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Biomedical subjects

J B Cheng

Publications and source records attributed to J B Cheng.

At least 37 records · Page 2Linked to original sources
PubMed1994

Vortex flow filtration of mammalian and insect cells.

The use of vortex flow filtration for harvesting cells or conditioned medium from large scale bioreactors has proven to be an efficient, low shear method of cell concentration and conditioned medium clarification. Several 8-10 L batches of the human histiocytic lymphoma U-937 cell line (ATCC CRL 1593) were concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane. An aggressive filtration regimen caused a 17% loss of cell viability and a 32% loss of IL-4 receptor binding capacity when compared to a batch centrifuged control. A reduction of the rotor speed from 1500 to 500 RPM and reduction of system back pressure from 10 to 0 PSIG resulted in cell viability and IL-4 binding capacity comparable to the control. Several 10 L batches of baculovirus infected Sf-9 cells were also concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane. SDS-PAGE analysis of filtrate samples showed that aggressive filtration caused cell damage which led to contamination of the process stream by cellular lysate. When rotor speed was reduced to 500 RPM and system back pressure was reduced to 0 PSIG, the amount of contaminating lysate proteins in filtrate samples was comparable to a batch centrifuged control.

Animals↗
PubMed1993

Antigen-mediated pulmonary eosinophilia in immunoglobulin G1-sensitized guinea pigs: eosinophil peroxidase as a simple specific marker for detecting eosinophils in bronchoalveolar lavage fluid.

Eosinophil peroxidase (EPO) has been used previously to detect the number of eosinophils in the peritoneal exudate and bone marrow of mice. The present study was undertaken to determine 1) whether EPO activity may provide a measure of a change in eosinophils in bronchoalveolar lavage fluid (BALF) of guinea pigs, 2) whether immunoglobulin (Ig)G1 could play a role in pulmonary eosinophilia and 3) effects of pharmacological agents on the EPO response in an IgG1 passively sensitized animal model. The activity of EPO was assessed by the ability of cell lysates (0.1% Triton-100 treatment) to oxidize 1 mM o-phenylenediamine in the presence of 1 mM H2O2 for 5 min at 22 degrees C. The enzyme activity was found to be eosinophil dependent, inhibited by the EPO inhibitor 3-amino-1,2,4-triazole (IC50 = approximately 0.1 mM) and relatively resistant to heat treatment (no loss of activity after 2-hr preincubation at 56 degrees C). To determine antigen-dependent eosinophil and EPO responses, guinea pigs were passively sensitized i.p. with 0.5 mg/kg of an affinity-purified antiovalbumin (OA) IgG1. Two to 3 days later, the sensitized animals were injected with pyrilamine (5 mg/kg, i.p.) before OA aerosol challenge. Aerosolized OA (0.1%) caused a significant increase in both eosinophil number and EPO activity in BALF of sensitized guinea pigs at 18 to 24 hr post-challenge. At a given concentration of aerosolized OA, the enzyme activity increased as a function of the antibody dose and time post-OA challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Competition of leukotrienes and ICI-198,615 for [3H]LTD4 binding sites in guinea pig lung membranes suggests the involvement of two LTD4 receptor subtypes.

Pharmacological analysis of the effects of leukotriene D4 (LTD4) antagonists on contraction of guinea pig airway smooth muscle to leukotrienes reveals the presence of two subtypes of the LTD4 receptor. This finding is, however, inconsistent with [3H]LTD4 equilibrium binding results, which show no evidence of a heterogeneity of pulmonary [3H]LTD4 binding sites. It is possible that LTD4 binds to the two receptor subsets with equal affinity, and the pharmacological difference between them lies in the relative ability of leukotriene (LT) agonists and antagonists to interact at the receptor sites. This study was, therefore, undertaken to determine the rank order of potency of LTs and ICI-198,615 in competing with [3H]LTD4 for their respective binding sites in guinea pig lung membranes. To determine precisely the inhibitory constant (Ki) of LTC4, we used the irreversible gamma-glutamyl transpeptidase inhibitor, acivicin (AT-125), to prevent LTC4 metabolism. Incubation of lung membranes with 5 mM AT-125 for 120 min at 25 degrees C resulted in greater than 98% recovery of LTC4. Unlike L-serine-borate complex, AT-125 failed to inhibit pulmonary [3H]LTD4 binding. These results suggest that AT-125 can be used in this study. Nonlinear least squares analysis of the results of LTD4/[3H]LTD4 or ICI-198,615/[3H]LTD4 competitive binding reveals that either LTD4 (Ki = 0.49 nM) or ICI-198,615 (Ki = 6.89 nM) interacts at a single homogenous population of [3H]LTD4 binding sites. However, the data of competitive binding results of LTC4 (in the presence of AT-125) or LTE4 are best fitted for its interaction with high- and low-affinity [3H]LTD4 binding sites, designated as LTD4 alpha and LTD4 beta sites, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Antigen-dependent leukotriene synthesis and histamine release from IgG1 passively-sensitized guinea pig lungs ex vivo: relationship between serum levels of antigen-specific IgG1 and mediator synthesis/release.

Naive guinea-pigs were passively sensitized with varying amounts of affinity column purified, homologous, anti-ovalbumin IgG1 (anti-OA IgG1) and then examined for a) the capacity of lung tissue to release mediators (histamine and LTB4/LTD4) in response to antigen-challenge ex vivo and b) the attendant circulating levels of anti-OA IgG1. Intraperitoneal administration of anti-OA IgG1 (0.125-0.75 mg/kg) to guinea-pigs facilitated the synthesis of LTB4 (8-25 ng/g lung) and LTD4 (18-80 ng/g) and the release of histamine (1-7 ug/g) from lung tissue after exposure to 10 micrograms/ml of ovalbumin for 20 min ex vivo. Peak levels of mediators were found using 0.5 mg/kg anti-OA IgG1 with an ED50 = 0.35 mg/kg. LTD4/LTB4 synthesis and histamine release were both antigen concentration- and time-dependent, and LT synthesis was observable in non-perfused lungs and in lungs perfused free of blood. Maximum sensitization occurred at 1-2 days post i.p. administration of anti-OA IgG1 and was maintained up to 7 days. Measurement of anti-OA IgG1 using an enzyme-linked immunosorbent assay demonstrated that circulating antibody levels were 2-6 micrograms/ml at the doses which caused sensitization. The level of anti-OA IgG1 found in passively sensitized animals was at least 100-fold less than that found in actively-sensitized guinea-pigs despite the similar magnitude in LTD4/LTB4 synthesized and the amount of histamine released. Using purified antibody, the results demonstrate that in guinea-pigs, IgG1 can play a prominent role in regulating lung LT synthesis and histamine release, and that microgram per ml circulating levels of this antibody are sufficient to sensitize naive lungs.

Animals↗
PubMed1990

Evidence that peptidoleukotriene is a prerequisite for antigen-dependent thromboxane synthesis in IgG1-passively sensitized guinea pig lungs.

Lungs from guinea pigs passively sensitized with an affinity-purified IgG1 antibody produce both leukotriene (LT)D4 and thromboxane (Tx)B2 upon ex vivo antigen challenge. This study was undertaken to determine the possibility of endogenously generated peptido-LTs being a prerequisite for Tx synthesis. In immunoglobulin G1-sensitized lungs, exogenous LTD4 induced TxB2 production with a median effective dose of 4.1 nM, whereas the response to LTE4, LTB4 or platelet-activating factor was relatively weak. Although LTC4 was as potent as LTD4 in stimulating TxB2 generation, LTC4's dose-response curve was shifted significantly to the right by AT-125, an irreversible gamma-glutamyl transpeptidase inhibitor, suggesting that at least a part of LTC4 sensitized lungs with antigen (0.01-30 micrograms/ml ovalbumin) for 20 min precipitated a significant amount of LTD4 production. The levels of LTD4 range from 8 to 26 nM (without taking LTD4 recovery into consideration). This level is 2- to 7-fold greater than the median effective dose value observed with exogenous LTD4. Moreover, pretreatment of sensitized lungs with ICI-198,615 a specific LTD4 antagonist, blocked equally both antigen (IC50 = 0.01 microM)- and LTD4 (IC50 = 0.017 microM)-induced TxB2 production. When sensitized lung fragments were treated with 5 mM AT-125, ICI-198,615 was effective in preventing not only antigen-but also LTC4-dependent production of TxB2 (IC50 = 0.018 and 0.021 microM, respectively). In contrast, neither WEB-2086, a platelet-activating factor antagonist, nor pyrilamine, a histamine antagonist, inhibited antigen and LTD4 responses (IC50 greater than 30 microM). Unlike its effect on antigen response, ICI-198,615 was unable to block Ca2+ ionophore-induced TxB2 production.2

Animals↗
PubMed1989

Involvement of peptidoleukotrienes in antigen-dependent thromboxane (TX) synthesis in IgG1-sensitized guinea pig lungs.

Lungs from IgG1-sensitized guinea pigs synthesize both leukotrienes (LTs) and thromboxane (Tx) upon ex vivo antigen challenge. This study was undertaken to investigate whether antigen-dependent Tx synthesis could result from prior formation of LTD4. In IgG1-sensitized lungs, LTD4 effectively induced Tx formation (ED50 = 2-4 nM). In these lungs, the levels of antigen-dependent formation of LTD4 (8-26 nM formed by 0.01-10 micrograms/ml antigen challenge) were 2-7 X greater than the ED50 value of LTD4-stimulated Tx synthesis. In addition, incubation of the sensitized lungs with ICI-198,615, a LTD4 antagonist, prior to antigen-challenge prevented Tx formation (IC50 = 0.01 microM). Our results indicate that LTD4 generated from IgG1-sensitized lungs could play a prominent role in stimulating Tx synthesis. LTC4 may also be involved because of rapid formation of LTC4 upon antigen-stimulation and its capacity to induce Tx synthesis.

Animals↗
PubMed1988

The development of a sensitive and specific radioreceptor assay for leukotriene B4.

To establish a simple and sensitive quantitation of leukotriene B4 (LTB4), we developed a radioreceptor assay (RRA) using a highly specific [3H]leukotriene B4[( 3H]LTB4) binding to a guinea pig spleen homogenate. The assay detected LTB4 levels as low as 0.12 pmol per tube. Fifty percent inhibition of bound [3H]LTB4 was obtained by 2.5 nM of unlabeled LTB4. [3H]LTB4 competition studies indicated that 20-hydroxy-LTB4 was 8 times, 6-trans-LTB4 was 640 times and 20-carboxy-LTB4 was 1000 times less effective than LTB4. The peptide leukotrienes C4, D4 and E4 showed no effect on [3H]LTB4 binding. Recovery rates averaged 97% after ethanol extraction and evaporation of known amounts of LTB4. The intra-assay coefficients of variation for three samples were 2.4%, 7.2% and 8.4%, respectively. This assay was validated by measuring LTB4 released from human granulocytes stimulated with calcium ionophore A23187. The LTB4 level was maximal at 10 min (156.8 +/- 36.2 pmol/3 x 10(6) cells) and decreased rapidly after 15 min. This radioreceptor assay for leukotriene B4 is highly sensitive and is comparable to the reported sensitivity by radioimmunoassay. The method is simpler and less expensive than other methods such as high pressure liquid chromatography and is suitable for routine measurement of leukotriene B4.

Animals↗
PubMed1987

A simple and sensitive radioreceptor assay for leukotrienes.

A simple and sensitive radioreceptor assay (RRA) for leukotrienes (LTs) was developed using a highly specific [3H]leukotriene D4 (LTD4) binding to guinea pig lung membrane homogenates. The assay can detect down to 0.15 pmol of LTD4. The values for fifty percent inhibition of bound [3H]LTD4 was 1.5 nM for LTD4, 45 nM for LTC4 and 24 nM for LTE4. LTB4 at 3.0 X 10(-5)M had no effect on [3H]LTD4 binding. The RRA for LTs in the absence of serine-borate complex was bi-specific for both LTC4 and LTD4. However, in the presence of 20 mM serine-borate this method was highly specific for LTD4. Recovery rate averaged 87.2% after ethanol extraction and evaporation of known amounts of LTD4. When the radioreceptor assay and radioimmunoassay data for leukotriene levels in the samples were compared to each other, an excellent correlation was observed with a correlation coefficient 'r' of 0.992. The assay was also validated by quantitation of Lts released from human granulocytes stimulated with calcium ionophore, A23187. The method is simpler, less expensive, and more specific for LTD4 than the other methods such as high pressure liquid chromatography and radioimmunoassay and is suitable for routine measurement of either LTD4 specifically or LTC4 plus LTD4 simultaneously in one cell system.

Animals↗
PubMed1987

Comparison of antigen and Ca++-ionophore-induced peptidoleukotriene release from guinea pig lung preparations using high-performance liquid chromatography.

This study was undertaken to investigate antigen-induced peptidoleukotriene release from lungs of sensitized guinea pigs using a recently developed solid-phase extraction and high-performance liquid chromatography assay system. This release was compared to the response due to Ca++-ionophore (A23187) challenge. Incubation of lung fragments (0.6 g) from actively sensitized guinea pigs with ovalbumin (3 micrograms/ml) for 20 min at 37 degrees C resulted in the release of 40 to 60 ng of leukotriene (LT)D4 detected in the extracted filtrate (40-50% recovery of LTD4). The amount of LTD4 determined using the high-performance liquid chromatography assay correlated well with the quantity determined by a LTC4 radioimmunoassay. LTD4 release was saturable and was optimal at a tissue concentration of 0.6 g/2.5 ml of buffer. Kinetic analysis of LT generation showed that after antigen challenge, LTC4 levels peaked at 3 min and declined rapidly with time; LTD4 levels then increased significantly, reaching a maximum at 15 min and decreased slightly at 60 min. LTE4 was not detected until 30 min after antigen challenge after which it increased slowly. The kinetic results permit an estimation of the rate of LTD4 and LTE4 formation to be 5 and 0.17 ng/min/g of lung, respectively. In contrast to antigen challenge, LTD4 release from Ca++-ionophore-stimulated lung fragments was not saturable and was biphasic with increasing amounts of tissues. Moreover, LTD4 produced by Ca++-ionophore stimulation could not be detected during the first 10 min but thereafter increased linearly with incubation time.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

[3H]leukotriene B4 binding to the guinea-pig spleen membrane preparation: a rich tissue source for a high-affinity leukotriene B4 receptor site.

Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [3H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compete with [3H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [3H]LTB4 binding, but NaCl and KCl decreased it. Spleen [3H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25 degrees C was 0.47 nM-1 min-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg of protein. Moreover, the LTB4/[3H]LTB4 competition study performed at 4 or 25 degrees C revealed the inhibitory constant (Ki) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [3H]LTB4 binding was: LTB4 (Ki = 2.8 nM) greater than 20-hydroxy-LTB4 (23 nM) greater than LTA4 (48 nM) greater than LTA4 methyl ester (0.13 microM) greater than 20-carboxy-LTB4 (greater than 6.6 microM) greater than or equal to arachidonic acid (0.15mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihyropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [3H]LTB4 binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Tissue distribution and functional correlation of [3H]leukotriene C4 and [3H]leukotriene D4 binding sites in guinea-pig uterus and lung preparations.

To determine the distribution of leukotriene (LT) C4 and LTD4 receptors and functional significance of each receptor, we used [3H]LTC4 and [3H]LTD4 to measure the binding activity in various tissue homogenates and to correlate the relative ability of the LT agonists to inhibit binding with their contractile responses in guinea-pig uterine or lung parenchymal preparations. Guinea-pig brain contained the highest binding activity of 1 to 2 nM [3H] LTC4 followed by small intestine, heart, lung, kidney, uterus, etc., whereas guinea-pig lung had at least 2.7-fold greater [3H] LTD4 binding activity than any other tissues tested. In the brain and uterine homogenates, the rank order of potency for the compounds in competing with [3H]LTC4 for binding sites was LTC4 much greater than LTD4 greater than LTE4 greater than FPL-55712 = arachidonic acid. The in vitro functional study showed that the ability of the LT agonists to produce uterine contraction was in the order of LTC4 greater than LTD4 greater than LTE4, which is compatible with their relative effect for inhibition of uterine or brain [3H]LTC4 binding. In the lung homogenate, either LTC4, LTD4 or LTE4 inhibited effectively [3H]LTD4 binding and the potency order for the [3H]LTD4 competition study was LTD4 greater than LTE4 greater than LTC4 much greater than FPL-55712 greater than arachidonic acid. These LT agonists also produced lung contraction effectively and the difference among their contractile ability was not significant. We conclude that 1) there are distinct functional LTC4 and LTD4 receptors, 2) activation of the LTC4 receptor could account for the uterine contraction due to the LT agonists and 3) the lung contraction induced by LTC4, LTD4 and LTE4 is at least mediated partly by the LTD4 receptor.

Animals↗
PubMed1985

Comparison of the effects of verapamil and nifedipine on airway muscarinic reactivity and receptors.

Previous studies have shown inconsistent results of the effect of verapamil and nifedipine on airway reactivity. This study was undertaken to determine and compare their effects on the activity of airway muscarinic receptors by contraction experiments and by radioligand receptor binding assays. We found that verapamil (10(-5)-10(-4) M) reduced contraction at rat tracheal muscle to methacholine, a finding consistent with its inhibition on binding of (3H)QNB to bovine tracheal muscle membranes. Unlike verapamil, nifedipine (10(-5)-3 X 10(-5) M) inhibited neither methacholine response nor (3H)QNB binding. Verapamil but not nifedipine decreased the affinity of binding sites. Neither of them affected the concentration of (3H)QNB binding sites. We conclude that the effect of verapamil on airway muscarinic receptor binding sites differs qualitatively from that of nifedipine, and suggests that its effect on the binding sites could account for the inhibitory action of verapamil on airway muscarinic response.

Airway Resistance↗
PubMed1984

Evidence for a similar receptor site for binding of [3H] leukotriene E4 and [3H] leukotriene D4 to the guinea-pig crude lung membrane.

To determine whether the action of leukotriene E4 is mediated by its cross reaction with leukotriene D4 receptor sites, we compared [3H] leukotriene E4 and [3H]leukotriene D4 binding activities in selected tissues as well as their competition results in the guinea-pig crude lung membrane. We demonstrated good correlation of [3H]leukotriene E4 and [3H]leukotriene D4 binding activities among the tissues studied. A significant correlation was demonstrated between the ability of leukotriene C4, D4 and E4, FPL-55712 and arachidonic acid to inhibit lung [3H]leukotriene E4 and [3H]leukotriene D4 binding. These correlations suggest that leukotriene E4 binds to a site which is similar to or close to the leukotriene D4 receptor.

Animals↗
PubMed1984

Identification of calcium antagonist receptor binding sites using (3H)nitrendipine in bovine tracheal smooth muscle membranes.

(3H)Nitrendipine binding to the bovine tracheal muscle membrane at 25 degrees C was rapid, saturable (Bmax = 14.8 +/- 3.9 fmol/mg protein) and of high affinity (Kd = 0.15 +/- 0.04 nM). The rank order of Ca2+ antagonists competing for airway (3H)nitrendipine binding was nitrendipine not equal to nisoldipine not equal to nifedipine much greater than verapamil. Cromolyn, however, neither inhibited nor increased the binding.

Animals↗
PubMed1984

Effect of the serine-borate complex on the relative ability of leukotriene C4, D4 and E4 to inhibit lung and brain [3H] leukotriene D4 and [3H] leukotriene C4 binding: demonstration of the agonists' potency order for leukotriene D4 and leukotriene C4 receptors.

To define the potency order of the leukotrienes for inhibition of [3H] leukotriene D4 and [3H] leukotriene C4 binding, we investigated leukotriene C4, D4 and E4 competition with and without the serine-borate complex in guinea pig lung and brain homogenates. Without it, the rank order of their potency for inhibition of lung [3H] leukotriene D4 or [3H] leukotriene C4 binding was leukotriene C4 = leukotriene D4 greater than leukotriene E4. Presence of the complex altered the potency order for both competition studies: for the [3H] leukotriene D4 competition it was leukotriene D4 greater than leukotriene E4 = leukotriene C4 and for the [3H] leukotriene C4 competition it was leukotriene C4 much greater than leukotriene D4 greater than or equal to leukotriene E4.

Animals↗