eliminated. Similar studies with adrenal mitochondria indicated that ABT had no effect on mitochondrial P450 concentrations or on mitochondrial steroid metabolism. The results demonstrate that the in vitro actions of ABT on adrenal cytochromes P450 are highly selective for those isozymes that catalyze xenobiotic metabolism. Therefore, ABT should serve as a useful probe for further characterization of adrenal xenobiotic-metabolizing P450 isozymes.

Adrenal Glands↗

PubMed1994

Melting and premelting transitions of an oligomer measured by DNA base fluorescence and absorption.

Incorporation of 2-aminopurine (2AP) in place of adenine gives an optical probe of local and global DNA conformation. The temperature dependence of the absorption of the duplex d[CTGA(2AP)-TTCAG]2 DNA decamer shows that the helix has approximately an all-or-none melting transition. Absorbance at wavelengths of 260 and 330 nm monitors the average normal base conformation and the 2AP base local conformation, respectively. From this measure, the 2AP base melts less than 1 degree C below the other bases. Temperature-dependent lifetime measurements of 2AP also mirror the melting transition. Absorption spectra show that below Tm most 2AP's are H-bonded. Fluorescence intensity and excitation spectra measurements show, on the other hand, that the most highly-fluorescent states correspond to non-H-bonded 2AP's which sense conformational changes of the helix. The temperature dependence of the fluorescence spectral shift shows the conformation and/or dynamics of the 2AP base changes 10 degrees C or more below Tm. The data suggest a premelting transition which is purely dynamic in nature--transient exposure of most 2AP's to water increases, while the average conformation remains B-helical.

2-Aminopurine↗

PubMed1994

ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1.

Endothelin-1 (ET-1), a 21-residue vasoactive peptide, is produced in vascular endothelial cells from the 38-residue inactive intermediate big endothelin-1 via a specific cleavage at Trp-21-Val-22. The protease that catalyzes the conversion, endothelin-converting enzyme (ECE), constitutes a potential regulatory site for the production of the active peptide. We report the identification of ECE-1, a novel membrane-bound neutral metalloprotease that is expressed abundantly in endothelial cells in vivo and is structurally related to neutral endopeptidase 24.11 and Kell blood group protein. When transfected into cultured cells that normally secrete only big ET-1, the ECE-1 cDNA conferred the ability to secrete mature ET-1. In transfected cells, ECE-1 processes endogenously synthesized big ET-1 as well as exogenously supplied big ET-1, which interacts with ECE-1 on the cell surface. ECE-1 may provide a target for pharmacological intervention to alter ET-1 production.

Amino Acid Sequence↗

PubMed1994

A non-activating "humanized" anti-CD3 monoclonal antibody retains immunosuppressive properties in vivo.

OKT3, a mouse anti-human CD3 mAb, is a potent immunosuppressive agent used in clinical transplantation to prevent or treat allograft rejection. Associated with this therapy is the systemic release of several cytokines that result in a series of adverse side effects. This release of cytokines is dependent on the cross-linking mediated by OKT3 between T cells and the Fc gamma R-bearing cells. To generate an anti-human CD3 mAb with reduced activating properties as compared with OKT3, we have transferred the complementary determining regions of OKT3 onto human IgG frameworks and then performed point mutations that reduce the affinity of the "humanized" anti-CD3 mAbs for Fc gamma Rs. Initial, in vitro, studies showed that whereas OKT3 and the parental humanized anti-CD3 mAbs activated T cells similarly, a humanized Fc variant failed to do so. Both the Fc variant and the activating anti-CD3 mAbs induced comparable modulation of the TCR and suppression of cytolytic T cell activity, in vitro. In the current study, we exploited an experimental model in which human splenocytes from cadaveric organ donors were inoculated into severe combined immunodeficient mice (hu-SPL-SCID mice) to test the activating and immunosuppressive properties of these anti-human CD3 mAbs in vivo. Unlike injection of OKT3 or of the parental humanized mAb, administration of the Fc variant did not result in T cell activation in vivo, as evidenced by the lack of induction of surface markers of activation, and of systemic human cytokines, including IL-2. Importantly, similar prolongation of human allograft survival was achieved with all anti-CD3 mAbs, indicating that the nonactivating anti-CD3 mAbs retained significant immunosuppressive properties in vivo. Thus, the use of an Fc variant in clinical transplantation should result in fewer side effects than observed with OKT3, while maintaining its clinical efficacy.

Animals↗

PubMed1994

Rapamycin-induced long-term allograft survival depends on persistence of alloantigen.

In this study the mechanism of rapamycin-induced long-term allograft tolerance was investigated in a rat model. We have demonstrated that the tolerance is strain specific, but is not organ specific. The tolerized rats failed to generate high levels of donor-specific cytotoxic Ab and cytotoxic cells in vivo. Removal of the alloantigen from the tolerized rats with or without concomitant thymectomy could break down the status of tolerance, and the rats regained the capability to reject the grafts and to develop specific cytotoxic Ab and cytotoxic cells. These results clearly indicate that the maintenance of the rapamycin-induced long-term tolerance to allografts depends on the persistence of alloantigens. Mechanistically, we have shown that the reduced IL-2 production and the reduced antigenicity of the graft in the tolerized rat contribute to, but are not solely responsile for, the tolerance. The results of adoptive transfer experiments suggest that regulatory cells or suppressive serum factors are not involved in the tolerance. The fact that the removal of the alloantigen in the thymectomized rat could reverse the tolerance, indicates that there is no clonal deletion. We propose that chronic desensitization due to prolonged engagement of TCR by persistent alloantigens is the major mechanism at the late stage of the rapamycin-induced allograft tolerance.

Animals↗

PubMed1994

Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells.

The balance between T helper type 1 (Th 1) and T helper type 2 (Th2) cells determines the outcome of many important diseases. Using cloned murine T cell lines, evidence is provided that Th1, but not Th2, cells can be activated by specific antigens or a T cell mitogen, concanavalin A, to produce large amounts of nitric oxide (NO). Furthermore, NO can inhibit the secretion of interleukin (IL)-2 and interferon-gamma by Th1 cells but has no effect on IL-4 production by Th2 cells. Th1 and Th2 cells can, thus, be distinguished by their differential production of and susceptibility to NO. NO exerts a self-regulatory effect on Th1 cells which are implicated in immunopathology.

Animals↗

PubMed1994

Inhibition of nitric oxide synthesis by interleukin-4 may involve inhibiting the activation of protein kinase C epsilon.

The murine macrophage cell line, J774, when activated with interferon-gamma (IFN-gamma), expressed high level of inducible nitric oxide synthase (iNOS) and bound significantly more [3H]-phorbol-dibutyrate (PBu2) compared to non-activated cells. The increased PBu2 binding to the particulate fraction of the cells is a measure of activation and translocation of protein kinase C (PKC). Both the expression of iNOS and the enhanced. PBU2 binding in the activated J774 cells were significantly inhibited by the pretreatment of the cells with murine recombinant interleukin-4 (IL-4). Stimulation of J774 cells by IFN-gamma and lipopolysaccharide results in the translocation predominantly of the epsilon isoform of PKC (PKC-epsilon), and this is inhibited by IL-4. The inhibition of PKC activation was also evident by measuring the PKC activity in the cytosolic-fraction of the IL-4-treated cells. Activated J774 cells pretreated with IL-4 or a PKC-specific inhibitor (RO31-8220) failed to express mRNA of iNOS analyzed by PCR. These results, therefore, suggest that the inhibition of nitric oxide synthesis in activated murine macrophages by IL-4 is at the transcriptional level and may involve the inhibition of the activation of PKC-epsilon.

Amino Acid Oxidoreductases↗

PubMed1994

Long-term ethanol feeding selectively impairs the attachment of rat perivenous hepatocytes to extracellular matrix substrates.

BACKGROUND/AIMS: We have previously shown that long-term ethanol consumption by rats results in a profound decrease in hepatocyte attachment to various extracellular matrix substrates. The present study investigated whether differences in attachment exist between cells isolated from either the periportal or perivenous regions of the liver. METHODS: Rats received long-term ethanol, and hepatocytes were selectively isolated by the digitonin-collagenase perfusion method. The ability of periportal and perivenous cells isolated from ethanol-fed and pair-fed control rats to attach to plates coated with either laminin, fibronectin, or type I collagen was then assayed. RESULTS: With all substrates, the attachment of perivenous hepatocytes isolated from ethanol-fed animals was significantly impaired. Time-course studies showed that although the rate of attachment of perivenous cells from ethanol-fed animals was only slightly reduced, a dramatic decrease in absolute attachment was observed. Furthermore, the perivenous cells isolated from ethanol-fed animals detached more readily from the substrate-coated plates than the corresponding periportal cells or either periportal or perivenous cells from pair-fed controls. CONCLUSIONS: Long-term ethanol consumption impairs hepatocyte-extracellular matrix interactions more severely in the perivenous region of the liver. This finding could be relevant to the pathological changes observed in alcoholic liver injury.

Animals↗

PubMed1994

Viscosity and flow properties of concentrated solutions of chitosan with different degrees of deacetylation.

The effects of the degree of deacetylation (DD) on the viscosity and flow behaviour of concentrated solutions of chitosan were investigated using 0.2 M CH3COOH and 0.2 M CH3COOH/0.1 M CH3COONa aqueous solutions as solvents. The results indicated that the viscosity and flow properties of the solutions differed with the DD of chitosan. The solution viscosities and the non-Newtonian flow properties as well as the flow activation energies E gamma increased with the increasing DD of chitosan. However, the additional salt decreased the viscosities and the non-Newtonian flow properties of the solutions of chitosan, but did not change the flow activation energies E gamma of the solutions.

Acetylation↗

PubMed1994

Genetic vaccination against leishmaniasis.

The gene encoding for a major surface glycoprotein, gp63, of Leishmania major was cloned into the eukaryotic expression plasmid pCDNAI with CMV or RSV promoters. The highly susceptible Balb/c mice were injected intramuscularly with 100 micrograms/mouse of the purified plasmid. The plasmids were found to be stable in vivo for at least 40 days after injection and expressed significant levels of gp63, demonstrable by immunohistological staining with specific antibody. The immunized mice developed significant resistance against L. major infection compared to controls similarly immunized with the empty plasmid. Spleen cells from the immunized mice produced significant levels of IL-2 and IFN-gamma but no detectable IL-4 when cultured with leishmanial antigens in vitro.

Animals↗

PubMed1994

Experimental nerve thermal injury.

Thermal injury to rat sciatic nerve was produced by local heating (47 and 58 degrees C) and studied physiologically and morphologically. Unmyelinated nerve fibres showed a greater direct vulnerability to hyperthermia; first manifest as a reversible conduction block of C fibre action potentials and at higher temperatures by immediate and selective axonal degeneration. By contrast, lower grade nerve thermal injury resulted in delayed, selective loss of myelinated fibres. Evidence from this study suggests that this is secondary to a heat-induced angiopathy, immediately and diffusely manifest in the vasa nervorum and giving rise to a progressive and ultimately severe reduction in nerve blood flow. The relative sparing of unmyelinated fibres is likely to be a result of their greater resistance to ischaemia. The pathological vulnerability of unmyelinated fibres to thermal injury, coupled with the susceptibility of large myelinated nerve fibres to secondary ischaemia, largely resolves previous contradictions in the literature.

Action Potentials↗