Levels of selected growth factors in viable and necrotic regions of xenografted HCT-8 human colon tumours

Xenografted tumours were produced in nude mice by injection of HCT-8 human colon tumour cells. At average volumes of about 750 mm³, animals were injected with fast green vital dye, and 20 min later, tumours were excised and dissected into viable (stained) and necrotic portions (unstained). Viable and necrotic regions were then examined for cell yields, colony forming efficiencies, and levels of basic fibroblast growth factor (FGF-2), transforming growth factors-β₁ and -α (TGF-β₁, TGF-α), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) using enzyme-linked immunoassay (ELISA) procedures. Levels in the viable and necrotic regions were compared to levels in unseparated tumours. The average extent of necrosis in HCT-8 tumours of this size was 64%. The data for cell yields, colony forming efficiencies FGF-2, VEGF, TGF-β₁ and TGF-α indicated that values determined in the unseparated tumours could be understood on the basis of the weighted average between viable and necrotic tissue, with the higher values occurring in the viable tissue. Low levels of FGF-2 and VEGF were found in the necrotic portions of the tumour while no measurable levels of TGF-β₁ and TGF-α could be determined. PDGF levels were, however, equivalent in both the viable and necrotic regions indicating that necrotic tissue could be an important reservoir for this growth factor.     

CYTOKINETICS OF TUMOUR AND ENDOTHELIAL CELLS AND VASCULARIZATION OF LUNG METASTASES IN C3H/HE MICE

A model of lung metastases was developed using intravenous injection of tumour cell aggregates of spontaneous C3H/He mammary tumours in syngeneic mice. the growth rate of lung tumours decreased with increasing tumour volume, with mean host survival of 46 days. the cytokinetics of individual tumours ranging between 0.004 and 4.2 mm³ in volume were studied. the labelling index (LI) ranged between 12 and 17%, the DNA synthesis time (T_s) being 9–10 hr. the growth fraction (GF) ranged between 26 and 38%. the cell cycle time (T_c) was found to be 18–19 hr. the LI and the GF decreased with increasing tumour volume doubling time (T_d). No correlation was found between the tumour volume and T_c. the LI of endothelial cells within these tumours, ranging between 0.004 and 4.2 mm³ in volume was 14–15% and endothelial cell proliferation was not affected by tumour growth. Vascular parameters were also determined for these tumours as a function of tumour volume. Vascular volume increased with increasing tumour size while the percentage of capillary vessels decreased. the cellular volume to capillary volume ratio increased with increasing tumour volume. Necrosis was observed in 0.27 mm³ tumours and increased with increasing tumour size. The results from these studies suggested that the age-dependent decrease in proliferative activity of tumour cells growing in the lung is related to change in effective vascularity.     

THE CELL PROLIFERATION KINETICS OF THE EMT6/M/AC MOUSE TUMOUR AT FOUR VOLUMES DURING UNPERTURBED GROWTH IN VIVO

The cell proliferation kinetics of the EMT6/M/AC mouse tumour were determined at four different volumes between 1–5 mm³ and 175 mm³. The decrease in the growth rate between these volumes was mainly due to a decrease in the rate constant for cell production. A small increase in the rate constant for cell loss occurred, but this was thought to be insignificant. The cell loss factor increased from 40% at 1–5 mm³ to over 70% in the 175 mm³ tumours. An increase in the median cell cycle time, from 14-1 hr to 18-5 hr was also found between these same volumes. Results obtained for the NCTC fibrosarcoma and the R-l rhabdomyosarcoma indicate that there may be a threshold volume in these sarcomas below which little or no cell loss takes place. This was not found in the EMT6/ M/AC tumour.     

SERIAL TRANSPLANTATION, HISTOLOGY AND CELLULAR KINETICS OF A RAT ADENOCARCINOMA

Data are reported on the growth, histology and cell population kinetics of a spontaneous mammary tumour in the rat at the 1st and 35-40th transplantation generation. With serial transplantation subcutaneously in isologous animals, the macroscopic growth characteristics of the tumour did not alter. The tumour had a volume doubling time of approximately 1.6 days at 400 mm³, increasing to about 7 days at 20,000 mm³. The histology, however, changed dramatically from that of a well differentiated papillary adenocarcinoma with a well defined vascular pattern and minimal necrosis to become by the 35th generation a poorly differentiated pleomorphic cellular tumour in which stromal elements and associated vascular supply were minimal and necrosis prominent. These changes were not manifested in the median cycle time of its cells, 16–18 hr, but by an increase in the growth fraction from 0.36 to 0.59 with a commensurate increase in the cell loss factor from 0.04 to 0.30.     

Cell loss from viable and necrotic tumour regions measured by 125I-UdR

Loss of cells from vital and necrotic areas of the syngeneic mammary adenocarcinoma EO 771 in male C57 BL/6J mice may be measured by use of 125I-labelled 5-iodo-2'-deoxyuridine (125I-UdR). Later than 50 hr after an intraperitoneal injection of 20 muCi 125I-UdR the incorporated activity of the entire tumour was externally measured and found to decrease with time after injection. The injected amount was neither chemo- nor radiotoxic. By injecting the vital dye 'light green', unstained necrotic and stained viable regions were separately excised and measured for loss of activity throughout the natural development of the labelled tumour. With the appearance of necrotic regions, labelled viable cells became necrotic, and activity was slowly eliminated. With increasing proportions of necrosis during tumour growth, the rate of loss of activity of the whole tumour decreased. Loss of activity from viable tumour regions reflected cell death and exceeded the loss rates of the whole tumour by a factor of 2 to 3. The data show that loss of activity from the whole tumour results from a superposition of different elimination rates of viable and necrotic tumour regions and is not an immediate consequence of cell death in the course of undisturbed tumour development.     

T Lymphocyte Density and Distribution in Human Colorectal Mucosa,and Inefficiency of Current Cell Isolation Protocols

Mucosal tissues are critical immune effector sites containing complex populations of leukocytes in a tissue microenvironment that remains incompletely understood. We identify and quantify in human distal colorectal tissue absolute mucosal CD3⁺ lymphocytes, including CD4⁺ and CD8⁺ subsets, by direct visualization using immunohistochemistry (IHC), immunofluorescence (IF), and an automated counting protocol (r²=0.90). Sigmoid and rectal mucosal tissues are both densely packed with T lymphocytes in the mucosal compartment. Both compartments had similar densities of CD3⁺ T lymphocytes with 37,400 ± 2,801 cells/mm³ and 33,700 ± 4,324 cell/mm³, respectively. Sigmoid mucosa contained 57% CD3⁺CD4⁺ and 40% CD3⁺CD8⁺ T lymphocytes which calculates to 21,300 ± 1,476/mm³ and 15,000 ± 275/mm³ T lymphocytes, respectively. Rectal mucosa had 57% CD3⁺CD4⁺ and 42% CD3⁺CD8⁺ or 21,577 ± 332, and 17,090 ± 1,206 cells/mm³, respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm³ and 2,708 ± 245/mm³ CD4+ and CD8+ T lymphocytes. These data suggest only ~20.7% recovery compared to IHC results for these markers. Further studies will determine if this reflects a selective bias in only CD3⁺, CD4⁺ and CD8⁺ T cells or can be generalized to all flow-analyzed cells from mucosal tissues for phenotyping and functional testing.     

Cell kinetic studies on the JB-1 ascites tumour of the mouse

Abstract. The FLM method, modified by double labelling with [³H]- and [¹⁴C]-thymidine, has been applied to the 4-day old JB-1 ascites tumour of the mouse. It results in well separated waves of purely [³H]- and purely [¹⁴C]-labelled mitoses, which show a remarkable asymmetry with long tails to the right. The following values for the mean transit times of the cells have been derived from this FLM curve, for a tumour age of 4–6 days: T_C= 32.5 hr, T_S= 16.7 hr, T_G1= 3.7 hr, T_G1= 11.0 hr and T_M= 1.1 hr. A further evaluation of the FLM curve, however, is difficult, due to the non-stationary growth of the tumour. A number of other experimental findings (growth curve, decrease of the labelling and mitotic index with increasing tumour age, two single-labelled FLM curves starting 4 and 6 days after tumour inoculation) indicate that the cell cycle time increases during the experimental period of the double-labelled FLM curve (about 2 days). A lengthening of the cycle time should result in an increasing enlargement of the areas under the waves of the modified FLM curve. However, such an increase in area has not been found; the areas are constant. All the results of the present cell kinetic studies would be consistent if it were postulated that the cell cycle time lengthens with increasing tumour age up to about 4 days after inoculation, then remains relatively constant at between 4 and 6 days and thereafter increases again. Short-term double labelling experiments suggest that this is actually the case. Under the assumption of nearly constant phase durations during the 5th and 6th day of tumour growth further conclusions can be drawn from the modified FLM curve. In particular, it follows that the transit times of the cells through successive cycle phases are uncorrelated and the variances of the transit times through a cycle phase are proportional to the duration of this phase.     

Uptake of 36Cl and 22Na by the Brain-Cerebrospinal Fluid System: Comparison of the Permeability of the Blood-Brain and Blood-Cerebrospinal Fluid Barriers

Abstract: Transport and permeability properties of the blood-brain and blood-CSF barriers were determined by kinetic analysis of radioisotope uptake from the plasma into the CNS of the adult rat. Cerebral cortex and cerebellum uptake curves for ³⁶Cl and ²²Na were resolved into two components. The fast component (t_½ 0.02–0.05 h, fractional volume 0.04–0.08) is comprised of the vascular compartment and a small perivascular space whereas the slow component (t_½ 1.06–1.69 h, fractional volume 0.92–0.96) represents isotope movement across the blood-brain barrier into the brain extracellular and cellular compartments. Uptake curves of both ³⁶Cl and ²²Na into the CSF were also resolved into two components, a fast component (t_½ 0.18 h, fractional volume 0.24) and a slow component (t_½ 1.2 h, fractional volume 0.76). Evidence suggests that the fast component represents isotope movement across the blood-CSF barrier, i.e., the choroid plexuses, whereas the CSF slow component probably reflects isotope penetration primarily from the brain extracellular fluid into the CSF. The extracellular fluid volume of the cerebral cortex and cerebellum was estimated as ?13% from the initial slope of the curve of brain space versus CSF space curve for both ³⁶Cl and ²²Na. Like the choroid plexuses, the glial cell compartment of the brain appears to accumulate Cl from 2 to 6 times that predicted for passive distribution. The relative permeability of the blood-CSF and blood-brain barriers to ³⁶Cl, ²²Na, and [³H]mannitol was determined by calculating permeability surface-area products (PA). Analysis of the PA values for all three isotopes indicates that the effective permeability of the choroidal epithelium (blood/CSF barrier) is significantly greater than that of the capillary endothelium in the cerebral cortex and cerebellum (blood-brain barrier).     

The Oropharyngeal Airway in Young Adults with Skeletal Class II and Class III Deformities: A 3-D Morphometric Analysis

Objectives

1) To determine the accuracy and reliability of an automated anthropometric measurement software for the oropharyngeal airway and 2) To compare the anthropometric dimensions of the oropharyngeal airway in skeletal class II and III deformity patients.

Methods

Cone-beam CT (CBCT) scans of 62 patients with skeletal class II or III deformities were used for this study. Volumetric, linear and surface area measurements retroglossal (RG) and retropalatal (RP) compartments of the oropharyngeal airway was measured with the 3dMDVultus software. Accuracy of automated anthropometric pharyngeal airway measurements was assessed using an airway phantom.

Results

The software was found to be reasonably accurate for measuring dimensions of air passages. The total oropharyngeal volume was significantly greater in the skeletal class III deformity group (16.7 ± 9.04 mm³) compared with class II subjects (11.87 ± 4.01 mm³). The average surface area of both the RG and RP compartments were significantly larger in the class III deformity group. The most constricted area in the RG and RP airway was significantly larger in individuals with skeletal class III deformity. The anterior-posterior (AP) length of this constriction was significantly greater in skeletal class III individuals in both compartments, whereas the width of the constriction was not significantly different between the two groups in both compartments. The RP compartment was larger but less uniform than the RG compartment in both skeletal deformities.

Conclusion

Significant differences were observed in morphological characteristics of the oropharyngeal airway in individuals with skeletal class II and III deformities. This information may be valuable for surgeons in orthognathic treatment planning, especially for mandibular setback surgery that might compromise the oropharyngeal patency.     

Functional units in rainbow trout (Salmo gairdneri, Richardson) liver: III. Morphometric analysis of parenchyma, stroma, and component cell types

Hepatic stroma and parenchyma with its component cell types were quantitatively described in adult male and female actively-spawning 5-year-old rainbow trout (Salmo gairdneri, Richardson). Point-count morphometry of glycol methacrylate sections estimated volume compartments for stroma and parenchyma. Veins composed 85% of the stroma while arteries and bile ducts occupied approximately 6-7% each. Parenchyma accounted for 95% of hepatic volume. Point-count morphometry of transmission electron micrographs estimated volume compartments as well as numerical and surface density measurements for parenchymal components. Within the hepatic parenchymal compartment, hepatocytes occupied 85% and showed significant sex differences. Female hepatocytes were significantly more numerous but were smaller, only 60% of the volume of male hepatocytes. Since hepatocyte nuclear volume was equal in both sexes, differences were due to reduced cytoplasmic volume in females. Perisinusoidal macrophages of females occupied larger volumes of their respective parenchymal compartments, and their larger mean cytoplasmic volumes suggested activation. Biliary epithelial cells of preductules and ductules were numerous. Ratios of numerical density of hepatocytes to biliary epithelial cells were consistent with a tubular arrangement of hepatocytes. Factors possibly mediating the sexual dimorphism are discussed.     

A simple model for cell volume and developmental compartments in nutrient limited cyclostat cultures of algae

Daily light-dark cycles can entrain cell growth and division cycles in populations of algae growing in nutrient limited continuous cultures, or cyclostats. In this study a simple model for the flux of cells between discrete developmental stages is formulated for periodic cyclostat cultures of algae. Cell growth, in terms of volume, was set as being constant within a given developmental compartment, but variable between compartments. Growth within a given compartment or transition between compartments was restricted to specific intervals of the subjective day. The model was calibrated to phosphate limited cyclostat growth of Euglena gracilis, with the intervals for transition between compartments fixed at the times relative to the subjective dawn corresponding to critical transition points in the phased cell cycle of this organism. The model output for mean population volume per cell agreed well with experimental data. Although greatly simplified, the periodic behavior of the model volume frequency distributions for the discrete compartments provide reasonable approximation of experimentally determined distributions.     

PERTURBED CELL PROLIFERATION KINETICS OF THE EMT6/M/AC TUMOUR IN MICE TREATED WITH ANTI-MOUSE LYMPHOCYTE SERUM

The cell proliferation kinetics of the EMT6/M/AC tumour were determined at two volumes, 6-3 mm³ and 180 mm³, in mice treated with anti-mouse lymphocyte serum, AMLS. Comparison of the growth curve with that obtained in non-AMLS treated animals showed a marked increase in the growth rate at all volumes in the treated group. In contrast, the cell cycle time and the intermitotic phase times were not significantly different in the treated and untreated groups at comparable volumes. The increase in the growth rate in AMLS treated mice was obtained in spite of decreases in both the rate constant for cell production and the growth fraction, and was due to a marked decrease in the rate constant for cell loss.     

Oscillating growth patterns of multicellular tumour spheroids

The growth kinetics of 9L (rat glioblastoma cell line) and U118 (human glioblastoma cell line) multicellular tumour spheroids (MTS) have been investigated by non-linear least square fitting of individual growth curves with the Gompertz growth equation and power spectrum analysis of residuals. Residuals were not randomly distributed around calculated growth trajectories. At least one main frequency was found for all analysed MTS growth curves, demonstrating the existence of time-dependent periodic fluctuations of MTS volume dimensions. Similar periodic oscillations of MTS volume dimensions were also observed for MTS generated using cloned 9L cells. However, we found significant differences in the growth kinetics of MTS obtained with cloned cells if compared to the growth kinetics of MTS obtained with polyclonal cells. Our findings demonstrate that the growth patterns of three-dimensional tumour cell cultures are more complex than has been previously predicted using traditional continuous growth models.     

The Growth of the Emt6 Tumour In the Lungs of Balb C Mice Following Intravenous Inoculation of Tumour Cells From Culture

The growth of the EMT6 tumour in the lungs of Balb C mice has been studied following intravenous inoculation of different numbers of tumour cells taken from culture. At various times after injection of cells into mice, cell suspensions have been prepared from pairs of lungs and the number of in vitro colony forming cells assayed by plating into petri dishes. Following intravenous injection of 10⁵ cells, the time required for doubling of the number of clonogenic tumour cells appearing in the cell suspension is around 17 hr until such time that the total tumour cell population per set of lungs reaches 10⁸ cells (at 10–12 days). This doubling time has to be corrected for changes in ability to extract cells from the lungs into the cell suspension at various times and also for possible changes in plating efficiency in vitro. When these correction factors are applied, the most likely value for the doubling time of clonogenic tumour cells in the lungs is in the range 20–24 hr. This is a similar figure to that previously deduced for the EMT6 flank tumour during its microscopic period of growth. After reaching a total size of 10⁸ tumour cells, the time for doubling of the number of clonogenic tumour cells in the lung increases. During the later stages of tumour growth a good correlation is seen between total lung tumour weight and the number of clonogenic cells present. For the final 3–4 days of the initial period of rapid tumour growth, it is possible to carry out a haemocytometer count of tumour cells in the lung suspension and hence surviving fraction experiments may be carried out after various forms of treatment. In this way the response to treatment of microscopic tumour foci may be determined.     

The effect of external calcium and lanthanum on platelet calcium content and on the release reaction

Calcium compartments in calf platelets were studied using a lanthanum washout procedure to distinguish between surface-bound calcium and intracellular calcium. The calcium content of calf platelets ranges from 20 to 60 nmol/10⁹ platelets and is sensitive to the calcium concentration of the suspending medium. With 1 mM calcium in the medium, calcium uptake is rapid and reaches steady state within 1–2 min. Results obtained with the lanthanum procedure indicate that it is the surface compartment which is most affected by the extracellular calcium concentration. The surface compartment appears to be saturable and is highly exchangeable. Although the total calcium as well as the calcium content of the surface and internal compartments are variable, the ratio of calcium in either compartment to the total saturated calcium is quite constant. The data indicate that 68–85% of the platelet calcium is located internally. Thrombin produces an immediate release of platelet calcium and labeled serotonin and an increase in the ⁴⁵Ca²⁺ uptake of both the surface and internal compartments. The release reaction is not dependent upon exogenous calcium or an influx of exogenous calcium since it occurs even in the presence of $\text{ethyleneglycol-bis-(β-aminoethylether)}\text{-N,N′-}\text{tetraacetic}$ acid. Lanthanum, however, inhibits the release reaction possibly by blocking surface calcium site and reducing the mobility of endogenous platelet calcium.     

Size structure and morphological composition of the Pechora Sea winter bacterioplankton and conditions of its formation

Analysis of conditions of formation of microplanktonic microbial communities (MC) was carried out for the Pechora Sea using the data of winter 2002–2005. The patterns of distribution of different MC groups in different water types are discussed. Regular variations of the indices of abundance in a gradient of physicochemical variables were revealed. The estimated average bacterial numbers for under-ice conditions were 234 × 10³ cells/mL, with the average cell volume of 0.58 μm³ and total cell area ～550 mm²/L. The estimated total microbial biomass in the 0–25 m water horizon of the Pechora Sea is 187 × 10⁹ g wet biomass.     

The influence of CO2 enrichment, phosphorus deficiency and water stress on the growth, conductance and water use of Pinus radiata D. Don

Abstract. Seedlings of Pinus radiata D. Don were grown in growth chambers for 22 weeks with two levels of phosphorus, under either well-watered or water-stressed conditions at CO₂ concentrations of either 330 or 660mm³ dm^?3. Plant growth, water use efficiency and conductance were measured and the relationship between these and needle photosynthetic capacity, water use efficiency and conductance was determined by gas exchange at week 22. Phosphorus deficiency decreased growth and foliar surface area at both CO₂concentrations; however, it only reduced the maximum photosynthetic rates of the needles at 660 mm³ CO₂ dm^?3 (plants grown and measured at the same CO₂ concentration). Water stress reduced growth and foliar surface area at both CO₂ concentrations. Increases in needle photosynthetic rates appeared to be partly responsible for the increased growth at high CO₂ where phosphorus was adequate. This effect was amplified by accompanying increases in needle production. Phosphorus deficiency inhibited these responses because it severely impaired needle photosynthetic function. The relative increase in growth in response to high CO₂ was higher in the periodically water-stressed plants. This was not due to the maintenance of cell volume during drought. Plant water use efficiency was increased by CO₂ enrichment due to an increase in dry weight rather than a decrease in shoot conductance and, therefore, transpirational water loss. Changes in needle conductance and water use efficiency in response to high CO₂ were generally in the same direction as those at the whole plant level. If the atmospheric CO₂ level reaches the predicted concentration of 660 mm³ dm^?3 by the end of next Century, then the growth of P. radiata will only be increased in areas where phosphorus nutrition is adequate. Growth will be increased in drought-affected regions but total water use is unlikely to be reduced.     

Spatio-temporal tumour model for analysis and mechanism of action of intracellular drug accumulation

We have developed a one-dimensional tumour simulator to describe the biodistribution of chemotherapeutic drugs to a tumoral lesion and the tumour cell’s response to therapy. A three-compartment model is used for drug dynamics within the tumour. The first compartment represents the extracellular space in which cells move, the second corresponds to the intracellular fluid space (including cell membrane) which is in direct equilibrium with the extracellular space, and the third is a non-exchangeable compartment that represents sequestered drug which is trapped in the nucleus to damage the cellular DNA, directly triggering cell death. Analytical and numerical techniques (Finite Element Method) are used to describe the tumour’s response to therapy and the effect of parameter variation on the drug concentration profiles in the three compartments.     

CELL PROLIFERATION IN THE ERYTHROID COMPARTMENT OF GUINEA-PIG BONE MARROW: STUDIES WITH 3H-THYMIDINE

Single and multiple injections of ³H-TdR have been used for measuring the rate of proliferation in morphologically defined cell populations of guinea-pig bone marrow that are committed to erythroid differentiation. The conclusions are based on the analysis of absolute cell numbers in the maturational compartments, the labeling and mitotic indices, labeled mitotic curves, pulse and chase grain counts over dividing and interphase cells, and on the rate of labeling during multiple, repeated injections of ³H-TdR. The average duration of S and the rate of cycling is similar in all maturational compartments of the erythron. The majority of cells progress to the next maturational compartment by the time they divide for the second time. All proerythroblasts and basophilic erythroblasts are in cycle. Polychromatic erythroblasts incapable of incorporating ³H-TdR reach the orthochromatic population in the span of 5–6 hr. The orthochromatic population is renewed every 20–24 hr. The number of divisions between the proerythroblast and orthochromatic erythroblast does not exceed four and some cells may undergo only two divisions during the maturation pathway. Cell input from a progenitor cell population contributes to the maintenance of the erythron. The kinetic behavior of progenitor cells is similar to that of proerythroblasts. By the time of their second division, progenitor cells may reach either the proerythroblast or basophilic erythroblast compartments. The kinetic behavior of basophilic transitional cells corresponds to the predicted behavior of the erythroblast progenitor cell pool. Several of the conclusions are based on the assumption that grain count halving is the result of cell division. In view of the evidence discussed, this assumption in the present studies seems justified.