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Biomedical subjects

J Carlsson

Publications and source records attributed to J Carlsson.

377 records · Page 21Linked to original sources
PubMedDate not supplied

Development and in vitro studies of epidermal growth factor-dextran conjugates for boron neutron capture therapy.

A delivery molecule for directed boron neutron capture therapy against epidermal growth factor (EGF) receptor-rich tumors, such as gliomas, squamous carcinomas, and breast cancers, is presented. EGF and sulfhydryl boron hydride (BSH) were covalently coupled to an allylated 70 kDa dextran chain to form a conjugate. Conjugates with low and high substitution rates of BSH, as well as without BSH, were investigated. The conjugate with a low amount of boron had approximately 6 BSH (72 boron atoms) per dextran, while the conjugates with higher amounts had an average substitution of 55 BSH (660 boron atoms) per dextran. The maximum substitution of boron to dextran in a single experiment was over 800 boron atoms. Binding, retention, and internalization of 125I-labeled conjugates were investigated on cultured human glioma cells. Binding of the conjugates was EGF receptor specific, but the amount of BSH coupled to dextran affected specificity, more than the presence of dextran. The nonspecific binding of the conjugates increased with the amount of attached boron. This was partly due to nonspecific adhesion to the plastic in the culture dishes. [125I]EGF-allyldextran with 6 BSH had a binding maximum after 4 h of continuous incubation and thereafter decreased in binding, while [125I]EGF-allyldextran with the higher substitution rate had a slow increase of binding during 24 h. Over 93% of the radioactivity bound to the cells was internalized, but the retention was quite poor. Only one-third of the cell-bound activity was still associated to the cells 4 h after incubation had ended. In conclusion, it is possible to load the conjugates produced with high amounts of boron, and they retained specificity for the EGF receptor and internalized into cultured cells. Theoretical calculations show that about 10(3) boron atoms per EGF-based conjugate are needed to give a satisfactory therapeutic response. These conjugates are within reach of that level.

Boron Neutron Capture Therapy↗
PubMedDate not supplied

Chemical modification of surface active poly(ethylene oxide)-poly (propylene oxide) triblock copolymers.

A general route has been developed to chemically modify a series of poly(ethylene oxide)-poly(propylene oxide) triblock copolymers with molecular weights from 6500 to 14600. It is initiated by the introduction of p-nitrophenyl groups; such nitrophenyl conjugated copolymers are stable in an organic milieu and in a dry state but are seen to react easily with amino-containing molecules including small peptides. Among them, introduction of 2-pyridyl disulfide groups after coupling with 2-(2-pyridyldithio)ethylamine enables the selective attachment of thiol-containing molecules. The released thiopyridone in such thiol-disulfide reactions can be used to quantify the content of 2-pyridyl disulfide groups. In addition, a new type of modified copolymers was developed for the radioisotope (125I) labeling purpose that consists of a reaction of nitrophenyl conjugated copolymers with hydrazine and a subsequent coupling with N-succinimidyl 3-(4-hydroxyphenyl)propionate (Bolton-Hunter reagent). Adsorption studies of 125I-labeled and 2-pyridyl disulfide conjugated copolymers on polystyrene particles are consistent with previous determinations of surface coverage using other technologies, in turn indicating that this new chemical modification does not alter their surfactant properties on hydrophobic solid phase. The coating of common hydrophobic surfaces with 2-pyridyl disulfide conjugated copolymers has been demonstrated as a general and robust immobilization method to generate a high-sensitivity bioactive surface with low nonspecific binding. The optimal space between immobilized ligands can also be controlled by incubating the solid phase with solutions containing mixtures with different ratios of unmodified and modified copolymers.

Chemical Phenomena↗
PubMedDate not supplied

Preparation and purification of an end to end coupled mEGF-dextran conjugate.

The amino terminus of mouse epidermal growth factor (mEGF) was coupled directly to the aldehyde end of dextran through a reductive amination procedure. The highest coupling efficiency was approximately 80% and could be reached after approximately 24 h of reaction time at pH 8. Gel filtration on Sephadex G-50 Fine removed free mEGF from the conjugate. Preparative polyacrylamide gel electrophoresis was used to separate the conjugate from excess noncharged dextran. The conjugate bound specifically to the EGF receptor on cultured glioma cells as shown in displacement tests with free mEGF. The conjugate was stable in the pH interval 4-9, in 2 M sodium chloride, in 7 M urea, and in human serum and could still bind to the EGF receptor after such treatments. The conjugates are candidates for targeted nuclide therapy.

Amination↗
PubMedDate not supplied

Closo-dodecaborate(2-) as a linker for iodination of macromolecules. Aspects on conjugation chemistry and biodistribution.

Boron-containing compounds like closo-dodecaborate(2-) are in theory suitable for radioactive labeling with halogens. The boron-halogen bond is stronger than carbon-halogen bond and is not likely to be recognized by deiodinating enzymes in vivo. Peptides and proteins may be conjugated with various closo-dodecaborate(2-)-containing ligands, and thereafter, the conjugate can be iodinated. Since closo-dodecaborate(2-) is more avidly iodinated than tyrosine in moderately acidic media, such conjugates may be directly labeled on the boron part with radioisotopes of iodine using the standard Chloramine-T procedure. Mercapto-undecahydro-closo-dodecaborate(2-) (BSH) was reacted with the double bond of allyldextran to form a boronated dextran compound of the molecular size of about 70 kDa. This compound, in the text denoted as Dx-BS, and cesium dodecahydro-closo-dodecaborate(2-) were labeled using iodine-125. The two compounds were administered to rats in order to study their in vivo stability. The results indicate that iodinated Dx-BS is stable for about 20 h in vivo. The degradation rate, as indicated by thyroid uptake, was found low. [125I]Iodo-closo-dodecaborate(2-), which is a possible degradation product of [125I]Dx-BS-I, was rapidly excreted in urine without significant accumulation in any organ.

Animals↗
PubMedDate not supplied

Effects of dextranation on the pharmacokinetics of short peptides. A PET study on mEGF.

The effects of dextranation on the biodistribution of mouse epidermal growth factor (mEGF, 6 kDa) were assessed. By reductive amination, mEGF was coupled to 13 and 46 kDa dextran. The two dextranated conjugates and free mEGF were labeled with the positron-emitting nuclide (76)Br (T(1/2) = 16 h). After intravenous administration to Sprague Dawley rats, the radioactivity biodistribution was evaluated by positron emission tomography (PET) and by measurements of dissected tissues. The dextranation prolonged the retention time in blood, especially when the dextran chain was long. [(76)Br]mEGF-dextran conjugates were shown to have significantly, more than 5 times, lower kidney accumulation than the nonconjugated [(76)Br]mEGF. In conclusion, dextranation affects the biodistribution of mEGF in vivo giving a prolonged circulation time, a decreased uptake in kidney, and an increased spleen accumulation.

Animals↗
PubMedDate not supplied

Effects of dextranation on the uptake of peptides in micrometastases: studies on binding of EGF in tumor spheroids.

Four different types of radiolabelled dextranated EGF were added to spheroids consisting of the human glioma cells U-343MGaCl2:6. Binding was analysed both in the peripheral well-nourished regions and in the deeper regions containing mainly quiescent cells. The substances had different molecular weights, and they were characterized regarding hydrophilic properties and isoelectric points. Two of the analysed conjugates, 125I-EGF-dextran (CDAP) and 125I-EGF-allyldextran-BSH, gave very low 125I binding in the deeper regions even after 24 h of incubation while better binding in these regions was found for 125I-EGF-dextran-DTPA, 125I-EGF-allyldextran and for the reference substance 125I-EGF. The molecular weight seemed not to be of major importance for the binding properties and there were no clear relationships between binding and the hydrophilic properties or the isoelectric point values. The obtained differences could not be explained by differences in molecular weight or easily measured physicochemical parameters such as hydrophilic properties or isoelectric point values. Thus, other explanations must be found.

Biological Transport↗
PubMedDate not supplied

Oculomotor disturbances in patients with tension headache.

Forty-seven female patients with tension headache were studied with oculomotor tests. The test results were compared with those of a control group consisting of 38 healthy women. The peak velocity of the saccades was lower in the patient group than in the control group (p greater than 0.01). There were no differences between the patient group and the control group regarding accuracy and latency of the saccades. The velocity gain values of the smooth pursuit eye movements were lower in the patient group than in the control group (p greater than 0.01). The differences in both the number and the amplitude of the corrective saccades between the two groups were significant (p greater than 0.01). The number of square waves superimposed on the smooth pursuits was the same in both groups.

Adult↗
PubMedDate not supplied

Heterogeneity in cell loss and frequency of slow growing colonies of human glioma cell lines. Some effects of radiation.

Changes in cell loss and in the relative number of slowly growing colonies were analysed after irradiation of five human glioma cell lines using a micro-colony assay. The cells were seeded at low density on small palladium covered islands which were separated by agarose barriers. The cell density at seeding was chosen to give, on the average, one cell per island. The size of each island allowed growth to a maximum number of 10-15 cells before confluency was reached. Each palladium island was individually identified in a coordinate system and cell detachment was prevented by a thin overlay of agarose. The number of fast growing colonies was, after irradiation, similar for all the cell lines studied. Large variations were, however, seen in radiation-induced cell loss and in the relative number of slowly growing colonies. Cell lines with a high cell loss tended to produce few slowly growing colonies. The variations in cell loss and number of slowly growing colonies strongly influenced the overall growth of the cell populations and there was no correlation between the decrease in the number of fast growing colonies and the decrease in the overall growth rate directly after irradiation. Further studies of tumour cell heterogeneity, with the micro-colony assay, could perhaps reveal at least some reasons for the varying response often seen between tumours when exposed to therapeutical agents.

Cell Division↗
PubMedDate not supplied

Effects of epidermal growth factor receptor blocking in cultured glioma spheroids.

A concentration as high as 1 microgram/ml of non-radioactive epidermal growth factor, EGF/was necessary to inhibit effectively the binding of 125I-EGF in glioma U-343MGaC12:6 cells. This concentration blocked the available EGF receptors within 30 minutes in monolayers, while 24 hour treatments were required in spheroids. The effects on growth, incorporation of radioactive thymidine, cell density and on extracellular pH were analysed in spheroids after exposure to 1 microgram/ml EGF. The high EGF concentration did not significantly modify the growth curves for monolayers and small spheroids but increased the volume growth of large spheroids. The increase was partly due to lower cell density and partly to increased proliferation. The EGF treatment gave an increased incorporation of thymidine in spheroids, for at least up to 5 days after the administration, while no effect was seen in monolayers. The cell density decreased after the EGF treatment as seen from morphometric analysis in histological sections and by counting the number of cells per volume unit after trypsinization. The capacity to take up radiolabelled dextran increased, probably due to the decreased cell density. Other EGF-induced changes were also recognized, such as a reduction in extracellular pH by 0.1 units in the central regions of spheroids and an increase in intracellular pH by 0.47 units in analysed monolayer cells. The results showed that it is not possible to block the EGF-receptors without imposing changes in growth and metabolism.

Cell Count↗
PubMedDate not supplied

Cell type dependent effectiveness of tumour cell inactivation by radiation with increased ionisation density.

Radiation with increased ionization density, LET, will in the near future be applied in targeted radiotherapy and has already, to some degree, been applied in external radiotherapy with accelerated ions. There are indications from the literature that different types of cells are sensitized differently when the LET is increased. Radiation with three different ionisation densities was applied in the present study; 0.8 keV/microns photons from a reference 60Co source, 6 keV/microns helium ions from the entrance region of a monoenergetic helium ion beam and 25 keV/microns from a range modulated helium beam. Three types of cultured cells were analysed: human colon carcinoma LS-174T, human glioma U-343MG and hamster embryonic lung V79-379A. There were interesting differences between the cell types; the LS-174T cells showed strong sensitization already at an LET of 6 keV/microns and there was nearly no difference between the shape of the survival curves after irradiation with 6 or 25 keV/microns. The V79-379A cells were less sensitive to the 6 keV/microns radiation quality and the survival curve was, in this case, very similar to the reference 60Co survival curve. However, the V79-379A cells were sensitized when exposed to the 25 keV/microns helium ions. The U-343MG cells were intermediate in the sense that the 6 keV/microns curve fell in between the 60Co and the 25 keV/microns curves. These variations indicate that different types of cells react differently to changes in LET and that this is probably of special interest for intermediate LET radiation. Some cells might be easily sensitized by intermediate LET qualities while others might be more resistant and this is of importance for the future optimization of radiation treatment with both targeted agents and accelerated ions.

Alpha Particles↗
PubMedDate not supplied

Leukotriene and 5-lipoxygenase inhibitor induced variations in thymidine uptake in a human glioma cell line cultured as monolayers or as multicellular spheroids.

Effects of exogenous leukotrienes and/or treatment with the 5-lipoxygenase inhibitor, AA-861, on the 3H-thymidine uptake were investigated in a human glioma cell line, U-343MGa, growing as monolayers or multicellular spheroids. The spheroids contained about four times more endogenous leukotrienes than the monolayers. Administration of 0.1 microM exogenous leukotriene D4 increased the 3H-thymidine uptake in the spheroids while it gave a decrease in the monolayers. Inhibition of 3H-thymidine uptake was induced in the spheroids by 10 microM AA-861 and this inhibition was only seen during the period at which central necrosis develops in the spheroids. This drug showed an inhibitory effect on monolayers one day after subculture but gave no measurable effect on the monolayers two days later. It seemed that AA-861 exerted an inhibitory action at culture conditions associated with "cellular stress" such as subculture (trypsinization) of monolayers and induction of necrosis in spheroids. Induction of "cellular stress" with heat or exposure to a Ca(2+)-ionophore also gave an inhibitory action of AA-861 and the inhibition could be counteracted by administration of exogenous B4 leukotrienes. The observed effects are probably related to the activation of the arachidonic acid cascade and indicate that leukotrienes in some way interact with "cellular stress" and induce acute changes in 3H-thymidine uptake. Further research is necessary to reveal the detailed molecular mechanisms.

Antineoplastic Agents↗
PubMedDate not supplied

Growth of prostatic cancer cells, DU 145, as multicellular spheroids and effects of estramustine.

The human prostatic carcinoma cell line DU 145 was grown as multicellular spheroids in vitro. The volume doubling time during the early exponential growth phase was about 5 days. The saturation volume, in the plateau phase of the growth curve, was in the order of 1.4 mm3. The spheroids developed a central degenerative region surrounded by a 0.1-0.3 mm layer of viable cells. The DU 145 spheroid system is planned to be used as a model in studies on chemotherapy and targeted radiotherapy of micrometastases of prostatic cancer. Some effects of the drug estramustine, EM, a conjugate of estradiol and nornitrogen mustard, were analysed in this introductory study. Tritium-labelled estramustine, 3H-EM, bound both in the viable cell layers and in the degenerative region of the spheroid already after 1 hour of incubation which indicated good penetration. The viable cells bound only low levels of 3H-EM while the degenerative region bound 3H-EM to a higher extent. The amount of bound 3H-EM increased after incubation for 24 hours. The binding was nonspecific since it could not be inhibited by pretreatment with an excess of non-radioactive EM. Furthermore, 3H-EM bound to a similar extent in glioma and colon carcinoma spheroids used for comparison. Incubation of DU 145 spheroids for 24 hours with EM (20 mg/ml) induced a growth delay of 6-7 days and a transient increase in the volume of the extracellular spaces for a few days following the treatment. The results showed that the binding of EM to prostate DU 145 cells growing as spheroids was not specific and that the toxic action was limited. An interesting result was that EM works as an extracellular space expander. This might be exploited in combination treatments with other agents.

Autoradiography↗
PubMedDate not supplied

The non-variation in radiosensitivity of different proliferative states of human glioma cells.

The radiosensitivity of the human glioma cell line U-343MGa, while growing as spheroids and as conventional monolayers, was studied. The spheroids were first irradiated with 60Co photons, and the radiosensitivity was then analyzed in different cell layers with varying proliferative activity. The different cell layers were isolated by automated sequential trypsinization. A relatively high radioresistance was found in all analysed cell layers, and the inner, mainly quiescent cells, were as resistant as the outer proliferating cells. It was, in parallel experiments on monolayers, shown that serum starved quiescent cells were as resistant as proliferating cells, and that direct cellular cloning did not give lower cell survival values than delayed cloning, as has been shown for other cell types. Thus, the studied glioma cells were equally radioresistant under all tested proliferative conditions when conventional low-LET radiation was applied. The spheroids were then irradiated with cyclotron accelerated helium ions with an intermediate ionization density, LET approximately equal to 55 keV/microns, and this gave a clear decrease in survival in all cell layers.

Calibration↗
PubMedDate not supplied

Adjuvant 131I-anti-CEA-antibody radioimmunotherapy inhibits the development of experimental colonic carcinoma liver metastases.

Adjuvant radioimmunotherapy (RIT) for human colonic cancer was performed in a nude rat model of experimental liver metastases. Thirty-three rats were injected intraportally through a mesenteric vein with 5 x 10(6) cells from the human colonic cancer cell line LS174T. Within half an hour, 20 MBq (n = 2), 75 MBq (n = 5), or 150 MBq (n = 10) of the 131I-labelled anti- carcinoembryonic antigen (CEA) monoclonal antibody (MAb) 38S1 was administered intravenously (i.v.), whereas control groups received either i.v. saline injections (n = 12) or 150 MBq of the irrelevant 131I-labelled MAb 79C (n = 4). Decay corrected whole-body data showed that more than 80% of the initially MAb-bound radioiodine was excreted during the first 2 weeks. Whole- body clearance and blood clearance of 131I-38S1 and 131I-79C were essentially similar. At sacrifice 5-7 weeks after administration, neither 20 MBq nor 75MBq 131I-38S1 significantly prevented the development of liver metastases. By contrast, with 150 MBq, no metastases formed in the animals treated with MAb 131I-38S1 or 131I-79C. A radiation induced effect on the haematopoietic system was found in the 150MBq dosage groups. It is concluded that the inhibition of tumour induction was not strictly dependent on a radiation dose delivered by a tumour-specific MAb. Since a non-tumour-specific 131I-MAb, in a smaller group of animals, proved equally efficacious in preventing tumour growth, the total body 131I dose was probably the major contributing factor.

Animals↗