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Biomedical subjects

A S Walia

Publications and source records attributed to A S Walia.

At least 19 recordsLinked to original sources
PubMed1998

Clamp for coronary artery operations.

Aortocoronary bypass grafting is an accepted procedure for ischemic heart disease. Proper visualization of the coronary artery is mandatory for good surgical anastomosis. This is essential when a coronary operation is performed without cardioplegia or in surgical procedures without bypass support. For better visualization of a coronary artery, we are presenting a coronary artery clamp. We have used this clamp in minimally invasive coronary artery operations to achieve a bloodless field.

Anastomosis, Surgical↗
PubMed1994

Improved gas chromatography/mass spectrometry analysis of barbiturates in urine using centrifuge-based solid-phase extraction, methylation, with d5-pentobarbital as internal standard.

Effective solid-phase extraction, derivatization, and GC/MS procedures are developed for the simultaneous determinations of butalbital, amobarbital, pentobarbital, and secobarbital, using a deuterated pentobarbital (d5-pentobarbital) as the internal standard. Buffered (pH 7) urine samples were extracted with Bond Elute Certify II cartridge. Iodomethane/tetramethylammonium hydroxide in dimethylsulfoxide was used for methylation, while a HP 5970 MSD equipped with a 13 m J & W DB-5 column (5% phenyl polysiloxane phase) and the Thru-Put Target software package were used for GC/MS analysis and data processing. This protocol was found to be superior, in both chromatographic performance characteristics and quantitation results, over a liquid-liquid extraction procedure without derivatization using hexobarbital as the internal standard. Extraction recoveries observed from control samples containing four barbiturates range from 80% to 90%. Good one-point calibration data are obtained for all four barbiturates in the 50 to 3200 ng/mL range. Interestingly, the one-point calibration data for pentobarbital are inferior to the other three barbiturates--due to interference from the internal standard (d5-pentobarbital). The calibration data of pentobarbital are best described by a hyperbolic curve regression model. Precision data (% CV) for GC/MS analysis, over-all procedure, and day-to-day performance are approximately 2.0%, 6.0%, and 8.0%, respectively. With the use of a 2 mL sample size, the attainable detection limit is approximately 20 ng/mL.

Amobarbital↗
PubMed1993

Evaluation of enzyme immunoassay performance characteristics--phencyclidine example.

Four reagent formulations (three provided by a manufacturer; one prepared in-house by mixing equal volumes of two commercial reagents) are used for the assay of phencyclidine (PCP) in urine samples. Performance characteristics evaluated included assay precision and sensitivity at and near the assay cutoff concentration. Data resulting from the reagent prepared in-house are better than those using then commercially available formulations, and are comparable with those obtained using the recently available new commercial formulation.

Enzyme Multiplied Immunoassay Technique↗
PubMed1991

Enzymatic digestion, solid-phase extraction, and gas chromatography/mass spectrometry of derivatized intact oxazepam in urine.

Enzymatic digestion with beta-glucuronidase (EC 3.2.1.31) was used to release intact oxazepam from urine samples containing the d5-analog internal standard. The resulting specimens were extracted with Du Pont PREP Type W cartridge (processed by a PREP Automated Sample Processor), Bond Elut Certify, and J.T. Baker "spe" columns for comparison of the columns' extraction recovery and overall effectiveness. Methyl iodide/tetrahexylammonium hydrogen sulfate and N,O-bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (10 g/L) were used for the methylation and trimethylsilylation studies. We used a Hewlett-Packard HP 5790 mass-selective detector equipped with a 13-m J & W DB-5 column (5% phenyl polysiloxane phase) for gas chromatography/mass spectroscopy (GC/MS) analysis and the Thru-Put Target software package for data processing. After several exploratory experiments, we adopted the Du Pont PREP system methylation procedure because of its effective recovery, the superior stability of the derivatization product, the possibility of incorporating a clean-up step, and the potential for high throughput. The extraction recovery from a set of control samples was 87%. Coefficients of variation obtained for six replicates of GC/MS analysis and for the overall procedure were 1% and 3%, respectively. Excellent linearity was established in the 50-8000 micrograms/L concentration range studied. With the use of 3-mL samples, a 20-microL final reconstitution volume, oxazepam at 50 micrograms/L was easily detected under the adopted operation conditions.

Benzodiazepines↗
PubMed1991

Simultaneous analysis of amphetamine, methamphetamine, and 3, 4-methylenedioxymethamphetamine (MDMA) in urine samples by solid-phase extraction, derivatization, and gas chromatography/mass spectrometry.

A rapid and effective solid-phase extraction procedure using Bond Elute Certify bonded silica sorbent cartridges was adopted to extract amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) from urine samples. The extract was derivatized with trichloroacetic anhydride prior to gas chromatography/mass spectrometry (GC/MS) analysis with selected ion monitoring of the following ions: 190, 91, 188; 204, 91, 202; 162, 135, 202; 194, 123; and 211, 209 for the derivatized amphetamine, methamphetamine, MDMA, d5-amphetamine, and d9-methamphetamine, respectively. The first of the ions listed for each compound was used for quantitation. The compound d5-amphetamine was used as the internal standard for amphetamine, and d9-methamphetamine was used for methamphetamine and MDMA. Results showed a higher than 65% recovery and a reproducibility with less than a 5% coefficient of variation. When a sample size of 2 mL was used, the lowest detectable concentration was about 50 ng/mL, and a near-perfect fit can be obtained (within the 250 to 4000-ng/mL concentration range studied) using a second-order polynomial model.

3,4-Methylenedioxyamphetamine↗
PubMed1991

Evaluation of immunoassay methods for the screening of cocaine metabolites in urine.

Immunoassay kits for urine cocaine (and metabolite) screening, obtained from two commercial sources, were examined for correlation of their results, expressed in terms of equivalent benzoylecgonine concentration, with the gas chromatography/mass spectrometry (GC/MS) concentration of benzoylecgonine. The correlation coefficients obtained, based on 62 (out of a total sample population of 3295) highly relevant samples, were 0.467 and 0.766 for Abuscreen (ARIA) and TDx (TDX), respectively. The preliminary screen cutoff values, which correspond to 150 ng/mL benzoylecgonine (as determined by GC/MS), were calculated based on the resulting regression equations and found to be 380 and 190 ng/mL for ARIA and TDX, respectively. With these cutoff values, ARIA generates 5 false negatives and 16 unconfirmed presumptive positives, while TDX results in 3 false negatives and 6 unconfirmed presumptive positives.

Binding, Competitive↗
PubMed1991

Simultaneous gas chromatography/mass spectrometry assay of methadone and 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine.

An efficient extraction and gas chromatography/mass spectrometry (GC/MS) procedure has been developed for the simultaneous determination of methadone and 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine in urine samples. The merits of this procedure include (1) effective high-volume sample processing; (2) excellent gas chromatography characteristics; (3) high precision for quantitative methadone determination--1.0% coefficient of variation (CV) for GC/MS injection replicates and 1.2% for extraction replicates; (4) excellent linearity within the range (0 to 1200 ng/mL) studied; and (5) adequate detection limits (50 ng/mL) for most practical purposes. The detection limit for methadone may be improved 40-fold by using a different internal standard.

Gas Chromatography-Mass Spectrometry↗
PubMed1989

Effects of retinoids on human thymus-dependent and thymus-independent mitogenesis.

The effects of all-trans-retinoic acid (RA), 13-cis-retinoic acid, and N-(4-hydroxyphenyl)retinamide on the mitogenic responses of various populations of human lymphocytes have been evaluated. Superoptimal concentrations of mitogens allowed the greatest RA-induced potentiation of lymphocyte proliferation. All three retinoids at concentrations as low as 5 x 10(-14)M significantly potentiated the proliferation of adenoidal and tonsillar lymphocytes stimulated by pokeweed mitogen (PWM). However, the responses of adenoidal and tonsillar lymphocytes to Staphylococcus aureus Cowan strain A were not potentiated by retinoids. Retinoids also caused significant potentiation of proliferation of PWM-stimulated peripheral blood lymphocytes (PBL). However, endpoint concentrations of retinoids required to significantly potentiate PBL proliferative responses to PWM were much higher than required for potentiation of adenoidal or tonsillar lymphocytes. PBL responses to concanavalin A (Con A) were significantly potentiated by retinoid concentrations as low as 10(-8) to 10(-10) M. Retinoid-potentiated responses were also observed wi Con A-stimulated thymocytes, but the endpoint concentrations required for significant potentiation were 10-fold higher than required to potentiate PBL responses to Con A. These data indicate that the sensitivity of lymphocytes to the retinoid-mediated potentiation of mitogenesis depends on the lymphoid compartment from which the cells are obtained. Tonsillar and adenoidal lymphocytes were the most responsive of the lymphocytes tested to the retinoid-induced potentiation of PWM responses. In addition, retinoids appear to selectively potentiate T cell-dependent proliferative activity.

Adenoids↗
PubMed1989

In vitro effect of acetaldehyde on cell-mediated cytotoxicity by murine spleen cells.

The effects of acetaldehyde in vitro on the lytic capacity of murine spleen cells have been evaluated in three systems: antibody-dependent cell-mediated cytotoxicity (ADCC), natural killer (NK) activity, and alloimmune cytotoxic T lymphocyte (CTL) activity. Acetaldehyde had a biphasic effect on ADCC. Concentrations less than 1 mM acetaldehyde potentiated ADCC. Concentrations greater than 1 mM produced a progressive decrease in lysis. The inhibitory effects were at the effector cell level and were partially irreversible. Preincubation experiments showed that inhibition of ADCC was both concentration and time-dependent. Preincubation of the spleen cells for short periods of time produced potentiated lysis by concentrations of acetaldehyde up to 10 mM. However, potentiation of lysis in preincubation and short term (4h) lytic assay experiments was more variable than longer term (18h) experiments in which the acetaldehyde was not removed by washing. NK activity and alloimmune CTL-mediated lysis were also inhibited by acetaldehyde. Concentrations of acetaldehyde up to 20 mM did not significantly decrease lymphocyte viability as determined by trypan blue exclusion. Acetaldehyde was lost from the reaction mixtures by first order kinetics with a rate constant of 0.5/hr. Thus, the final concentrations were 64-99.99% lower than the starting amounts.

Acetaldehyde↗
PubMed1989

Ethanol inhibition of cell-mediated lysis of antibody-sensitized target cells at a calcium-dependent step.

Ethanol inhibits antibody-dependent cell-mediated cytotoxicity in a dose-dependent manner. The inhibitory effect of ethanol was reversed by the addition of excess calcium or calcium ionophore A23187. Excess calcium at 4-8 mM concentrations was required to reverse 50% of the inhibition caused by ethanol. In seven of nine experiments, 16 mM excess calcium completely reversed the inhibition and produced greater lysis than the control. Excess calcium in the absence of ethanol induced a dose-dependent increase in lytic activity by the spleen cells. However, the reversal of inhibition by ethanol could not be attributed to a simple additive effect resulting from the increased cytolytic capacity of the lymphocytes in the presence of excess Ca2+. Ionophore A23187 at 1 microM also partially reversed the inhibitory effect caused by ethanol. Ionophore alone did not potentiate lytic activity. When target cells were not sensitized with antibody, excess calcium had no effect on the lysis of target cells in the presence of ethanol-treated or untreated lymphocytes. These data suggest that ethanol inhibits antibody-dependent cell-mediated cytotoxicity at a calcium-dependent step.

Animals↗
PubMed1989

In vitro effects of ethanol and acetaldehyde on lymphoid cells as determined by 31P NMR spectroscopy.

We have used NMR spectroscopy to test the direct in vitro effects of ethanol and its metabolite acetaldehyde on a human leukemic T cell line (Molt-4) and normal murine spleen cells. The metabolic status of phosphate metabolites (phosphomonoesters including sugar phosphate, inorganic phosphate and ATP's) in cell suspension was monitored by 31P NMR spectroscopy. Spectral changes were observed in the intensity of inorganic phosphate and ATP. Addition of high concentrations of ethanol (400-1600 mM) to Molt-4 cells resulted in very little spectral change. However, the addition of 40 microM acetaldehyde resulted in substantially increased inorganic phosphate (Pi) signals, and decreased phosphomonoesters and ATP signals. Similar changes, but to a lesser degree, were observed with normal mouse spleen cells.

Acetaldehyde↗
PubMed1988

Effects of retinoids on macrophage function and IL-1 activity.

The effects of three retinoids, all-trans-retinoic acid (RA), 13-cis-retinoic acid (cRA), and N-(4-hydroxyphenyl) retinamide (4-HPR), on macrophage function were evaluated. In vitro, RA, cRA, and 4-HPR caused a greater than twofold increase in phagocytosis of IgG-sensitized bovine erythrocytes (IgG-ORBC) by a mouse macrophage cell line (RAW). Significant increases in phagocytosis were produced by retinoid concentrations as low as 2 x 10(-10) M. RA also significantly increased phagocytosis of IgG-sensitized ORBC by BALB/c peritoneal macrophages in vitro. The ability of RAW macrophages to bind IgG-ORBC was significantly increased by 10(-6) to 10(-14) M RA. The potentiation of mitogenic responses of spleen cells to Con A and PWM by RA was relatively independent of macrophage function, i.e., splenocytes that were macrophage-depleted were responsive to the potentiating effects of RA. The effects of retinoids on T-cell-dependent B-cell mitogenesis induced by PWM appeared not to be dependent on their previously reported capacity to alter prostaglandin synthesis. Treatment of spleen cells with 10(-6) M indomethacin did not abolish the potentiating effects of RA. However, RA in a dose-dependent fashion increased IL-1 activity at the level of the target T-cell. The greatest potentiation of IL-1 activity was at 10(-8) M RA. These results show that retinoids can modulate macrophage function at two different levels: potentiation of phagocytosis and potentiation of IL-1 activity at the level of the T-cell.

Animals↗
PubMed1987

In vitro effects of retinoids on murine thymus-dependent and thymus-independent mitogenesis.

The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated. The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids. However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited. All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses. In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation. Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR. These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol. Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells. Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.

Animals↗
PubMed1987

In vitro effect of ethanol on cell-mediated cytotoxicity by murine spleen cells.

The effects of ethanol on murine spleen cell-mediated lysis have been studied. Concentrations of 5.5-176 mM ethanol produced progressive inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC). Binding of spleen cells to antibody-sensitized target cells was not inhibited by comparable concentrations of ethanol. Kinetic analysis revealed decreased rates of lysis with increasing concentrations of ethanol. Changes of effector to target cell ratios revealed an inhibition of maximum lysis and decreased lytic efficiency in the presence of 88 mM ethanol. Preincubation experiments showed the inhibitory effect of ethanol to be reversible. Macrophage-depleted spleen cells appeared to be as susceptible to inhibition by ethanol as unfractionated spleen cells. Ethanol also inhibited natural killer and alloimmune cytotoxic T cell activity. The ADCC data were analysed by using a mathematical model which incorporates the kinetics of lysis, dose-response relationships, heterogeneity of the lytic effectors, reversibility of inhibition and ethanol loss during incubation. An inhibition constant (KI) of 373 mM-2 when two ethanol molecules interact with the site of inhibition was calculated. 50% inhibition of lysis is produced by 52 mM (0.24%) ethanol. The results are consistent with a model which assumes that lysis is due to a critical number of interactions which ultimately trigger the lytic event. Alcohol interferes with lysis by reacting with sites which are required for triggering the lytic event. Although the molecular details of the mechanism of inhibition are as yet undefined, we infer that ethanol inhibits ADCC at the programming for lysis or the lethal hit stages.

Animals↗
PubMed1987

Monoclonal IgM antibodies that inhibit primary Moloney murine sarcoma growth.

Monoclonal IgM antibodies with specificity for Moloney murine sarcoma virus (M-MuSV)-Moloney murine leukemia virus (M-MuLV) from two hybridoma clones have been isolated and characterized. The monoclonal antibodies have specificity for a cytoplasmic and cell surface Friend-Moloney-Rauscher group-specific antigen. Immunoelectron microscopy revealed antibody binding to the surface of virus-expressing cells but not to the budding virus particles. Treatment of M-MuSV-injected mice with monoclonal IgM anti-M-MuSV significantly inhibited tumor growth compared to virus-inoculated animals receiving either saline or MOPC 104E. Nude mice exhibited delayed tumor induction following treatment with the monoclonal antibodies but ultimately died from tumor growth. Virus-injected euthymic mice that were treated with monoclonal IgM anti-M-MuSV generated a potentiated spleen cell-mediated cytotoxicity against Moloney sarcoma cells compared to virus-infected treated with saline. This potentiation of cytotoxicity remained after trypsinization of the spleen cells and thus was probably not due to passively adsorbed monoclonal antibody. The antibodies alone or in the presence of complement did not neutralize M-MuLV. The IgM antibodies induced specific tumor cell cytotoxicity in vitro mediated by complement spleen cells, lymph node cells, or thymus cells. In conclusion, two monoclonal IgM anti-M-MuSV antibodies that bind to the tumor cell surface did not neutralize virus can inhibit primary M-MuSV-induced tumor growth in vivo. The regression event appeared to involve heterogeneous mechanisms. Complete regression remained thymus dependent even with passive antibody therapy, but significant tumor growth inhibition was produced independent of T-cells. In vitro these IgM antibodies induced complement and cell-mediated cytotoxicity.

Animals↗
PubMed1986

Simultaneous expression of multiple immune complex receptors on murine thymocytes and spleen cells.

Mixed rosette studies were performed to evaluate the coexpression of IgG Fc. IgM Fc, and complement receptors (C3R) by thymocytes obtained from mice 7 days after cortisone injection and by spleen cells. Indicator cells coated with IgM, IgG, or C3 independently were mixed and could be distinguished by morphology or by a fluorescein label. In double-marker studies, 36% of spleen cells formed rosettes with IgG- and/or IgM-sensitized red blood cells. Among this population there was a 24% overlap of cells binding IgM and IgG complexes simultaneously. Of the spleen cells, 78% bound IgM- and/or C3-sensitized cells. Of the spleen cells forming rosettes with IgM and C3 indicator cells, 15% coexpressed these receptors. With IgG and C3 indicator cells, 58% of spleen cells bound to one or both kinds of complexes with an 18% overlap. Of cortisone-resistant thymocytes, 14% formed rosettes with IgM- and/or IgG-sensitized red blood cells; within this population there was an overlap of 21%. With IgM- or C3-sensitized cells, 19% of cortisone-resistant thymocytes bound to one or both, among which there was a coexpression of 21%. With IgG- or C3-sensitized cells, there was a 14% overlap of rosette-forming cells binding both. In triple-marker studies 79% of spleen cells formed rosettes with C3-, IgG-, and/or IgM-sensitized indicator cells, out of which 11% coexpressed IgM and IgG FcR, 20% coexpressed IgG and C3R, and 10% coexpressed IgM FcR and C3R. Of rosette-forming cells, 13% coexpressed all three receptors. With cortisone-resistant thymocytes, 19% bound one or more kinds of immune complexes. Among these, 9% coexpressed IgG FcR and C3R, 14% coexpressed IgM FcR and C3R, and 14% bound IgG and IgM complexes. We could not detect the simultaneous expression of all three receptors on cortisone-resistant thymocytes. Using Isopaque-Ficoll fractionation of cells binding C3-sensitized cells, cortisone-resistant thymocytes were enriched and depleted of C3-receptor-bearing cells and their Lyt phenotypes were determined by immunofluorescence microscopy. The C3-receptor-enriched population contained 56% C3R+ cells which were 79% Lyt-1 positive and 100% Lyt-2 positive. The C3R-depleted population contained 1.3% C3R+ cells with 10% Lyt-1 positive and 22% Lyt-2 positive among the total. Surface phenotypic expression of normal and cortisone-resistant thymocytes was also evaluated by direct and indirect fluorescence by fluorescence-activated cell sorter (FACS).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Immune response to polyoma tumor cells in mice--III. Stimulation of tumor cell growth in vitro by spleen cells from immunized animals.

We have evaluated the stimulation of target cell growth in vitro by spleen cells from mice which were immunized with polyoma-transformed cells and other tumor and non-tumor antigens. Stimulation was particularly seen under conditions of immunization that were suboptimal for the production of specific cytotoxicity. Significant stimulation of polyoma target cell growth was observed by lymphocytes from mice immunized against 10(5) Py 4198 tumor cells. This stimulation of target cell growth was not confined to polyoma-transformed cells only. Cells transformed by SV40, H-MuSV and non-transformed cells like 3T3 and embryo fibroblasts were also stimulated. Immunization of mice with syngeneic embryo fibroblasts also resulted in stimulation of tumor cell growth by the spleen cells from the immunized mice. However, the growth stimulation was less consistent and did not occur in all target cells tested. The specificity of immunostimulation was further studied with the Moloney sarcoma virus (M-MuSV) system; an antigenically distinct tumor system. Spleen cells from M-MuSV tumor-bearing mice stimulated cell growth in vitro not only against MuSV-transformed cells but also with SV40-transformed and polyoma-transformed cells as targets. Significant stimulation of target cell growth was also observed by spleen cells from mice that were immunized against 'non-pertinent' antigens, e.g. sheep red blood cells and allogeneic (C57B 1/6) spleen cells.

Animals↗