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Biomedical subjects

M T Madsen

Publications and source records attributed to M T Madsen.

At least 19 recordsLinked to original sources
PubMed1999

The human antibacterial cathelicidin, hCAP-18, is bound to lipoproteins in plasma.

Cathelicidins are a family of antibacterial and lipopolysaccharide-binding proteins. hCAP-18, the only human cathelicidin, is a major protein of the specific granules of human neutrophils. The plasma level of hCAP-18 is >20-fold higher than that of other specific granule proteins relative to their levels within circulating neutrophils. The aim of this study was to elucidate the background for this high plasma level of hCAP-18. Plasma was subjected to molecular sieve chromatography, and hCAP-18 was found in distinct high molecular mass fractions that coeluted with apolipoproteins A-I and B, respectively. The association of hCAP-18 with lipoproteins was validated by the cofractionation of hCAP-18 with lipoproteins using two different methods for isolation of lipoproteins from plasma. Furthermore, the level of hCAP-18 in delipidated plasma was <1% of that in normal plasma. Immunoprecipitation of very low, low, and high density lipoprotein particles with anti-apolipoprotein antibodies resulted in coprecipitation of hCAP-18. The binding of hCAP-18 to lipoproteins was mediated by the antibacterial C-terminal part of the protein. The binding of hCAP-18 to lipoproteins suggests that lipoproteins may play an important role as a reservoir of this antimicrobial protein.

Amino Acid Sequence↗
PubMed1999

Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases.

We have developed a specific and efficient method for complete removal of polyhistidine purification tags (HisTags) from the N-termini of target proteins. The method is based on the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the HisTag is cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain. Some sequences, however, are resistant to DPPI cleavage and a number of mature proteins have nonsubstrate N-termini which protects them against digestion. For such proteins, HisTags composed of an even number of residues can be cleaved off by treatment with DPPI alone. When the target protein is unprotected against DPPI, a blocking group is generated enzymatically from a glutamine residue inserted between the HisTag and the target protein. A protein with a HisTag-Gln extension is incubated with both DPPI and GCT. As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented. The desired sequence is finally obtained by excision of the pyroglutamyl residue with PGAP. All the enzymes employed can bind to immobilized metal affinity chromatography (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our aminopeptidase-based method for specific excision of HisTags and use of subtractive IMAC for removing processing enzymes. Typical recoveries were 75-90% for the enzymatic processing and subtractive IMAC. The integrated process holds promises for use in large-scale production of pharmaceutical proteins because of a simple overall design, use of robust and inexpensive matrices, and use of enzymes of either recombinant or plant origin.

Amino Acid Sequence↗
PubMed1998

Active recombinant rat dipeptidyl aminopeptidase I (cathepsin C) produced using the baculovirus expression system.

An active form of rat dipeptidyl aminopeptidase I (DPPI, cathepsin C) was obtained by heterologous expression in insect cells. Baculoviruses carrying a cDNA sequence encoding the entire rat DPPI precursor was used to infect High Five cells in a serum-free medium. Recombinant DPPI (rDPPI) was secreted into the medium from which it was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography (HIC), and ion-exchange chromatography. A polyhistidine-tagged form of the enzyme (HT-rDPPI) was purified from the medium by immobilized metal affinity chromatography (IMAC). In vivo activation of native rat DPPI involves at least three chain cleavages per subunit and the ability of the expression system to imitate this processing was investigated. Both rDPPI and HT-rDPPI were secreted into the medium as unprocessed and inactive proenzymes and gradually converted into their active forms in the medium. This process was not completed at the time of harvest but mature enzyme processed similarly to native rat and human DPPI could be obtained by incubating the eluates from the HIC and IMAC columns at pH 4.5 and 5 degrees C for 18-40 h. The yield of purified and matured enzyme was approximately 50 mg/liter, and it was shown that rDPPI and HT-rDPPI were active against both a dipeptide-p-nitroanilide substrate and human growth hormone N-terminally extended with an Ala-Glu dipeptide.

Animals↗
PubMed1998

Changing bone marrow micro-environment during development of acute myeloid leukaemia in rats.

The Brown Norwegian rat transplanted with promyelocytic leukaemic cells (BNML) has been used as a model for human acute myeloid leukaemia. We have previously shown that both the blood supply to the bone marrow and the metabolic rate decrease in relation to the leukaemic development in these rats. Here we have investigated how the development and progression of this leukaemia affect oxygenation, pH and proliferation of normal and leukaemic cells in vivo. Bone marrow pH was measured by a needle electrode. Nitroimidazol-theophylline (NITP) was used to identify hypoxic cells, and we applied bromodeoxyuridine (BrdUrd) to identify DNA replicating cells. The leukaemia progressed slowly until day 27 after which a rapid deterioration could be observed leading to severe changes over the following 5 d. In whole blood there was evidence of progressing metabolic acidosis. In bone marrow the fraction of leukaemic cells increased to > 90% and the pH dropped to about 6.5. The fraction of NITP+ cells increased to > 80% in bone marrow and to about 40% in blood. The fraction of BrdUrd+ cells was unchanged in blood, but decreased in bone marrow both for normal cells (from about 20% to 5%), and for leukaemic cells (from about 45% to 25%), evidently as a result of the severely changed microenvironment. In this study we have demonstrated in vivo the development of an acidic and hypoxic bone marrow hampering normal haemopoiesis during leukaemic growth. Our data support the notion of BNML as a valuable tool for studying leukaemogenesis.

Acute Disease↗
PubMed1997

A method for quantifying SPECT uniformity.

Field uniformity is an important parameter for monitoring the performance of SPECT imaging systems. However, it is difficult to apply objective measures of uniformity because of the large variance associated with reconstructed images. In the proposed method, annular sampling of the SPECT uniformity image is used to reduce the noise level without decreasing the magnitude of uniformity artifacts. The reconstructed uniformity image is sectioned into annular rings centered on the center of rotation to match the expected distribution of uniformity artifacts. Statistical fluctuations are reduced by averaging the counts within the annular rings, allowing the use of objective measures of field uniformity such as integral uniformity. Application of the annular sampling technique on simulated and phantom uniformity images showed that the technique could reliably quantify SPECT uniformity artifacts at acceptable count levels. As a result this method can be used to objectively evaluate SPECT field uniformity in systems which utilize parallel collimation and circular orbits.

Biometry↗
PubMed1996

The primary structure and enzymic properties of porcine prochymosin and chymosin.

Preliminary investigations by N-terminal sequence analysis showed that pig and calf chymosin possessed 80% amino acid sequence identity but showed considerable differences in their enzymatic properties. A comparison of their structures may therefore contribute to an understanding of the significance of the amino acid residues responsible for the differences in these properties. Pig chymosis was extracted from the stomachs of pigs of less than 3 weeks of age, and was purified by ion exchange chromatography. Half of the primary structure was determined by amino acid sequencing and the complete structure was deduced from a cloned chymosin cDNA. Results showed that the zymogen showed 81% sequence identity with calf prochymosin and 57% identity with pig pepsinogen A. The size of the propart and location of the residue which becomes the N-terminus in the active molecule were the same in the prochymosins. The maximum general proteolytic activity at pH 3.5 of pig chymosin was 2-3% of that of the activity of pig pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of pig chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.3 of stomach extracts of individual pigs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or N-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and pig chymosin differ in the following positions (pig pepsin numbering, subsites in parentheses): Ser 12 Thr (S4), Leu 30 Val (S1/S3), His 74 Gln (S'2), Val 111 Ile (S1/S3), Lys 220 Met (S4). With regard to the low general proteolytic activity of pig chymosin, the substitution Asp 303 Val relative to calf chymosin may contribute to an explanation of this.

Amino Acid Sequence↗
PubMed1995

Blocking of tissue factor pathway inhibitor (TFPI) shortens the bleeding time in rabbits with antibody induced haemophilia A.

Tissue factor (TF)/FVIIa initiates coagulation by activating factor IX (FIX) and factor X (FX). Tissue factor pathway inhibitor (TFPI)-FXa complexes form and inhibit TF/FVIIa. Blocking of TFPI may facilitate haemostasis initiated by FVIIa/TF thereby compensating for impaired FIX/FVIII-dependent coagulation. This hypothesis was tested in a study using rabbits made temporarily haemophilic by the injection of antibodies against FVIII. These rabbits were given i.v. injections of anti-TFPI IgG antibodies and 40 min later bleeding was initiated by cutting the nail including the apex of the cuticle. Injection of anti-TFPI IgG shortened the bleeding time significantly from 26 min to 11 min (normal mean bleeding time in non-haemophilia rabbits: 5 min). Treatment with anti-TFPI IgG also resulted in a shortening of the haemophilic aPTT to a level slightly longer than the normal aPTT. In addition, the prolonged dilute TF clotting time was shortened by the anti-TFPI IgG treatment. Thus, both bleeding and coagulation parameters indicated that blocking of TFPI may be potentially haemostatic in haemophilia.

Animals↗
PubMed1995

Quantitation of SPECT performance: Report of Task Group 4, Nuclear Medicine Committee.

A comprehensive performance testing program is an essential ingredient of high-quality single-photon emission computed tomography (SPECT). Many of the procedures previously published are complicated, time consuming, or require a special testing environment. This Task Group developed a protocol for evaluating SPECT imaging systems that was simple, practical, required minimal test equipment, and could be performed in a few hours using processing software available on all nuclear medicine computers. It was designed to test rotational stability of uniformity and sensitivity, tomographic spatial resolution, uniformity and contrast, and the accuracy of attenuation correction. It can be performed in less than three hours and requires only a Co-57 flood source, a line source, and a tomographic cylindrical phantom. The protocol was used 51 times on 42 different cameras (seven vendors) by four different individuals. The results were used to establish acceptable ranges for the measured parameters. The variation between vendors was relatively small and appeared to reflect slight differences in basic camera performance, collimation, and reconstruction software. Individuals can use the tabulated values to evaluate the performance of individual systems.

Cobalt Radioisotopes↗
PubMed1995

The AAPM/RSNA physics tutorial for residents. Introduction to emission CT.

The article explores the fundamentals of emission computed tomography (CT) from a nonmathematical approach. Tomographic images reveal the internal distributions of radioactivity in three-dimensional objects, and thus allows anatomic localization and improves contrast. Tomography requires a stable distribution of radionuclides, uniform detector response, an accurate center of rotation, and a complete set of projections. In emission CT, a large number of measurements, called projections, are collected at various angles about the patient during the examination. This information is organized by the angles of acquisition into a stack, called a sinogram. Each projection is modified by applying a reconstruction filter (eg, ramp or windowed reconstruction filters). These modified projections are backprojected to form the transverse tomographic images. The quality of tomographic images generated from filtered backprojection depends on the underlying assumptions about the projections. Typical artifacts that result from violations of these assumptions include motion, uniformity, and attenuation artifacts. In addition, an inaccurate center of rotation, insufficient angular sampling, and errors in selection of pixel size can result in poor-quality reconstructed images.

Artifacts↗
PubMed1995

Cranial osteomyelitis: diagnosis and follow-up with In-111 white blood cell and Tc-99m methylene diphosphonate bone SPECT, CT, and MR imaging.

PURPOSE: To assess the usefulness of indium-111 white blood cell and technetium-99m methylene diphosphonate bone single photon emission computed tomography (SPECT), computed tomography (CT), and magnetic resonance (MR) imaging in cranial osteomyelitis. MATERIALS AND METHODS: Twenty-six cases (25 patients: 13 male, 12 female; mean age, 55 years) of suspected osteomyelitis were evaluated. Sixteen were postoperative. Final diagnosis was established by means of bone culture in 18 cases and clinical follow-up in eight. RESULTS: Of 35 CT scans, 10 were true-positive (TP); three false-negative (FN); 13, true-negative (TN); one, false-positive (FP); and eight, equivocal. Of 36 SPECT scans, 19 were TP; 13, TN; one, FP; one, FN; and two, equivocal. Of 11 MR images four were TP; five, TN; and two, FN. CONCLUSION: CT is best for differentiation between soft-tissue and bone infection. MR imaging is best for assessment of the calvaria and skull base. SPECT is best for assessment of altered bone and may be the best technique for follow-up.

Adolescent↗
PubMed1995

Scintigraphic evaluation of small bowel transit in healthy subjects: inter- and intrasubject variability.

OBJECTIVES: This study was performed to evaluate inter- and intrasubject variability of small bowel transit time and the effect of age and sex on transit in the small bowel. METHODS: Ten young subjects (five females) and nine elderly subjects (four females) were studied. Small bowel transit time was assessed by scintigraphic method using 99mtechnetium-sulfur colloid-labeled eggs. To evaluate intrasubject variability, 10 subjects underwent a second study on a later occasion. RESULTS: Mean small bowel transit time was 220.9 +/- 49 min with a range of 131 to 322 min, with no significant difference between the young and elderly groups or between sexes. There was a good intraobserver and interobserver reproducibility within the same test (r = 0.92 and r = 0.85, respectively). There was a significant biological variability between the first and the second studies, which was true whether the studies were processed independently (r = 0.43) or whether identical regions of interest were applied (r = 0.50). CONCLUSION: The normal range of small bowel transit time is wide in normal subjects; and there is a significant intrasubject biological variability for small bowel transit time. These findings should be kept in mind when using scintigraphic techniques for assessment of patients with motility disorders or when using the test repeatedly in the same subject in the evaluation of therapeutic measures. The results do not seem to be affected by age or sex.

Adult↗
PubMed1994

Positron emission tomographic measurement of bone marrow blood flow to the pelvis and lumbar vertebrae in young normal adults.

Ten young normal adults had pelvic and lumbar vertebral body bone marrow blood flow examined using [15O]water and positron emission tomography (PET) in a study designed to assess the feasibility and reproducibility of the PET technique for measuring marrow blood flow to various marrow regions. The procedure was well tolerated. Repeated blood flow measurements obtained from two consecutive [15O]water exams on each individual subject were highly reproducible. In addition, there was minimal variation in marrow blood flow from individual to individual and no gender differences were noted. In contrast, mean +/- SD bone marrow blood flows (expressed as milliliters per minute per 100 g) at selected anatomical sites were significantly different and were as follows: lower lumbar vertebral bodies, 17.6 +/- 3.1; most posterior and superior pelvis (conventional site of percutaneous bone marrow biopsy), 14.3 +/- 3.1; and total superior pelvis, 11.1 +/- 2.0. We conclude that PET is a relatively noninvasive, simple, and reproducible technique for measuring bone marrow blood flow. Marrow blood flow is consistent between normal young subjects, but varies significantly between different anatomic regions of the marrow.

Adult↗
PubMed1994

The safety and pharmacokinetics in adult subjects of an intravenously administered 99mTc-labeled 17 amino acid peptide (CYT-379).

A phase I study was designed to evaluate the safety and pharmacokinetics of a novel platelet reactive peptide, peptide acetyl-SYGRGDVRGDFKCTCCA-amide (CYT-379), which binds to the fibrinogen receptor of activated platelets and also binds to 99mTc. Eleven subjects with suspected deep venous thrombosis had 0.1, 0.5 or 1.0 mg of the peptide infused intravenously. Pharmacokinetics were determined by assaying blood samples in 6 of the 11 subjects and by urine sampling in 5 of these 6 subjects. Plasma and whole blood time-activity curves demonstrated an initial fast component with half-time clearance of 0.2 +/- 0.01 and 0.2 +/- 0.02 h and a slow component with half-time clearance of 2.8 +/- 0.3 and 2.7 +/- 0.2 h (mean +/- SEM for plasma and whole blood, respectively). Urine clearance was 22.6 +/- 3.3 and 10.8 +/- 1.6 mL/min when normalized to body surface area. The cumulative excretion of 99mTc-CYT-379 in the urine was 16.6 +/- 3.6, 45.6 +/- 16.9 and 45.6 +/- 1.8% of the administered dose over 0-2, 0-12 and 0-24 h after radiopharmaceutical injection, respectively. Images obtained in 11 subjects immediately, at 1-2, and 4-6 h after injection were evaluated for abnormalities and were compared with duplex Doppler ultrasonography. 99mTc-CYT-379 images were positive in only 3 of 7 subjects who had a positive duplex Doppler examination in at least one lower extremity. One subject with negative duplex Doppler had also negative 99mTc-CYT-379 scintigraphy. One subject with negative scintigraphy and two other subjects with positive scintigraphy had no other imaging studies of the deep venous system performed. No adverse reactions were observed during or after the infusion of 99mTc-CYT-379. 99mTc-CYT-379 appears to be a safe radiopharmaceutical and demonstrates rapid clearance from plasma in human subjects.

Adult↗
PubMed1994

Captopril-enhanced 99Tcm-MAG3 renal scintigraphy in subjects with suspected renovascular hypertension.

This pilot study was undertaken to generate preliminary data on the accuracy of captopril-enhanced renal scintigraphy with a relatively new radiopharmaceutical, 99Tcm-mercaptoacetyltriglycine (99Tcm-MAG3) for detecting significant renal artery stenosis. Truth data was based either on arteriographic or outcome criteria (blood pressure response to therapy). Twenty-seven subjects with suspected renovascular hypertension were studied with baseline and captopril-enhanced 99Tcm-MAG3 renal scintigraphy and renal arteriography. Scan interpretations were expressed as a probability of a significant renal artery stenosis. Scan interpretations were compared with renal arteriographic results, renal vein renin levels, blood pressure values after renal artery repair, and blood pressure control after 4-26 months of clinical follow-up. Using > or = 50% luminal obstruction on arteriography as the reference standard for renal artery stenosis and a high probability scan representing a positive test, the test sensitivity and specificity were 33 and 97%, respectively (using high or indeterminate probability to represent a positive scan, the test sensitivity and specificity were 67 and 83%, respectively). The negative predictive value of a low probability scan for renal artery stenosis was 80%. However, including a measure of renovascular hypertension (blood pressure response to renal artery repair) as the reference standard, the accuracy of the scan improves, with the negative predictive value of a low probability scan for renovascular hypertension increasing to 97%. Scintigraphic results were also positively correlated with renal vein renin values in a statistically significant fashion (two-tailed Fisher exact test statistic = 6.43, P = 0.0219). Captopril-enhanced 99Tcm-MAG3 renal scintigraphy is a moderately accurate technique for detecting renal artery stenosis. More importantly, our preliminary findings suggest that the scintigraphic technique using 99Tcm-MAG3 appears to predict the blood pressure response to renal artery repair in subjects with suspected renovascular hypertension, thereby separating subjects with haemodynamically insignificant renal artery stenosis from those with renovascular hypertension.

Angiography↗
PubMed1993

Dual isotope brain SPECT imaging for monitoring cognitive activation: physical considerations.

The physical considerations of using dual isotope brain SPECT imaging to monitor blood flow changes during cognitive activation studies were investigated. These factors included field uniformity, spatial resolution and crosstalk. Serial dual isotope single photon emission computed tomographic (SPECT) studies of a test tube phantom and an anthropomorphic brain phantom filled with 99Tcm and 123I were made over a 10 h period. The reconstructed counts in the 99Tcm and 123I windows were corrected for crosstalk and were plotted as a function of time. The plotted data from each window decreased over time with a half-life characteristic of each radionuclide. The relative difference between true 123I and 99Tcm region counts has to be of the order of 10% to be statistically significant at the P < 0.05 level.

Brain↗
PubMed1993

Dual isotope brain SPECT imaging for monitoring cognitive activation: initial studies in humans.

A dual isotope, single photon emission tomography (SPECT) technique using 99Tcm-hexamethylpropyleneamine oxime (HMPAO) and 123I-iodoamphetamine (IMP) was investigated to determine its suitability for assessing regional cerebral blood flow (rCBF) changes resulting from cognitive activation. The similarity of the 123I-IMP and 99Tcm-HMPAO distributions under the same physiological conditions was first investigated by administering the two agents to human subjects (n = 8) either simultaneously or at different times but during the performance of the same task. Normalized ratio images generated from the 99Tcm and 123I data showed that the two tracers distributed similarly in the left and right cerebral hemispheres when administered under similar physiological conditions. There was, however, a significant anterior/posterior gradient that appears to be the result of partial volume effects due to small differences in spatial resolution of the two agents. In two subjects, 99Tcm-HMPAO was administered during a resting period with eyes-closed and 123I-IMP was injected later during visual checkerboard stimulation. Ratio images showed a localized increase in the occipital lobes during the visual stimulation consistent with the expected increase in rCBF. The dual isotope strategy appears promising for study of changes in rCBF due to cognitive activation.

Adult↗
PubMed1993

An emission-based technique for obtaining attenuation correction data for myocardial SPECT studies.

In this paper, a technique is described for obtaining the information needed to perform attenuation correction in the thorax entirely from an emission study. This technique is based on the observation that the variation in soft tissue and lung attenuation coefficients is small among individuals. Thus only the outer contours of the body and lungs need be determined for obtaining the attenuation map. The contours are determined by using 99Tcm-macroaggregated albumin (MAA) to locate the lungs and an external source wrapped about the chest to locate the body boundary. Simulation studies were performed to investigate how errors in the presumed tissue attenuation coefficients affect the accuracy of the correction. Body and lung attenuation coefficients were varied from 20% less to 20% more than the coefficients used in the corrections. Over this range, there was less than a 15% alteration in the relative distribution of counts in the left ventricle. To test clinical feasibility, seven patients referred for clinical myocardial perfusion studies were scanned before and after the placement of the body source and the administration of 99Tcm-MAA. Reconstructed images from these studies showed clear demarcation of all body and lung boundaries. The presence of 99Tcm-MAA in the lungs had no significant effect on the clinical interpretation of the single photon emission computed tomographic (SPECT) studies. It is concluded that this technique is feasible for clinical application and that it offers important advantages over other current methods.

Adult↗