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Biomedical subjects

J T Wu

Publications and source records attributed to J T Wu.

At least 55 records · Page 3Linked to original sources
PubMed1999

Development of a microplate assay for serum chromogranin A (CgA): establishment of normal reference values and detection of elevated CgA in malignant diseases.

Chromogranin A (CgA), a marker for neuroendocrine cells, is associated with poor prognosis when detected by immunohistochemical technique in prostate tumors. We have developed an ELISA on microplates for serum CgA and established the normal reference range. We also attempted to find out whether elevated serum CgA levels could be found in patients with various malignant diseases. Because of non-Gaussian distribution, both medians and 97.5 percentiles of serum CgA levels for men and women of four different age groups were determined. For women, the median and 97.5 percentiles are 20.7 and 63.9 ng/mL for ages 20 to 50, and 32 and 93.8 for 50 to 80 years of age, respectively; for men, they are 27.9 and 78.4 ng/mL for ages 18 to 40 and 41.6 and 92 for 40 to 80 years old, respectively. Elevated serum concentrations of CgA were detectable in patients with prostate cancer not undergoing hormonal treatment, and in patients with various malignant diseases including nonendocrine carcinomas. Most elevated serum CgA levels were associated with sera containing highly elevated serum tumor markers. Drugs targeting neuroendocrine cells should be administered for cancer patients with elevated serum CgA levels.

Adult↗
PubMed1998

On-line analysis by capillary separations interfaced to an ion trap storage/reflectron time-of-flight mass spectrometer.

The interface of high-resolution capillary separation methods to time-of-flight mass spectrometry (TOF-MS) has generated considerable interest since TOF can provide the rapid and sensitive detection required by high resolution separations. In recent years, our laboratory has developed a variety of high-resolution capillary separation methods interfaced to TOF-MS via an ion trap storage/reflectron time-of-flight (IT/reTOF) instrument. Using this hybrid configuration, detection of fast separations at very low detection levels has been successfully performed for on-line separations of peptides and protein digests using electrospray ionization. In this report, we review the current status in our laboratory of interfacing high-performance liquid chromatography, capillary electrophoresis, and capillary electrochromatography to an IT/reTOF-MS instrument and various applications that have been developed involving this technology.

Chromatography, High Pressure Liquid↗
PubMed1998

Development of an immunoassay specific for the PSA-ACT complex without the problem of high background.

We have developed an assay specific for the PSA-ACT (PSA-alpha 1-antichymotrypsin) complex that effectively diminishes the problem of high assay background commonly reported by other investigators. The assay follows a two-site ELISA format. Polyclonal anti-PSA antibodies were coated on the microplate to capture the PSA complex from the serum, whereas the biotinylated anti-ACT polyclonal antibodies and HRP-conjugated streptavidin were used for detection. The high background ordinarily associated with this assay was greatly reduced when milk casein was added in addition to albumin for blocking and when the Super Block was also included in the diluents for sample dilution and dilution of enzyme conjugated detecting antibodies. The assay has a sensitivity of 0.05 ng/mL. The within-run precision ranges from 4.2-7.2% and the between-run precision falls between 5.8-8.5%. Cross reactions with ACT and free PSA (fPSA) are 0.0001% and 0.02%, respectively. The highest concentration of PSA-ACT complex in the maternal sera was < 0.4 ng/mL by this assay, much less than reported in the literature. Using this improved assay, the sum of fPSA and PSA-ACT concentrations were less than that of their corresponding total PSA (tPSA) most of the time. We believe that this improved assay should be used to replace the current tPSA assay for screening, monitoring, and managing patients with prostate cancer.

Enzyme-Linked Immunosorbent Assay↗
PubMed1998

Serum chromogranin A: early detection of hormonal resistance in prostate cancer patients.

We monitored both chromogranin A (CgA) and neuron specific enolase (NSE) in serial serum specimens from 14 patients with prostate cancer (CAP patients) showing resistance to hormonal treatment. Elevated serum CgA was detected in 10 out of these 14 patients (71%) during treatment, and an early appearance of elevated serum CgA was found in 6 of 14 (43%) of these patients when serum tPSA levels were still in the normal range. If patients with radical prostatectomy were not included, the percentage of patients showing an early appearance of elevated serum CgA would have been much higher. Elevated serum CgA levels also were found in patients not subject to hormonal therapy. Serial specimens from two out of three prostate cancer patients, randomly selected, contained elevated serum CgA. Serum NSE was not detectable in any of the serial specimens we studied, suggesting that CgA, not NSE, should be used as a marker for neuroendocrine differentiation. We also compared the serum CgA in random serum specimens between patients with BPH (benign prostate hyperplasia) and with prostate cancer in the concentration range of serum tPSA between 3-15 ng/mL. Although serum CgA concentrations in BPH patients overlapped considerably with those levels in patients with prostate cancer, levels > 100 ng/mL should suggest prostate cancer. The early appearance of elevated serum CgA allows an early change of therapy to be made and can lead to the effective prevention of any further development of metastases.

Androgen Antagonists↗
PubMed1998

Monitoring percent free PSA in serial specimens: improvement of test specificity, early detection, and identification of occult tumors.

We have measured the serum concentration of prostate specific antigen (tPSA) and determined the percent free PSA (% fPSA) in serial specimens from 64 patients with prostate cancer, 35 patients with benign prostate hyperplasia (BPH), and 3 patients with prostitis. We found that the % fPSAs were not a constant for individual patients during the course of the disease. When we compared only the % fPSA of the first specimen of serial specimens from individual patients, who were largely untreated, 37% of BPH specimens were above 22%, whereas only 1.6% cancer samples were above that value. We also found that 67% of cancer specimens and 14% of BPH samples were below 8%, respectively. Although % fPSA distribution pattern remained similar between two types of specimens, less differentiation was found between BPH and prostate cancer in random specimens compared to the study using first specimens of an individual patient's serial samples. Percent fPSA apparently are affected by treatment. However, the most important benefit for the determination of % fPSA appears to be the sensitivity of % fPSA to identify occult tumors when the tPSAs were in the normal concentration range. Determination of % fPSA also seems to improve the specificity of tPSA, not only during screening for the differentiation between BPH and prostate cancer, but also during monitoring of treatment and recurrence.

Humans↗
PubMed1998

Advantages of replacing the total PSA assay with the assay for PSA-alpha 1-antichymotrypsin complex for the screening and management of prostate cancer.

Several advantages become immediately apparent when the prostate specific antigen (PSA, or tPSA) assay is replaced by the assay specific for the serum PSA-alpha 1-antichymotrypsin (PSA-ACT) complex. For instance, random contributions to the tPSA value by various serum minor PSA isoforms can be avoided, making possible the determination of a more accurate relation of the PSA-ACT concentration to the tumor activity. Discrepancies in percent free PSA (% fPSA) values from the same specimens due to the use of different commercial kits also can be eliminated, mainly because the PSA-ACT assay does not have the problems in antibody selection and calibrator preparation usually associated with the tPSA assay. We found that at the present time different cutoffs of % fPSA for the differentiation of BPH from prostate cancer must be established for each individual tPSA assay. Cutoffs established using values from one tPSA assay should not be used for making clinical decisions when their tPSA values are determined by a different kit. Moreover, when we monitored the patients during treatment with serum tPSA, specific fPSA, and specific PSA-ACT complex assays simultaneously, it was clear that any interpretation of the patient's clinical status based on tPSA values alone could be misleading. Because there is less PSA-ACT complex in BPH specimens relative to that found in cancer serum samples, expressing fPSA as "fPSA/PSA-ACT x 100" and measuring PSA-ACT complex concentrations instead of tPSA during screening improve the measurable contrast between BPH and prostate cancer. Although individually modest, collectively these advantages can add up to considerable improvements.

Humans↗
PubMed1998

Isolation of the intact molecule and ectodomain of C-erbB-2 oncoprotein from SK-BR-3 cells and development of immunoassays on microplate.

We isolated both the intact molecule (p185) and the ectodomain (p120) of c-erbB-2 oncoprotein from SK-BR-3 breast tumor cells. The p120 was extracted from the cells by 0.05 M phosphate buffer, pH 7.2, whereas the extraction of the p185 required the presence of a detergent, such as 1% Triton X-100 in 0.05 M Tris buffer. Protease inhibitors were also included in the extraction buffer during the isolation of p185 in order to prevent cleavage of p185 to p120 by an unknown protease apparently also present in the extract. In case there was any p120 in the p185 preparation, the p120 could be separated from p185 by chromatography on a Superose 12 column. Using the p120 and p185 as calibrators, we have established two microplate sandwich immunoassays: one measures both p185 and p120 (total assay) and the other is specific for the p185. Since capturing and detecting antibodies used in the total assay react against the extracellular domain of the c-erbB-2 oncoprotein, they can therefore be used to measure the p120 in serum and p185 in breast tumor tissue cytosol. On the other hand, the p185 specific assay uses the capturing antibody against the cytosolic domain of the oncoprotein and consequently can only measure p185 in breast tumor tissue cytosol.

Antibodies, Neoplasm↗
PubMed1998

Development of a three-dimensional topographic map display for capillary electrophoresis/mass spectrometry with an ion trap/reflectron time-of-flight mass spectrometer detector: applications to tryptic digests of isoforms of myelin basic protein.

A three-dimensional (3-D) contour map format has been developed to display the large amount of data continuously collected throughout an on-line capillary separation using an ion trap storage/reflectron time-of-flight detector (IT/reTOF). The resulting data are displayed on a single computer screen with a mass-to-charge ratio value-elution time-intensity representation. The intensity of various components is represented by 16 different colors so that the mass-to-charge ratio value, the elution time, and the intensity can be conveniently determined for each component. In addition, the mass spectrum and total ion chromatogram or total ion electropherogram (TIE) are shown on the same screen as the 3-D map that enables the correlation of a single spot in the 3-D map to the peaks in the TIE and the corresponding mass spectrum. The 3-D map has been used to identify various posttranslational modification sites of bovine myelin basic protein charge isomers, where the datafiles of tryptic digests of proteins analyzed by capillary electrophoresis/mass spectrometry were processed by this software and a comparison could be performed among the isoforms. The feature of in-screen integration over both the separation domain and the mass domain makes the acquisition of the selected ion chromatogram very convenient and greatly improves the ability to detect modified components present in low amounts.

Amino Acid Sequence↗
PubMed1998

Induction of tumor-specific T cell response by cognating tumor cells with foreign antigen-primed Th cells.

Sufficient CD4+ T cell help is very important in generating specific cytotoxic T cell responses. The inadequate activation of tumor-specific Th cells leads to failure of antitumor immunity. In general, each individual consists of some primed Th cells responding to certain antigens. If these tumor non-specific pre-primed Th cells can provide sufficient help, the generation of tumor-specific T cells may be enhanced. In the present study, we tested this hypothesis by cognating and reactivating pre-primed ovalbumin (OVA)-specific Th cells with OVA-pulsed tumor cells which could simultaneously present both OVA and tumor-associated antigen on the same cell. We clearly demonstrated that immunization of OVA-sensitized mice with OVA-pulsed P388 cells, but not unpulsed P388 cells, led to the induction of P388-specific cytotoxicity and tumor resistance. Both CD4+ and CD8+ tumor-specific cytotoxic T cells were detected in vitro, but only CD8+ T cells played the major effector role in preventing the growth of challenged tumor in vivo. Taken together, our study demonstrated that the immunogenicity of tumor cells can be enhanced effectively by cognating pre-primed foreign antigen-specific Th cells with tumor cells. These findings have potential implications in developing methods to control tumor growth.

Animals↗
PubMed1998

Production of milligram concentrations of free prostate specific antigen (fPSA) from LNCaP cell culture: difference between fPSA from LNCaP cell and seminal plasma.

We have established a procedure for the production of milligrams of free PSA (fPSA) from LNCaP cells derived from a human carcinoma of the prostate. By growing LNCaP cells in a serum-free medium in the presence of a synthetic androgen (R1881) and taking advantage of the special design of the Micro-mouse Hollow Fiber Bioreactor, relatively pure fPSA could be obtained. We found that columns containing either Sephacryl S-100 or S-200 could be used to remove the small amount of bovine serum albumin (BSA) and PSA-alpha 1-antichymotrypsin complex (PSA-ACT) from the preparation. More than 90% of the PSA from LNCaP cell cultures are fPSA. Like fPSA from seminal plasma, two fractions of fPSA differing in protease activity can be separated by DEAE-Sepharose chromatography. Based on the band pattern exhibited on the Western blot following sodium dodecyl sulfate-polyacrylamide electrophoresis separation, fPSA from LNCaP contains more inactive PSA isoforms. This was confirmed by chromatofocusing: the isoelectric point (pl) of the major PSA isoforms from the LNCaP cell culture were higher (6.8 and 6.6) than that (6.4 and 6.1) of fPSA from seminal fluid. We conclude that the LNCaP cell culture is a reliable source for obtaining large quantities of pure fPSA both for the preparation of assay calibrators and controls and for studying the difference in fPSA between benign prostate disease and prostate cancer.

Animals↗
PubMed1998

Impact of assay parameters on the accuracy of free PSA test: source and stability of calibrator, calibration curve fitting, and level of total PSA in the serum.

The measurement of PSA is recommended for men over 50 years of age for screening of prostate cancer. However, proper differentiation of prostate cancer from benign prostate hyperplasia (BPH) relies on an accurate measurement of free PSA (fPSA) and a correct calculation of percent fPSA. Because of the extremely low concentration of fPSA in the serum, any slight deviation from its true value may produce large errors in percent fPSA calculated. Therefore, we undertook a study examining carefully those parameters of the fPSA assay which might affect the fPSA determination. We found that the integrity of the calibrator, the computer curve-fitting program selected, the source of the calibrator, and the total PSA or fPSA + PSA complexes (tPSA) concentration of the specimen all had an impact on the accuracy of the fPSA value assayed. We found that an examination of the slope of the calibration curve was important to reveal whether the calibrator had or had not been denatured during storage. We also found that the 4-parameter cure fitting program was best suited for plotting the fPSA calibration curve. The calibrator we isolated from LNCaP cells was acceptable for our assay because it had an affinity for the assay antibody very similar to that of serum fPSA. We also determined the effect of tPSA concentration on the fPSA determinations and found that within the concentration range of 4-10 ng/mL the impact on the percent fPSA calculated was not significant. We believe that our assay produces accurate fPSA values when all these assay parameters are well controlled.

Calibration↗
PubMed1998

Development of an assay for modulating anti-acetylcholine receptor autoantibodies using human rhabdomyosarcoma cell line.

Three types of autoantibodies against the acetylcholine receptors (AChR) of skeletal muscle are detectable in patients with myasthenia gravis including binding, blocking, and modulating anti-AChR antibodies. Modulating autoantibodies correlate best with the severity of the disease, but are also technically most difficult to measure because the assay generally requires fresh human muscle cells. We have developed an assay for the modulation of anti-AChR antibodies using a rhabdomyosarcoma (RD) cell line expressing AChR on the cell surface. By decreasing the FetalClone III serum from 10% to 0.5% in Eagles Minimal Essential Medium (EMEM) we were able to increase the number of AChR on RD cells to meet the need of sensitivity of the assay. The extent of modulation was determined as the percent of AChR internalized in the presence or absence of modulating autoantibodies. Less than 6% modulation was found with the normal serum (n = 42). The CVs of both the intra- and day-to-day precision were less than 20%. When clinical samples (n = 105) were assayed in our laboratory and also at Nichols Institute, a correlation coefficient of 0.816 was obtained. The selection of RD cell line, the success of increasing the expression of the AChR on RD cells and the use of 125I alpha-bungarotoxin of high specific activity allowed the establishment of an assay which can be used in routine clinical laboratory for the measurement of modulating anti-AChR autoantibodies for the management of patients with myasthenia gravis.

Autoantibodies↗
PubMed1997

Protein digest analysis by pressurized capillary electrochromatography using an ion trap storage/reflectron time-of-flight mass detector.

Pressurized capillary electrochromatography (pCEC) has been coupled to an ion trap storage/reflectron time-of-flight mass spectrometer for the analysis of peptide mixtures and protein digests. Taking advantage of the electroosmotic flow, high separation efficiency has been achieved in pCEC due to a relatively flat flow profile and the use of smaller packing materials. A supplementary pressure was used in these experiments which suppressed bubble formation and also allowed the tuning of the elution of peptides using the electrical field. In this work, a fast separation of a six-peptide mixture has been successfully performed. Using columns only 6 cm long, a tryptic digest of bovine cytochrome c was fully separated in around 14 min by properly tuning the applied voltage and the supplementary pressure. In addition, relatively complex protein digests, such as a tryptic digest of chicken ovalbumin, were analyzed using this pCEC/MS system, and more than 20 peaks were resolved in the total ion current chromatogram within 17 min. The use of an ion trap storage/reflectron time-of-flight mass spectrometer as an on-line detector further increased the resolving power of the pCEC by unambiguously identifying coeluting components. The nonscanning property of the time-of flight mass analyzer and the ion signal integration capability of the ion trap were successfully combined to provide rapid and sensitive full-mass range detection in these experiments.

Amino Acid Sequence↗
PubMed1997

Use of a polybrene capillary coating in capillary electrophoresis for rapid analysis of hemoglobin variants with on-line detection via an ion trap storage/reflectron time-of-flight mass spectrometer.

A polybrene capillary coating in capillary electrophoresis (CE) has been used for rapid analysis of hemoglobin variant digests. The use of the polybrene capillary coating has allowed sufficient separation to resolve the large number of digest products formed upon tryptic digestion of the whole protein, so that prior separation of the hemoglobin alpha and beta chains is not required. The resolution of the digest peaks obtained by CE is sufficient so that even single amino acid substitutions can easily be detected using UV absorption detection. The digest is further analyzed by capillary electrophoresis separation with on-line detection using electrospray ionization interfaced to the ion trap storage/reflectron time of flight device (CE/ESI-IT/reTOF), where a comparison of the total ion electropherograms and mass spectra of the mutant and normal hemoglobins can detect the presence of a mutation site. The CE separation and mass analysis can be accomplished in typically 10-15 min. The unique capability of the CE/ESI-IT/reTOF system for detection of fast separations with narrow peaks that may be under 1 s fwhm is demonstrated. The speed of this system is essential for resolution of the large number of peaks that are separated in a short time duration using CE separations.

Adult↗
PubMed1997

Hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans.

BACKGROUND: Hyperhomocyst(e)inemia is a risk factor for atherosclerosis and is prevalent in the elderly. The objective of this study was to determine whether hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans. METHODS AND RESULTS: High-resolution vascular ultrasonography was used to study endothelium-dependent and -independent vasodilation in a nonatherosclerotic peripheral conduit artery of 26 elderly hyperhomocyst(e)inemic subjects and 15 age- and sex-matched subjects with normal homocysteine levels. Flow-mediated, endothelium-dependent (nitric oxide-mediated) vasodilation was assessed by measuring the percent change in brachial artery diameter during reactive hyperemia. Endothelium-independent vasodilation was assessed after the administration of 0.4 mg sublingual nitroglycerin. Endothelium-dependent vasodilation was significantly impaired in the hyperhomocyst(e)inemic subjects compared with control subjects (3.7 +/- 0.6% versus 8.1 +/- 1.2%; P = .004), whereas endothelium-independent vasodilation was not different between the two groups (10.1 +/- 1.6% versus 9.3 +/- 1.5%; P = NS). In a linear regression analysis with serum homocysteine concentration, folic acid, age, sex, cholesterol (serum total, LDL, or HDL cholesterol), mean arterial blood pressure, use of antihypertensive medication, and baseline brachial artery diameter included as covariates, serum homocysteine concentration emerged as the only significant predictor of flow-mediated vasodilation. CONCLUSIONS: These data indicate that hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans and suggest that the bioavailability of nitric oxide is decreased in hyperhomocyst(e)inemic humans.

Aged↗