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Biomedical subjects

R M Sutherland

Publications and source records attributed to R M Sutherland.

At least 145 records · Page 8Linked to original sources
PubMed1983

Response of multicell spheroids to 1-MHz ultrasonic irradiation: cavitation-related damage.

The response of EMT6/Ro single cells and multicell spheroids (150-800 microns diameter) to 1-MHz ultrasonic irradiation was determined for intensities of 0-5 W/cm2 for 1-5 min. Damage was assessed by evaluating both lysis and survival of individual spheroid cells, growth of whole spheroids, and the physical integrity of whole spheroids by histological and scanning electron microscopic techniques. The surviving fraction of spheroid cells was higher than that of single cells, and several of the intensity-duration combinations showed a statistically significant correlation between resistance to ultrasound and increased spheroid size. This resistance with size was also found in spheroid growth experiments. Histological sections showed that both peripheral and internal damage occur. The relationship of spheroid damage to studies of effects on tissues and possible therapeutic use of nonthermal ultrasound are discussed.

Animals↗
PubMed1982

Immunoassay of blood spot TSH; development of a rapid two-site immunoradiometric assay and comparison with radioimmunoassay as a screening method for neonatal hypothyroidism.

The development of a two-site immunoradiometric assay (IRMA) for thyrotropin (TSH) eluted from dried blood filter paper discs is described and compared with a conventional TSH radioimmunoassay (RIA) as a screening procedure for neonatal hypothyroidism. The iodinated antibody for the IRMA is prepared by a reproducible four step procedure. Specific antibody is selected from whole TSH antiserum with TSH covalently linked to microparticulate cellulose, iodinated as the solid phase antigen-antibody complex, and eluted by repeated changes in pH from 3 to 10. The pooled eluted material is finally purified by Sepharose 6-B gel chromatography to recover labelled monomeric anti-TSH IgG. Solid phase antibody for the IRMA is prepared by covalently linking a partially purified IgG fraction of TSH antiserum to carbonyldiimidazole-activated microparticulate cellulose. Typically yields of 2-3 mg protein per 100 mg cellulose (2-3%, w/w) are obtained. The two-site IRMA involves a primary incubation of excess labelled TSH antibody and the blood disc for 16-18 h at pH 8 and a secondary 3 h incubation under agitation, with solid phase TSH antibody. Bound and free fractions are separated by a semi-automated washing procedure. Compared with the conventional RIA, the two-site IRMA is quicker (results available within 24 h compared to 3 days for RIA), more precise at clinically important TSH levels (25-30 mU TSH/l), and more sensitive (detection limit less than w mU/l compared to 6 mU/l for RIA). The two-site IRMA is technically simpler than RIA and has proved rugged in routine practice. TSH levels in filter paper blood spots prepared from whole blood with levels of standard TSH (MRC 68/38) within the range 6 to 200 mU/l, correlated well in both assays (r = 0.969). Recoveries of 98% and 104% were demonstrated for the two-site IRMA and RIA, respectively. It is concluded that the two-site TSH IRMA has advantages over conventional RIA in speed, sensitivity, precision and ruggedness and can be recommended as an efficient screening procedure for neonatal hypothyroidism.

Autoanalysis↗
PubMed1982

Nitrosourea-misonidazole combination chemotherapy: effect on KHT sarcomas, marrow stem cells and gut.

C3H/HeJ mice bearing i.m. transplanted KHT sarcomas were treated with varying doses of either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin; CHLZ) as single agents or in combination with 1 mg/g of the chemical radiosensitizer, misonidazole (MISO). Using an in vivo-in vitro tumour-excision assay, the administration of MISO simultaneously with or 3 h after low doses of BCNU (less than 20 mg/kg) was found to give a dose-modification factor (DMF) of approximately 1.65 relative to BCNU alone. At higher doses of BCNU, there was less enhancement of cell kill. The DMF for tumour growth delay was likewise dependent on BCNU dose, continuously decreasing with increasing BCNU dose. In contrast, the anti-tumour activity of CHLZ, assessed by both clonogenic cell survival and tumour-growth delay, was not significantly enhanced by the addition of MISO. The enhancement of gastrointestinal toxicity and haematotoxicity by BCNU-MISO combinations was assessed by LD50/7 and CFU-S assays, respectively. MISO enhanced BCNU marrow toxicity by a factor of 1.2-1.3, whilst gut toxicity was enhanced by a factor of approximately 1.2.

Animals↗
PubMed1982

Oxygen tensions in multicell spheroids of two cell lines.

O2 tensions (Po2) were measured with microelectrodes in multicellular spheroids from EMT6/Ro and V-79-171-B cells. The measurements were performed in spheroids kept in flowing growth medium that was equilibrated with 5% CO2 and air at a temperature of 37 degrees C and contained 5.5 mM glucose. The recorded Po2 profiles are characterized by a diffusion-depleted zone surrounding the spheroids and by a steep drop in Po2 within the spheroids over mean distance of 220 and 188 micrometer from the surface of EMT6/Ro and V-79-171B spheroids over mean distance of 220 and 188 micrometer from the surface of EMT6/Ro and V-79-171B spheroids respectively. Smaller spheroid exhibit parabolic Po2 profiles, larger ones show a central plateau. The region of the steep decrease in Po2 corresponds to the thickness of the viable rim: the plateau region is created by the absence of O2 consumption in the central necrotic area. Po2 in the centre of EMT6/Ro spheroids decreased from 66 mmHg at a diameter of 400 micrometer to 13 mmHg at a diameter of 1000 micrometer. Under the present conditions during growth and in the experiments, values below 5 mmHg were recorded only in spheroids 1200 micrometer. Comparably low Po2 was recorded in V-79 spheroids with diameters of 650 micrometer +. In spheroids of this cell type with a diameter of 400 micrometer, Po2 was 42 mmHg. The findings provide evidence that necrosis may arise at average Po2 of 57 and 42 mmHg in EMT6/Ro and V-79-171B spheroids, respectively, grown under the conditions described.

Cell Line↗
PubMed1982

A model for the growth of multicellular spheroids.

Based on biological observations and the basic physical properties of tri-dimensional structures, a mathematical expression is derived to relate the growth rate of multicellular spheroids to some easily measurable parameters. This model involves properties both of the individual cells and of the spheroid structure, such as the cell doubling time in monolayer, the rate of cell shedding from the spheroid and the depth of the external rim of cycling cells. The derived growth equation predicts a linear expansion of the spheroid diameter with time. The calculated growth rate for a number of spheroid cell types is in good agreement with experimental data. The model provides a simple and practical view of growth control in spheroids, and is further adapted to include parameters presumably responsible for the growth saturation in large spheroids.

Animals↗
PubMed1982

Isolation of quiescent cells from multicellular tumor spheroids using centrifugal elutriation.

A quiescent (nonproliferating) subpopulation was identified by flow cytometric analysis using two-step acridine orange staining in the EMT6/Rochester, N. Y. subline multicellular tumor spheroid, an in vitro culture system which provides a cellular microenvironment which mimics that of many of in vivo tumors. To isolate a viable quiescent cell subpopulation, centrifugal elutriation which allows for cell separation mainly on the basis of size was used. This technique provided single cells of relatively homogeneous cell volume which varied over a wide range (approximately 100 to 5000 cu microgram). Though the relatively small cell volume fractions were the most enriched (82%) in quiescent cells, such cells were also observed in significant numbers (congruent to 20%) even in the largest cell fractions. The cell clonogenicity of the various elutriation constant in fractions was also assessed and shown to be lowest (plating efficiency congruent to 20%) in the small spheroid cells but relatively constant in fractions containing intermediate and large cells (plating efficiency congruent to 50%). Continuous [3H]thymidine labeling indicated a slower rate of accumulation of labeled cells in the small spheroid cells, which may result from the transition of proliferating spheroid cells to the quiescent compartment during the course of labeling. These finding indicate the utility of centrifugal elutriation for quiescent cell characterization in in vitro tumor systems.

Animals↗
PubMed1981

Fibers in spinal nerves of tree frogs: Eleutherodactylus and Hyla.

This is a study on numbers and sizes of nerve fibers as determined by electron microscope mapping of the spinal nerves of two species (in different families) of free frogs. The study provides data for comparison of fiber populations in the grass frog, Rana pipiens. In both free frogs the greatest number of fibers in the ten pairs of spinal nerves are in the second and ninth rami. The combined counts of myelinated and unmyelinated fibers in the rami on one side of the cord in Eleutherodactylus and Hyla are respectively: to the forelimb, 3,147 and 4,460 fibers; to the trunk, 334 and 1,234 fibers; and to the hindlimb, 4,335 and 6,213 fibers. With a few notable differences this distribution of fibers to regional body parts correlates closely with that found in Rana. The arboreal frogs have a relatively smaller number of medium and especially large size fibers in comparison with the aquatic-terrestrial living grass frog. The importance is stressed of examining the spinal nerves of an entirely aquatic frog with different feeding habits, such as Xenopus, for further comparison.

Animals↗
PubMed1981

Shedding of mitotic cells from the surface of multicell spheroids during growth.

During the growth of EMT6/Ro mammary tumor multicell spheroids, a large number of cells are shed into the suspension medium. The rate of cell shedding was 218 cells per square millimeter of spheroid surface per hour, or up to 1.5% of the total spheroid cell content per hour. Shed cells had a clonogenic capacity equal to that of exponential monolayer cultures and were further characterized by volume distribution, mitotic index, flow cytofluorometry, and autoradiography. The results indicated that cells are released from the spheroid surface at mitosis, presumably due to a loosening of the cell-to-cell attachment during this cycle phase. These mitotic cells, when placed in monolayer culture, attached and grew synchronously with a cell cycle time of about 13 hours. Shed cells kept in suspension culture had a similar cell cycle time, but these cells reaggregated immediately after mitosis. The results indicated that cell shedding and reaggregation both occur near the time of mitosis and are intrinsic factors regulating the initiation and subsequent growth of multicell spheroids. Although these studies were done with spheroids cultured in vitro, shedding of mitotic cells may play an important role in the in vivo process of metastasis.

Animals↗
PubMed1981

In vivo response of KHT sarcomas to combination chemotherapy with radiosensitizers and BCNU.

Female C3H/HeJ mice bearing intramuscularly transplanted KHT sarcomas were treated with a single dose of 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU, 30 mg/kg, i.p.) alone or in combination with a single dose of misonidazole (MISO, 1.0 mg/g, i.p.) or its desmethylated metabolite Ro-05-9963 (2.0 mg/g, i.p.). The effectiveness of drug therapy was assessed by a tumour growth-delay assay (i.e. measuring the median time required for tumours to grow to treatment size x 4). The relative efficacy of administering the nitroimidazoles in various schedules ranging from 12 h before to 12 h after BCNU administration also was evaluated. Untreated control KHT tumours grew to the initial size x 4 in a median time of 4 days. No significant growth delay was seen in mice treated with either nitroimidazole alone, whilst treatment with BCNU alone produced a median growth delay of 7 days. Combination chemotherapy with 9963 administration 3 h after BCNU significantly increased the median tumour growth delay to 9 days. However, no significant growth delay was produced in any of the other combinations of these agents. The median growth delay was significantly reduced to 5 days when MISO was administered 3 h before BCNU, whereas MISO administered simultaneously 3,6, or 12 h after BCNU significantly enhanced delays ( 9 days). These results indicate that both MISO and 0063 may be combined with conventional therapeutic agents, in this particular case a nitrosourea, to produce an enhanced tumour response. The production of such a response appears to be nitroimidazole as well as schedule dependent.

Animals↗
PubMed1981

Neonatal screening for hypothyroidism in Scotland: results of a pilot study.

We have assessed the feasibility of screening for neonatal hypothyroidism based on thyroxine (T4) or thyrotrophin (TSH) assays on dried blood spots collected for the phenylketonuria (PKU) screening programme in Scotland. The TSH assay was both more sensitive and specific than T4 in detecting primary hypothyroidism and was sufficiently rugged and rapid for routine application. Using a labelled antibody assay for TSH, 99.7 per cent of the first 30,000 babies tested had values less than 25 mU/l, and 0.24 per cent had values between 25 and 50 mU/l. Of the 11 suspected positives (TSH greater than 50 mU/l), nine were confirmed on serum specimens as primary hypothyroidism (prevalence 1:3,300) and the two false positives (0.007% of total) were explicable by inappropriate time of sampling. No false negatives have so far been identified. The median time to detection by blood spot assay was 16 days (range 6 to 22 days). Five of the nine cases were not suspected clinically at the time of screening. It is concluded that TSH fulfils most of the criteria for an ideal screening test for primary hypothyroidism and a screening programme based on blood spot TSH will allow therapy to be commenced within one month of birth.

Congenital Hypothyroidism↗