Biomedical subjects

H Lilja

Publications and source records attributed to H Lilja.

At least 109 records · Page 6Linked to original sources

PubMed1995

Dual-label one-step immunoassay for simultaneous measurement of free and total prostate-specific antigen concentrations and ratios in serum.

We developed a simple one-step dual-label immunoassay for simultaneous measurement of the free, noncomplexed form of prostate-specific antigen (PSA) and total PSA. The assay is based on time-resolved fluorescence and includes a stable fluorescent chelate of Eu to label a monoclonal antibody (mAb) that detects only free PSA, whereas a second mAb labeled with a fluorescent chelate of Tb provides equimolar detection of both free PSA and PSA complexed to alpha 1-antichymotrypsin. A third mAb on a solid phase captures the free and complexed forms of PSA in an equimolar fashion. The simultaneous measurement of the free-to-total PSA ratio (F/T) with the one-step dual assay is not sensitive to variations in the sample volume. The discrimination between benign prostatic hyperplasia and prostate cancer patients, i.e., the area under the receiver-operating characteristic curve, increased from 0.64 (total PSA assay) to 0.78 and 0.81 when the F/T ratio was measured with single and dual assays, respectively.

Antibodies, Monoclonal↗

PubMed1995

Regulation of the enzymatic activity of prostate-specific antigen and its reactions with extracellular protease inhibitors in prostate cancer.

Prostate-specific antigen (PSA) is a tissue-specific serine protease similar in structure to the trypsin-like glandular kallikreins but which is unique inasmuch as the enzyme activity is similar to that of chymotrypsin. The active enzyme is a single chain glycoprotein of 237 amino acids. The major form of PSA in serum is complexed to alpha 1-antichymotrypsin (ACT). A small amount is free, non-complexed despite a large excess of ACT. This suggests that the form in serum lacks enzyme activity. Although serum PSA concentrations are regularly abnormally high (above 4 micrograms/L) in prostate cancer (CAP), the utility of PSA measurements in the early detection of CAP is limited, as many tumors are undetected at a cut-off of 4 micrograms/L. Also, 25% of all men with benign prostate hyperplasia (BPH) have serum PSA levels above 4 micrograms/L. Using assays specially developed to measure free and complexed forms of PSA in serum, we found the proportion of PSA-ACT complexes to be higher in CAP than in BPH, but the ratio of free-to-total PSA in serum to be lower. Using an abnormally low ratio of free-to-total PSA to detect CAP increases diagnostic specificity by 15 to 20%, compared to using a high serum PSA concentration. This suggests that the ratios of free-to-total PSA significantly increase the ability to distinguish BPH from localized CAP. The molecular basis is unclear, but may be related to the high incidence of prostate tumor cells producing both PSA and ACT. This is in contrast to the lack of ACT production in BPH epithelium. Possibly owing to lack of ACT production in BPH areas, conditions are not optimal for complex formation, whereas tumors producing both ACT and PSA may promote the formation of PSA-ACT complexes in CAP.

Enzyme Inhibitors↗

PubMed1995

Free and complexed prostate-specific antigen (PSA): in vitro stability, epitope map, and development of immunofluorometric assays for specific and sensitive detection of free PSA and PSA-alpha 1-antichymotrypsin complex.

Generation of 15 monoclonal antibodies (MAbs) allowed construction of epitope maps and specific two-site immunofluorometric assays for free prostate-specific antigen (PSA) and PSA complexed with alpha 1-antichymotrypsin (ACT). Close correlation of PSA concentrations obtained with the use of different assays of free PSA suggested extensive similarity in immunodetection of free PSA in serum. Assays of the PSA-ACT complex overestimated the concentration of PSA-ACT in serum because of nonspecific adsorbance of ACT or cathepsin G-complexed ACT to the solid phase. This interference was substantially decreased in the presence of heparin. In studying the stability of purified PSA and PSA-ACT complexes formed in vitro, we found that the free PSA was stable during storage for 4 weeks at 35 degrees C, whereas PSA-ACT complexes largely dissociated in these conditions. The instability of PSA-ACT complexes was counteracted by storage at low temperatures, by adjusting the pH of the storage buffer between 6.8 and 7.4, and through addition of 100-1000-fold molar excess of native ACT. The ease of calibration and the accuracy of free PSA assays in comparison with assays of the PSA-ACT complex suggest that measurements of free to total PSA most accurately reflect the inverse of the proportion of PSA complexed to ACT in serum.

Adsorption↗

PubMed1994

Expression of prostate specific antigen on the surface of a filamentous phage.

We have constructed two phagemid vectors containing the gene coding for human Prostate Specific Antigen (PSA) translationally fused to a pelB signal sequence and minor coat protein III of phage fd leading to phage particles that carry PSA on their tips. Phages were characterized by Western blotting and by panning, using biotinylated monoclonal anti-PSA antibodies immobilized onto streptavidin-coated microtitration wells. Binding of PSA-phages was 10(2)- to 5 x 10(3)-fold more effective than that of wild-type phages. Trypsin treatment of the phage abolished the specific binding. Competitive panning with different concentrations of added purified PSA demonstrated that binding was PSA specific. Finally, Western-blot analysis confirmed that full-length and shorter degradation products of the fusion between PSA and protein III were visible as probed with anti-PSA antibody.

Antibodies, Monoclonal↗

PubMed1994

Complex formation between protein C inhibitor and prostate-specific antigen in vitro and in human semen.

Protein C inhibitor (PCI), a serine-proteinase inhibitor first purified from human blood plasma, occurs at high concentrations (3-4 microM) in seminal fluid in both a high-molecular-mass and low-molecular-mass form. Immunochemical data have previously suggested that PCI in seminal plasma forms complexes with the most abundant serine proteinase in semen, prostate-specific antigen (PSA). To provide a structural characterization of the PCI target, immunodetected as PSA, a procedure was developed to isolate low-molecular-mass and high-molecular-mass-forms of PCI from seminal fluid. The high-molecular-mass form of PCI, recognized by monoclonal antibodies against PSA, was dissociated by alkaline treatment into the low-molecular-mass form of PCI and a 33-kDa protein identified as PSA by 25 conclusive steps of N-terminal sequence analysis. We developed a sensitive immunofluorometric assay (IFMA) to measure PCI-PSA complexes in body fluids and investigated the rate at which purified PSA may form complexes with purified PCI. Formation of complexes detected by this IFMA and the appearance of SDS-stable approximately 90-kDa complexes paralleled loss of PSA activity recorded with chromogenic substrates. The rate of complex formation was slow compared to that reported for PCI and activated protein C, but was enhanced up to sixfold in the presence of heparin. Less than 10% of the initial PSA activity remained after 3 h incubation with a sevenfold molar excess of PCI and in the presence of heparin. In freshly collected ejaculates, the rate of PCI-PSA complex formation measured by IFMA was similar to that observed between the purified proteins, and paralleled the appearance of SDS-stable complexes by immunoblotting. During gel dissolution in freshly collected ejaculates, approximately 40% of immunodetected PCI becomes complexed to PSA. Although PCI is a slow inhibitor of PSA, complexes between PCI and PSA are detected at levels that correspond to an inactivation of up to 5% of the PSA activity in the ejaculate.

Amino Acid Sequence↗

PubMed1994

The use of contraceptive methods among women seeking a legal abortion.

In a simple random sample study of 404 women 20 to 29 years of age, 201 applying for abortion and 203 continuing their pregnancies, all were interviewed personally and requested to complete a questionnaire. The aim of the study was to analyse the use and experience of contraceptives among the abortion applicants in this age group, and to compare their experience with that of women in a matched control group. The most important reasons given for desisting in the use of contraceptives were the experience of side effects, worry about the side effects, and the "human failure" factor involved even with methods of contraception such as pills, which generally are regarded as safe. This study demonstrates that women living alone without any permanent relationship, having a strained economy or having previously had an abortion, need particular and detailed contraceptive advice plus continued close follow-up. To improve the continuation rate, precise contraception counselling focusing on both positive and adverse effects to reduce worry seems essential.

Abortion Applicants↗

PubMed1994

Alpha 1-antichymotrypsin production in PSA-producing cells is common in prostate cancer but rare in benign prostatic hyperplasia.

OBJECTIVE: To investigate the distribution and production of alpha 1-antichymotrypsin (ACT) and prostate-specific antigen (PSA) in benign hyperplastic and malignant prostatic tissue, respectively. METHODS: Using monoclonal anti-ACT and anti-PSA IgGs for immunocytochemistry and alkaline phosphatase conjugated 30-mer oligodeoxynucleotide probes for nonradioactive in situ hybridization, tissue specimens were studied from 15 patients with benign prostatic hyperplasia after transurethral resection of the prostate (TURP) and from 9 patients with bladder cancer who underwent cystoprostatectomy. Cancer specimens were from 23 TURP patients and from ultrasound guided core biopsies in 14 patients. Prostate tumors were graded according to the Gleason system. RESULTS: We found no or only occasional small foci of immunostaining for ACT, and no ACT transcripts in the PSA-producing epithelium in areas with benign nodular hyperplasia. By contrast, a high proportion of cells expressed both ACT and PSA in prostate cancers with low Gleason score, as detected by immunocytochemistry and in situ hybridization. Poorly differentiated tumor cells manifested greater variation in immunostaining for both ACT and PSA. As compared to tumors of low Gleason score, high-score tumors less frequently manifested immunostaining for ACT than for PSA, and less frequently generated hybridization signals for both PSA and ACT transcripts. CONCLUSIONS: A significantly higher proportion of serum PSA has been reported to be complexed to ACT in patients with prostate cancer than in patients with benign prostatic hyperplasia. The presently reported lack of ACT production in PSA-containing BPH nodules may contribute to this by making conditions less optimal for complex formation between PSA and ACT. As opposed to this, production of both ACT and PSA in prostate cancers may enhance the complex formation between PSA and ACT.

Aged↗

PubMed1994

Comparability of the Tandem-R and IMx assays for the measurement of serum prostate-specific antigen.

OBJECTIVES: To assess the comparability of the Tandem-R and IMx serum prostate-specific antigen (PSA) assays across levels of the ratio of free-to-total serum PSA found in a community-based population of healthy men. METHODS: Banked serum samples from the baseline component of the Olmsted County Study of Urinary Symptoms and Health Status Among Men were thawed and analyzed using the Tandem-R and IMx PSA assays. Serum levels also were determined for the free, noncomplexed form of PSA, PSA complexed to alpha-1 antichymotrypsin, and total PSA with a research-based immunofluorometric assay. RESULTS: The results of the Tandem-R and IMx assays were strongly correlated at all levels of the ratio of free-to-total serum PSA. The Spearman correlation coefficients ranged from 0.87 to 0.98 (all p < 0.001). The relationship between the Tandem-R and IMx assays, however, differed at low levels of free-to-total serum PSA compared with high levels. In the lowest 10th percentile of the ratio of free-to-total serum PSA (0.04 to 0.18), the IMx assay read lower than the Tandem-R (slope +/- standard error = 0.92 +/- 0.04, intercept +/- standard error = 0.21 +/- 0.14); whereas in the upper 10th percentile of free-to-total ratio (0.46 to 0.65) the IMx assay yielded values higher than the Tandem-R assay (slope = 1.21 +/- 0.07, intercept = 0.14 +/- 0.05). In the middle 90%, the slope did not statistically differ from 1.0. CONCLUSIONS: For the majority of men, results of the Tandem-R and IMx PSA assays were virtually identical. The small differences found would not be of clinical significance for most men but should be considered when comparing results of different assays in sequential determinations for a specific man.

Adult↗

PubMed1994

Evaluation of stated motives for legal abortion.

In a study of 404 women (simple random sample), 20-29 years of age, 201 (group A) applying for abortion and 203 (group B) continuing their pregnancies, the women were given a questionnaire and in addition were interviewed. The aim of the study was to evaluate the spontaneous personal motives of women for abortion at a time when age is not supposed to be a common reason. The results showed that more than half of the women expressed that a bad relationship with the partner in one way or another was a motive for the abortion. Other important motives included characteristics of the women and their partners, mainly immaturity, work/studies and unsuitable life situation for having a child. Less common motives seemed to be economy, dwelling and medical and health factors. It is obvious that women in this study wanted to have a stable relationship to the child's father before they dared or wanted to have a child. Social networks in modern society seem to be too weak. The women do not want to face social and emotional problems as lonely mothers. Political decisions in the society, for example with parental benefit according to your income discourage women from continuing their pregnancies during their studies. It also seems important for the woman to feel mature enough to have a child. The provision and encouragement of methods for safer sex may be a possible way by which to reduce the number of abortions.

Abortion, Legal↗

PubMed1994

Tumor markers. Consensus Conference on Diagnosis and Prognostic Parameters in Localized Prostate Cancer. Stockholm, Sweden, May 12-13, 1993.

This chapter mainly deals with biochemical aspects on prostate specific antigen (PSA) and its clinical value. To a limited extent, also other tumor markers, which might be of importance in the evaluation of patients with prostate cancer are discussed. In serum, PSA exists in a free form or bound to antichymotrypsin. Interestingly, only 10% of PSA secreted from cancer cells seems to exist in a free form, as compared to 30% of PSA secreted from cells in benign prostatic hyperplasia (BPH). PSA seems to be closely, but not absolutely, related to tumor grade and stage. The mean value of PSA in patients with tumors dominated by Gleason grades 3 or below, was 10 ng/ml, compared to 29 ng/ml in those with higher grades. Patients with PSA values of 50 ng/ml or above almost exclusively had tumor of Gleason grades 4 or 5, and this limit usually reflected a generalized disease. Patients with PSA-values below 10 ng/ml almost exclusively had tumors confined to the prostate gland. In countries where screening for prostate cancer is believed in, it is important to understand that normal cut-off values are related to patient's age. The upper normal limit of males below 50 years of age should be set at 2.5 ng/ml, as compared to 6.5 ng/ml for men over 70 years of age. To improve the value of PSA determination and for scientific purposes, the standardization of the assay is urgently needed and under way. Prostate acid phosphatase (PAP) has in most centres been replaced by PSA. An elevated PAP value, as measured by the enzymatic method, invariably indicates a generalized disease and could thus be used as a complementary informative assay to PSA. Other markers have been used mainly to achieve additional prognostic information. In a multivariate analysis, the non-specific tumor marker neopterin, which reflects the host response to tumor antigens, was closely related to short-term prognosis. Neopterin was followed by thymidine kinase, a protein reflecting the cell turn-over and tumor grade. Also PSA at diagnosis seemed to add some prognostic information, whereas other markers did not.

Adult↗

PubMed1993

Structure, function, and regulation of the enzyme activity of prostate-specific antigen.

Prostate-specific antigen (PSA) and human glandular kallikrein 1 (hGK-1) are structurally similar products of the human glandular kallikrein gene locus on chromosome 19 that become selectively expressed by human prostate tissue. PSA is one of the most abundant prostate-derived proteins in the seminal fluid. The mature form of PSA, a single-chain glycoprotein of 237 amino acids, is a serine protease manifesting restricted chymotrypsin-like activity. PSA is mainly responsible for gel dissolution in freshly ejaculated semen by proteolysis of the major gel-forming proteins semenogelin I and II and fibronectin. PSA complexed to alpha 1-antichymotrypsin (ACT) is the predominant molecular form of serum PSA, although complex formation is slow between the purified proteins in vitro. PSA also forms stable complexes with alpha 2-macroglobulin (alpha 2M) in vitro, but as this results in encapsulation of PSA and complete loss of the PSA epitopes, the in vivo significance of this complex formation is presently unclear. A free, noncomplexed form of PSA constitutes a minor fraction of the serum PSA, although serum ACT occurs at large molar excess.

Genitalia, Male↗

PubMed1993

Power spectrum analysis of the fetal heart rate during noradrenaline infusion and acute hypoxemia in the chronic fetal lamb preparation.

In a chronically instrumented fetal lamb the effect on heart rate variability of noradrenaline as well as hypoxemia is studied by the use of power spectral analysis. Subsequent to both noradrenaline infusion and hypoxemia the very low frequency components of the variability are markedly decreased as compared with control conditions. After hypoxemia also a high frequency peak appears in the spectrum.

Acute Disease↗

PubMed1993

Production of alpha-1-antichymotrypsin by PSA-containing cells of human prostate epithelium.

Prostate-specific antigen (PSA) is a chymotrypsin-like serine protease exclusively produced by the prostate epithelium, and abundant in seminal fluid. In serum, PSA is predominantly complexed to a liver-derived serine protease inhibitor, alpha-1-antichymotrypsin (ACT). A higher proportion of serum PSA is complexed to ACT in prostate cancer than in benign prostate hyperplasia. Since the molecular basis for this is unclear, we have investigated whether or not ACT may be produced in the prostate gland. Immunocytochemistry, using either monoclonal or polyclonal IgGs, demonstrated specific immunostaining for ACT in normal PSA-containing prostate epithelium. Production of ACT in the normal PSA-producing prostate epithelium was demonstrated by means of nonradioactive in situ hybridization using 30-mer anti-sense DNA probes for ACT and for PSA. The ACT and PSA coding transcripts, as detected by in situ hybridization, were distributed perinuclearly, in contrast to the specific immunostaining for ACT and PSA which was most intense in the apical portion of the secretory cells. The results strongly suggest local production and release of ACT by the normal prostate epithelium that may be important for interaction between PSA and ACT in extracellular compartments.

Culture Techniques↗

PubMed1993

Serum prostate specific antigen complexed to alpha 1-antichymotrypsin as an indicator of prostate cancer.

Prostate specific antigen (PSA) in serum has recently been shown to occur in complex with alpha 1-antichymotrypsin and as an approximately 30 kDa. noncomplexed molecular form. We characterized PSA by 3 different assays in samples from 144 patients with benign prostatic hyperplasia (BPH) and 121 with carcinoma of the prostate. One of these noncompetitive assays measured total PSA by detecting PSA complexed to serine proteinase inhibitors and the noncomplexed molecular form, a second measured only PSA in complex with alpha 1-antichymotrypsin, whereas a third detected the noncomplexed form. PSA in complex with alpha 1-antichymotrypsin was the predominant form in all patient sera. Noncomplexed PSA constituted a minor fraction that was significantly smaller in patients with untreated prostate cancer than in those with BPH (p < 0.0001). The proportion of noncomplexed PSA does not correlate to the serum concentration of PSA or that of alpha 1-antichymotrypsin. In men with a serum PSA concentration of less than 10 micrograms./l. the combination of assays measuring total PSA immunoreactivity, the noncomplexed molecular form and PSA in complex with alpha 1-antichymotrypsin may facilitate discrimination between prostate cancer and BPH.

Biomarkers, Tumor↗

PubMed1993

Significance of different molecular forms of serum PSA. The free, noncomplexed form of PSA versus that complexed to alpha 1-antichymotrypsin.

Prostate specific antigen is an abundant prostate-derived serine protease in the seminal fluid. Low concentrations of the protein are normally released into blood, but above normal concentrations are frequently detected in prostate disease. The PSA-ACT complex is the predominant molecular form of serum PSA (up to approximately 95%) although complex formation is slow between the purified proteins in vitro. A free, noncomplexed form of PSA constitutes a minor fraction of the serum PSA, although serum ACT occurs in large molar excess. The free, noncomplexed form of serum PSA is reported to constitute a significantly smaller proportion of the PSA in untreated prostate cancer than in BPH. The molecular basis for this finding is unclear, but measurements of the proportion of the free form of serum PSA or the proportion of serum PSA-ACT may facilitate discrimination between prostate cancer and BPH.

Humans↗

PubMed1993

Europium-labeled oligonucleotides to detect point mutations: application to PIZ alpha 1-antitrypsin deficiency.

We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the polymerase chain reaction (PCR) and allele-specific oligonucleotides (ASO) with highly sensitive time-resolved fluorometry. ASO probes differing by a single base substitution and labeled with europium (Eu) chelates were hybridized in solution simultaneously with a biotinylated oligomer to a PCR-amplified nucleic acid fragment. The hybrids formed were then collected onto streptavidin-coated microtitration wells. Subsequently, the hybrids were washed under stringent conditions and the remaining ASO probe was measured in a time-resolved fluorometer. We discuss the strategy underlying the design of the Eu-labeled ASO probes for the solution hybridization assay. The method was applied to the detection of the Z-mutation in the alpha 1-antitrypsin gene. Evaluation of whole-blood samples spotted on Guthrie cards demonstrated successful accuracy of the method.

Base Sequence↗

PubMed1992

Gene structure of semenogelin I and II. The predominant proteins in human semen are encoded by two homologous genes on chromosome 20.

The genes for semenogelin I and II, the major protein constituents of the human seminal fluid, have been characterized by three overlapping clones in bacteriophage lambda, encompassing 31.5 kilobases (kb) of genomic DNA. The two genes are located 11.5 kb apart in the region q12-q13.1 on chromosome 20. Both genes are relatively compact, spanning only 2.7 and 3.1 kb, respectively. The transcription units are composed of three exons, of which the first encodes the signal peptide, the second encodes the secreted protein, while the third solely contains 3'-noncoding nucleotides. The nucleotide sequences exhibit a similarity of close to 90% in the exons and exceeding 80% in the introns and flanking nucleotides.

Amino Acid Sequence↗

PubMed1992

Prostate specific antigen predominantly forms a complex with alpha 1-antichymotrypsin in blood. Implications for procedures to measure prostate specific antigen in serum.

BACKGROUND: Prostate specific antigen (PSA) is a zymogen of a 33-kilodalton (kD) serine proteinase with extensive similarity to glandular kallikreins. The mechanism responsible for converting the zymogen into active proteinase has not been defined, but active PSA may be irreversibly inactivated in vitro by two of the major proteinase inhibitors in blood: alpha 1-antichymotrypsin and alpha 2-macroglobulin. METHODS: Procedures have been designed to characterize the different molecular forms of PSA in serum. One assay detects PSA epitopes available on both PSA binding to serine proteinase inhibitors and PSA not binding to a proteinase inhibitor. A second assay only detects PSA in complex with alpha 1-antichymotrypsin. A third assay mainly detects PSA not binding to a proteinase inhibitor. RESULTS: In serum samples, an 80-kD to 90-kD species of PSA in complex with alpha 1-antichymotrypsin is the predominant molecular form and a minor molecular form of serum PSA was an approximately 30-kD fraction not binding to a proteinase inhibitor. CONCLUSIONS: The benefits of detecting different molecular forms of serum PSA should be investigated regarding possibilities to facilitate differential diagnosis of carcinoma of the prostate (CAP) and benign prostatic hyperplasia (BPH).

Antigens, Neoplasm↗