Search PubMed⌕ Search

Biomedical subjects

T J Yang

Publications and source records attributed to T J Yang.

At least 37 records · Page 2Linked to original sources
PubMed1998

Physiologic-chemoattractant-induced migration of polymorphonuclear leukocytes in milk.

The somatic cell count (SCC; leukocytes and epithelial cells) in milk is used as an indicator of udder health status. A SCC above the regulatory standard is generally considered as an indication of mastitis. Therefore, milk with a SCC equal to or greater than the regulatory limit cannot be sold to the public because it is unsuitable for human consumption. This study was performed to determine whether SCC levels above the regulatory limit observed in goats during late lactation are a physiologic or a pathological response of the goat mammary gland. Differential counts of cells in nonmastitic goat milk samples during late lactation revealed that approximately 80% of the cells were polymorphonuclear leukocytes (PMNs). In addition, microchemotaxis assay results indicated that normal nonmastitic late-lactation-stage goat milk is significantly higher (P < 0.001) in PMN chemotactic activity than early-lactation-stage goat milk, with a mean chemotactic activity of 14.9 and 42.7/mg of protein for early and late lactation stages, respectively. Physicochemical analyses also suggest that the PMN infiltration observed in normal late-lactation-stage goat milk is due to a PMN chemotactic factor(s) that is different from the PMN chemotactic factor(s) present in mastitic milk. Interestingly, the PMN chemotactic factor in late-lactation-stage goat milk is highly acid resistant (pH 2), suggesting that the factor is able to survive the highly acidic gastric environment and may therefore be important in the augmentation of the immune systems of sucklings. These results indicate that the chemotactic factor(s) present in the milk of normal late-lactation-stage goats is nonpathological and may play a physiologic regulatory role in mammary gland involution. Hence, the regulatory standard for goat milk needs to be redefined in order to reflect this.

Animals↗
PubMed1998

Further characterization of the expression in liver and catalytic activity of CYP2B6.

Previous studies in this laboratory have determined the lack of specificity of several antibody and substrate probes of CYP2B6. The goals of the current study were to examine the expression of CYP2B6 in a bank of human liver microsome (HLM) samples using a new specific monoclonal antibody (MAb 49-10-20) and to further characterize the substrate specificity of CYP2B6. A 100-fold variability in expression of immunodetectable CYP2B6 was demonstrated in a bank of 19 HLM samples (0.7 pmol/mg protein to 71. 1 pmol/mg protein) using MAb 49-10-20. CYP2B6 levels were found to significantly (P < .0001) correlate with S-mephenytoin N-demethylation to nirvanol (r2 = 0.89), 7-hydroxy-4-trifluoromethylcoumarin formation (r2 = 0.81) and several markers of CYP3A levels and activity. The relationships between nirvanol formation and CYP3A levels or activity were found to depend on two HLM samples. Km (apparent) values were generated for benzyloxyresorufin O-deethylation (1.3 microM), benzphetamine N-demethylation (93.4 microM), 3-cyano 7-ethoxycoumarin O-deethylation (71.3 microM), midazolam 1'-hydroxylation (46.1 microM) and 4-chloromethyl-7-ethoxycoumarin O-deethylation (33.7 microM) using expressed CYP2B6. Testosterone 16beta-hydroxylation by expressed CYP2B6 resulted in atypical kinetics characteristic of substrate activation. The data best fit the Hill equation with a Km (apparent) of 50.5 microM and an n of 1.3 (n = number of sites bound by activator). In conclusion, the highly specific MAb 49-10-20 was used to provide further confirmation that S-mephenytoin N-demethylation to nirvanol is a CYP2B6 selective probe. Finally, some, but not all substrates of CYP2B6 demonstrate autoactivation.

Antibodies, Monoclonal↗
PubMed1997

Inhibitory monoclonal antibodies to human cytochrome P450 2D6.

Two monoclonal antibodies (MAbs) have been isolated that bind to human P450 2D6 and inhibit 2D6 catalyzed bufuralol 1-hydroxylation by 90%. One but not both of the MAbs immunoblotted 2D6. The MAbs were highly specific to 2D6 and did not cross-react with other P450s. Inhibitory monoclonal antibodies will be useful for determining the contribution of 2D6 to the metabolism of a wide variety of 2D6 and other P450 substrates in human tissues containing multiple P450s.

Antibodies, Monoclonal↗
PubMed1997

A monoclonal antibody inhibitory to human P450 2D6: a paradigm for use in combinatorial determination of individual P450 role in specific drug tissue metabolism.

Human cytochrome P450 2D6 metabolizes more than 50 common drugs and is polymorphically expressed, with 5-10% of the population lacking expression caused by mutant genes. This may result in a defective and toxic response in deficient individuals treated with 2D6 drug substrates. Baculovirus-expressed 2D6 was used to immunize mice for hybridoma production and two clones yielded monoclonal antibodies, that were positive against 2D6 by ELISA and inhibited 2D6 catalysed metabolism of bufuralol, dextromethorphan and phenanthrene by more than 90%. The inhibitory activity was highly specific to 2D6 and the monoclonal antibodies did not bind to 11 other P450s, nor inhibit seven human P450s tested. Analysis of eight human liver microsome samples showed that their basal bufuralol 1'-hydroxylase activity varied from 6.7-83.5 pmol min-1 nmol-1 P450. The monoclonal antibody 512-1-8 inhibited 2D6-dependent bufuralol 1'-hydroxylase in these samples by 10-70% indicating a widely variable role for 2D6 in human liver bufuralol 1'-hydroxylase activity and a role for other P450s in bufuralol metabolism. Independent analysis of several recombinant human P450s showed that 2D6, 2C8, 2C9, 2C19 and 1A2 exhibited bufuralol 1'-hydroxylase activity with 2D6 and 2C19 being the most active. Further analysis of three liver samples was made with individual inhibitory monoclonal antibodies. Inhibitory antibodies to 2D6, 2B6, 2E1, 2C8/9/19, 3A4 and 1A2 were added to the microsomes either singly or additively. Inhibitory activity of bufuralol 1'-hydroxylase was observed with antibodies to 2D6 (14-76%), 2C8/9/19 (24-69%) and 1A2 (2-25%) indicating a variable and different role for each of these P450s in the bufuralol 1'-hydroxylase of human liver. The monoclonal antibodies to 2B6, 2E1 and 3A4 were not inhibitory, indicating that these enzymes play no role in bufuralol 1'-hydroxylase metabolism. When the three antibodies to 2D6, 2C8/9/19 and 1A2, respectively, were all added, the total bufuralol 1'-hydroxylase of the liver samples was inhibited by more than 90%, indicating that the latter P450s catalyse all of liver bufuralol 1'-hydroxylase metabolism. These studies demonstrate that inhibitory monoclonal antibodies offer a simple and precise method for assessing the quantitative role of each P450 in the metabolism of a P450 substrate in a tissue, which include drugs, carcinogens, mutagens, toxic chemicals and endobiotics.

Animals↗
PubMed1997

Lactation stage-dependent changes of lymphocyte subpopulations in mammary secretions: inversion of CD4+/CD8+ T cell ratios at parturition.

PROBLEM: Determination of lactation stage-dependent changes in levels of lymphocyte subpopulations in milk. METHOD: Flow cytometric assay was used to identify and assay lymphocyte subpopulations in bovine milk at different stages of lactation. RESULTS: Lymphocyte subpopulations in mammary secretions of dairy cows change during the lactation cycle. In involuting glands (dry gland), approximately 80-90% of lymphocytes were CD2+ T cells. The proportion of CD2+ T cells, however, decreased to approximately 50% at the colostral stage an fluctuated between 50 to 60% in normal (mature) milk. Throughout the lactation stages, less than 5% were B cells as identified by the monoclonal antibodies against CD21 and MHC class II antigens. Subset analysis showed, however, that the proportion of CD5+ T cells decreased from 90% in involuting gland secretions to 75% in colostrum (peripartum stage), and to approximately 40-50% in the normal (mature) milk, CD4+ T cells constituted between 45 to 55% of lymphocytes in the dry gland secretion but decreased drastically at parturition and maintained at the level below 20% throughout normal lactation. In contrast, the proportion of CD8+ T cells in the dry gland secretion was low, between 30 to 40%, but increased steadily, in an inversely-related manner with that of CD4+ T cells, to approximately 40-50% at parturition and maintained at approximately 30-40% during the normal lactation stage thereafter. Two-color immunofluorescence study revealed further that practically all of the CD8+ cells in dry gland secretions were CD2+, and approximately 40% of them were CD5-. Throughout the lactation cycle, WC1+ gamma delta T cells comprised only 2 to 5% of lymphocytes in mammary secretions. CONCLUSIONS: T lymphocyte subpopulations change dynamically during stages of the lactation cycle. The selective migration of T lymphocyte subpopulations to and from the mammary gland, and their functional roles in the immune competence and regulation of the dam and sucklings remain to be elucidated.

Animals↗
PubMed1997

The regulatory role of transforming growth factor-beta in activation of milk mononuclear cells.

PROBLEM: Immunoregulatory role of milk mononuclear cells and cytokines during the involution of the mammary gland. METHOD OF STUDY: Bioassays were used to determine the levels of transforming growth factor beta (TGF-beta) and interleukin-2 (IL-2) and their effects on milk mononuclear cells (MMCs). RESULTS: MMCs collected from involuting glands were less responsive to Con A stimulation than peripheral blood mononuclear cells (PBMCs), and purified huTGF-beta 1 inhibited the activation of both MMCs and PBMCs by Con A. Furthermore, secretions collected over a period of approximately 4 weeks into the involution period contained high levels of active TGF-beta and extremely high levels of latent TGF-beta, MMCs stimulated with Con A produced higher levels of IL-2 than did the PBMCs of the same animals, and bovine milk TGF-beta and huTGF-beta 1 significantly inhibited the IL-2 production by MMCs. Mammary secretions of involuting glands did not contain any detectable levels of IL-2, and they inhibited the growth of the IL-2-dependent bovine lymphoblastoid T cell line (BLTC), even in the presence of 10 U/ml of rBo IL-2. CONCLUSIONS: The findings suggest that TGF-beta is a major immunoregulatory factor in the involuting mammary gland.

Animals↗
PubMed1997

[Effects of 16 drugs on immunological liver injury induced by BCG + lipopolysaccharides in mice].

AIM: To evaluate anti-hepatitis drugs. METHODS: Mice were injected i.v. with viable BCG 5 x 10(7) (live) bacilli/mouse, after 10 d, i.v. lipopolysaccharides (LPS) 5-10 micrograms/mouse. Serum aminotransferase levels and liver tissue were examined 10 h after i.v. LPS. Sixteen drugs were evaluated in this model. RESULTS: The level of AlaAT and AspAT were increased markedly. Submassive necrosis and infiltrations of granulocytes and lymphocytes were seen, which were not recovered till 14 d after i.v. LPS. Administration of interferon alpha-2a, Ara-AMP, hepatocyte growth factor, biphenyl dimethyl dicarboxylate, bicyclol, prednisolone, and cyclophosphamide for 10 d prior to i.v. LPS suppressed the elevation of serum AlaAT and AspAT. CONCLUSION: The model of immunological liver injury reproduced by i.v. BCG + LPS in mice may be used for evaluating anti-hepatitis drugs.

Animals↗
PubMed1996

2-(methylamino)-5-chlorobenzophenone imine: a novel product formed in base-catalyzed hydrolysis of diazepam.

Diazepam (1), a hypnotic and anxiolytic drug in worldwide use, formed an intermediate product in a mixture of ethanol and sodium hydroxide ([NaOH] > or = 1 M). The intermediate product slowly decomposed to form 2-(methylamino)-5-chlorobenzophenone imine (2) and 2-(methylamino)-5-chlorobenzophenone (3). The amount of 2 formed, relative to that of 3, increased with increasing NaOH concentration. Compound 2, a heretofore unknown derivative of 1, was characterized by high-performance liquid chromatography, ultraviolet-visible absorption, mass, and proton nuclear magnetic resonance spectral analyses.

Benzophenones↗
PubMed1996

Modulation of WC1, a lineage-specific cell surface molecule of gamma/delta T cells augments cellular proliferation.

WC1, also known as T19, is the only unique gamma/delta T-cell differentiation antigen described to date other than the gamma/delta T-cell receptor. We present evidence that modulation of WC1 results in augmented proliferation of gamma/delta T cells. Immobilized IL-A29, a monoclonal antibody (mAb) specific for WC1, augmented proliferation of gamma/delta T cells in the autologous mixed leucocyte reaction (AMLR) as well as proliferation induced by either anti-CD3 or anti-CD5 mAb. In contrast, anti-CD5 mAb did not increase proliferation in the AMLR even though both CD5 and WC1 are members of the scavenger receptor cysteine-rich family of proteins and are expressed by bovine peripheral blood gamma/delta T cells. IL-A29 did not induce proliferation when assessed alone or in the presence of either phorbol myristate acetate (PMA) or interleukin-2. IL-A29 also did not induce detectable calcium mobilization when evaluated in the presence of monocytes, PMA, or following cross-linking of IL-A29 with anti-immunoglobulin antibody. We conclude that WC1 is a gamma/delta T-cell lineage-specific cell-surface differentiation antigen which is involved in activation of gamma/delta T cells using an as yet unidentified pathway.

Animals↗
PubMed1994

Hydrolysis of temazepam in simulated gastric fluid and its pharmacological consequence.

Temazepam (TMZ), a hypnotic and anxiolytic drug, underwent hydrolysis in simulated gastric fluid (SGF; pH 1.2). The hydrolysis reaction of TMZ in acetonitrile:SGF (1:19 v/v) at 37 degrees C was an apparent first-order reaction, with a half-life of 5.47 +/- 0.17 h (i.e., approximately 12% of the remaining TMZ was hydrolyzed per hour). The predominant hydrolysis product (2'-benzoyl-4'-chloro-N-methyl-2-amino-2-hydroxyacetanilide) and a minor hydrolysis product [2-(methylamino)-5-chlorobenzophenone], derived from acid-catalyzed reaction of TMZ in an aqueous solution, were characterized by ultraviolet-visible absorption mass, infrared, and proton nuclear magnetic resonance spectra analyses. The kinetics of the hydrolysis reaction were studied as a function of acid concentration, temperature, and ionic strength and in deuterated solvent. Results indicated that the predominant hydrolysis reaction at pH approximately pKa (1.46) was caused by protonation at N4, followed by a nucleophilic attack by water at C5 of the C5-N4 iminium ion and a subsequent ring-opening reaction. Pharmacological activity tests in mice indicated that the predominant hydrolysis product of TMZ was inactive. The results suggest that a fraction of an orally taken TMZ may be inactivated by hydrolysis in the highly acidic gastric fluid.

Animals↗
PubMed1994

Lactation stage-dependent changes in levels of tumor necrosis factor/cachectin in milk.

PROBLEM: Determination of lactation stage-dependent changes in levels of tumor neurosis factor (TNF) in milk. METHOD: Bioassay and immunoblocking assay were used to identify and assay tumor necrosis factor (TNF; mostly TNF alpha) in bovine milk at different stages of lactation. RESULTS: TNF alpha levels in milk started to increase steadily after the onset of drying-off (weaning/involution), peaked at 4 to 6 wk prior to parturition and precipitously decreased to undetectable levels at parturition (colostrum). Thereafter, TNF alpha reappeared and maintained midlevel concentration in the mature (normal) milk throughout the rest of the lactation cycle. Analysis of cells in mammary secretions by flow cytometry revealed that elevated TNF alpha levels coincided with an increase in macrophages in the secretion from the dry period. CONCLUSIONS: These lactation stage-dependent changes in TNF alpha levels reflect differential effects that TNF alpha have on involution and prepartum remodeling of the mammary gland of the dam and on gastrointestinal development and immunoregulatory function of the suckling.

Animals↗
PubMed1993

Optimization of the L-M cell bioassay for quantitating tumor necrosis factor alpha in serum and plasma.

In attempting to quantitate TNF-alpha in body fluids using current bioassay protocols, we discovered several factors which adversely affect reliability, sensitivity and specificity. The objective of this study was to establish an optimum assay for use with serum and plasma samples. While adopting the commonly used L-M cell bioassay to test serum and plasma samples, we noted non-specific staining of cell debris and protein by crystal violet dye, even if the wells were washed prior to staining. However, when we replaced crystal violet with tetrazolium salts to measure cell viability, we discovered that both serum and plasma, from a variety of species, non-specifically reduced both MTT and XTT tetrazolium salts to a colored formazan product resulting in absorbance values significantly higher than those of medium and serum controls. This effect was particularly pronounced with fetal bovine serum which showed significant color development with as little as 6% serum in the test wells. Maximum sensitivity can only be obtained by eliminating these false negative readings. Therefore the serum or plasma containing supernatant must be removed from the test wells and replaced with fresh serum-free medium prior to addition of the substrate. This finding should be applicable to other body fluids such as urine, milk, and synovial fluid as well as any tetrazolium based assay for cell viability which uses serum-supplemented culture medium. Additionally, when the substrate XTT, which reduces to a water-soluble formazan product, was compared to MTT which reduces to a water-insoluble product, XTT was found to be superior since elimination of the solubilization process resulted in reduction of assay time. Also, overall absorbance readings using XTT were higher than with MTT, without any loss of sensitivity. There were no differences in detectability of TNF-alpha between serum and plasma and use of preservative-free heparin was found not to have adverse effect on TNF-alpha assay. Heat treatment of both serum and plasma seemed to inactivate factors that contributed to the non-specific lysis of the L-M cells when their concentrations exceeded 25%.

Animals↗
PubMed1992

An inhibitor of tumor cell growth from normal horse serum.

During our studies of cytostatic cytokines in the mixed leukocyte culture, we found that horse serum in the medium control contained a tumor cell growth-inhibitory factor. The fraction isolated by molecular sieving and ion exchange chromatography inhibited the growth and DNA synthesis of the primary culture and passaged cell line of the canine transmissible venereal sarcoma, murine T (L5178Y) and B (P3-X63-Ag8.653) lymphoid tumor cells, murine mammary tumor cells (RIII), bovine lymphoid tumor cells (BL3), and the nontransformed cell line of baby hamster kidney cells. Nontransformed cell lines such as African green monkey kidney (Vero) and Madin-Darby bovine kidney cells and normal mammary cells of the dog and goat were inhibited only at high concentrations. The active component is a protein with an Rf value of 0.09 upon electrophoresis in native 7.5% polyacrylamide gels. The eluate from the native gel could be further separated into three polypeptides with molecular weights of approximately 67, 65, and 55 kDa under reduced conditions in sodium dodecyl sulfate-polyacrylamide gels.

Animals↗
PubMed1992

Systemic profiles of antigen-specific lymphocytes in animals chronically exposed to staphylococcus antigen in the mammary region.

Systemic profiles of lymphocytes were assessed in goats exposed chronically with Staphylococcus antigens in the supramammary region. Animals were inoculated three times subcutaneously in the right supramammary region with heat-killed Staphylococcus aureus antigen (HKS) at 1 month intervals. Prior to immunization and 1 week following each injection, 3 and 6 day cultures of peripheral blood mononuclear cells were made to determine proliferative responses of lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). Peripheral blood lymphocytes responded significantly to both HKS and PHA in 3 day cultures after the second injection and showed peak responses after the final immunization, suggesting that repeated local injection of S. aureus antigen at the supramammary region, can induce an anamnestic response to the antigen in the peripheral blood of these animals with a concomitant increase in the responsiveness to the polyclonal mitogen, PHA. In contrast, initial antigen challenge induced little, if any, increase in responses to the specific antigen or mitogen when compared to pre-injection states. These data may also suggest that non-reactivity of peripheral blood lymphocytes to the HKS antigen immediately after the primary injection of antigen may be the result of local retention of antigen-reactive cells at the sites of infection.

Animals↗
PubMed1992

Elevated CD4 antigen expression among activated T cells in lymph nodes draining mammary regions chronically exposed to Staphylococcus aureus antigen.

We examined the cell cycle distribution and subset marker characteristics of mucosal-associated supramammary lymph node (MALN) T cell subpopulations at sites proximal and distal to the mammary region of animals repeatedly injected with Staphylococcus aureus antigen. Multiparameter flow cytometric analysis of draining MALNs showed that CD4+ T cells expressed significantly greater amounts of CD4 surface antigen than corresponding cell populations in non-draining MALNs. In contrast, the intensity of CD8 surface antigen expression among draining MALN CD8+ cells remained unchanged. Draining and non-draining MALNs contained nearly equal proportions of large CD4+ T cell subpopulations (CD4/CD8 ratio) with the former having greater cell numbers undergoing active DNA synthesis (S + G2M phase) in vivo. Similarly, draining MALNs had greater cell numbers of small CD4+ lymphocytes in the S + G2M phase, although with lower CD4/CD8 ratios of corresponding cell populations in non-draining MALNs. This study demonstrates that prolonged exposure with Staph aureus antigen enhances the cell number and expression of CD4 surface antigen among T lymphocyte subpopulations actively synthesizing DNA in draining MALNs. The role of the CD4 antigen in inflammatory responses at sites proximal (draining) and distal (non draining) to chronic infection within the mammary/mucosal immune network is discussed.

Animals↗
PubMed1992

Phenotypic and functional analysis of mucosal lymph node T cell subpopulations proximal and distal to chronic staphylococcal antigen challenge.

We investigated the functional and subset surface marker characteristics of supramammary lymph node T cell populations at sites proximal and distal to the mammary region of goats repeatedly injected with heat-treated Staphylococcus aureus antigen (HK-SAC). Flow cytometric studies showed quantitative differences in CD4+ and CD8+ T cell subsets among large and small lymphocyte populations in ipsilateral and contralateral supramammary lymph nodes of these animals. Although ipsilateral (draining) lymph nodes were enriched with CD4+ and CD8+ T cells, CD4/CD8 ratios were comparatively lower than those of contralateral (non-draining) lymph nodes (2.30 vs 2.60, respectively). Cell size analysis by flow cytometry showed that nearly 70% of the lymphocytes in ipsilateral nodes were of large cell phenotype with CD4/CD8 ratios of 2.52. In contrast, there were only 56.1% large lymphocytes in contralateral lymph nodes but with similar CD4/CD8 ratios of 2.55. The number of large lymphocytes in corresponding nodes of uninoculated control animals was significantly lower (50%) with much lower CD4/CD8 ratios (2.08). Alloantigenic responses of both ipsilateral and contralateral lymph node T cells were greater than those of uninoculated controls. Antigen-specific proliferation studies showed that ipsilateral lymph node T cells greatly enhanced both primed and non-primed lymph node B cell responses to HK-SAC, whereas those from contralateral lymph nodes were less stimulatory. In contrast, contralateral lymph node T cells had greater enhancing effects on PWM-induced polyclonal B cell responses. These studies indicate that repeated local infection with bacterial antigen induce changes in the numbers, ratios and antigen-specific and non-specific responses among ipsilateral (draining) and distal contralateral (non-draining) lymph node T cell populations in mucosal-associated immune systems such as the mammary gland.

Animals↗