Effects of ionizing radiation on the adhesive interaction of human tumor and endothelial cells in vitro.

A centrifugation assay was used to determine the effects of ionizing radiation on the adhesive interaction of A549 human lung adenocarcinoma tumor cells and human umbilical vein endothelial cells (HUVEC). The tumor cells were fluorescently labeled and divided into control (sham-irradiated) and irradiated groups. The irradiated groups were exposed to irradiation levels ranging from 5 to 20 Gy using a 137Cs source. A specified number of these A549 tumor cells were then delivered into each well of 96-well cell culture plates containing confluent monolayers of human umbilical cord vein endothelial cells (HUVEC), and were given time to adhere to the endothelial cells. The wells were then sealed and were exposed to an acceleration field varying from 1 to 42 g (0-500 rpm) for 10 min. Finally, the wells were drained, and the number of tumor cells adhering to the endothelial monolayer were counted using a fluorescent microscope system. Our results indicate that the irradiation of A549 tumor cells significantly increased their adhesive interaction with endothelial cells (number of adhering irradiated cells/number of adhering control cells = 1.0, 1.3, 1.9, 2.2 for 0, 5, 10, 20 Gy respectively). In contrast, when endothelial cells were irradiated, rather than tumor cells, adhesive interaction decreased with an increase in the radiation dose (irradiated/control = 1.0, 0.9, 0. 8, 0.5 for 0, 5, 10, 20 Gy respectively). Simultaneous irradiation of both the tumor cells and the endothelial cells did not alter their adhesive interaction significantly. These findings may have important implications for the metastatic ability of irradiated tumor cells.

Should direct measurements of tumor oxygenation relate to the radiobiological hypoxic fraction of a tumor?

PURPOSE: Numerous previous studies have attempted to relate the radiobiological hypoxic fraction (HF) to direct measures of tumor oxygenation such as HbO2 saturations, tumor pO2 levels, or hypoxic cell labeling. Although correlations have been found within tumor lines, no overall relationships were seen across tumor lines. The current objective was to examine the effect on HF of changes in the fractions of the oxygenated and anoxic tumor cells that remain clonogenic. METHODS AND MATERIALS: A mathematical model was developed that relates the HF to direct measures of tumor oxygenation. The primary assumptions were that: (a) the tumor is divided into distinct compartments of either fully oxygenated or fully anoxic cells, and (b) the survival of the oxygenated cells is negligible compared to that of the anoxic cells. Based on these assumptions, the HF is plotted as a function of the fractions of clonogenic or nonclonogenic, and oxygenated or anoxic cells. RESULTS: If all cells are clonogenic, then the HF equals the fraction of anoxic cells. If a higher fraction of anoxic than oxygenated cells are nonclonogenic, then the HF will be overestimated by the fraction of the tumor measured to be anoxic using direct measuring techniques. If a higher fraction of the oxygenated than anoxic cells are nonclonogenic, the HF will be underestimated by the fraction of anoxic cells. CONCLUSION: Correlations between the HF and direct measures of tumor oxygenation have been described within tumor lines evaluated under different physiological condition. However, such relationships can be totally unpredictable between different tumors if the fraction of the anoxic cells that is clonogenic varies substantially. Clearly, if tumor anoxia cannot be detected using direct measures, this is an accurate indication that the tumor is well oxygenated. When tumor anoxia is present, however, the conclusions are ambiguous. Even when a small fraction of the tumor is measured as anoxic, direct measures of tumor oxygenation may not be representative of the HF if a substantial proportion of nonclonogenic cells is present.

Late effects of ionizing radiation on the microvascular networks in normal tissue.

Damage to the microvascular networks constitutes one of the most important components of ionizing radiation damage to normal tissue. Previously, we have reported the early (3, 7 and 30 days postirradiation) effects of ionizing radiation on the structure and function of normal tissue microvascular networks. Here we report on the late effects of ionizing radiation on the structural and functional changes in microvascular networks in locally irradiated (single 10-Gy dose) hamster cremaster muscles observed 60, 120 and 180 days postirradiation; age-matched animals were used as controls. As in the previous study, intravital microscopy was used to measure structural and functional parameters in complete microvascular networks in vivo. A factorial design was used to examine the effects of radiation status, time postirradiation, and network vessel type on the structure and function of microvascular networks. Our results indicate that the progression of radiation-induced microvascular damage continues during the late times but that there is partial recovery from radiation damage within 6 months postirradiation. Red blood cell flux, red blood cell velocity, and capillary blood flow in irradiated networks at 180 days postirradiation were significantly greater than control levels. As at the early times, all vessel types were not damaged equally by radiation at every time.

Early effects of ionizing radiation on the microvascular networks in normal tissue.

Microvascular networks, which control the delivery of oxygen and nutrients and the removal of metabolic waste, are the most sensitive part of the vascular system to ionizing radiation. Structural and functional changes in microvascular networks were studied in locally irradiated (single 10-Gy dose) hamster cremaster muscles observed 3, 7 and 30 days post-irradiation. Networks were selected in reference to a well-defined location in the tissue to reduce heterogeneity due to spatial variations. Intravital microscopy was used to measure structural and functional parameters in vivo. A factorial design was used to examine the effects of radiation status, time postirradiation, and network vessel type on the structure and function of microvascular networks. While the diameter of microvessels in control animals increased significantly with age, vessel diameter in irradiated vessels decreased significantly with age. Red blood cell velocity in irradiated networks at 3 and 30 days postirradiation was significantly lower than in control networks. There was a significant decrease in capillary surface area and a significant increase in vessel hematocrit in irradiated animals. Blood flow in irradiated vessels was significantly lower than in control vessels. Changes in functional parameters were evident at 3 days postirradiation while changes in structural parameters occurred later. All vessel types were not damaged equally by radiation at every time examined.

Inherent cellular differences may explain the dissimilar survival of RIF-1 and KHT tumour cells under aerobic and hypoxic conditions.

Although previous work has shown striking differences in radiobiological hypoxic fraction between KHT and RIF-1 murine sarcomas, intravascular oxyhaemoglobin (HbO2 saturations have revealed less substantial variations. Using quantitative histological techniques, we have also found minor differences in the distributions of distances between tumour cells and the nearest bloods vessel for KHT versus RIF-1 sarcomas. We report here, the results of an investigation of the inherent ability of these tumour cells to withstand conditions of hypoxia by in vitro culturing under aerobic and anoxic conditions. Tumours were dissociated, seeded into culture dishes, and placed in air-tight aluminium chambers. These chambers were repeatedly evacuated and refilled with a mixture of 95% N2 and 5% CO2 over a 2.5-h period. Following anoxic exposure, cells were removed and replated, and the in vitro plating efficiency (PE) was determined using a colony survival assay. After normalizing to aerobic controls, KHT tumour cells had a significantly lower PE, following a 16-hour exposure to anoxic conditions (0.4), than RIF-1 (0.6). Increasing the hypoxic exposure to 40 h resulted in normalized PEs of 0.07 for KHT versus 0.4 for RIF-1. Although these results support the hypothesis that the two tumour lines have different inherent abilities to withstand hypoxia, they do not explain the failure of direct measures of tumour oxygenation to correlate with the radiobiological hypoxic fraction. Additional factors such as differences in oxygen diffusivity or oxygen consumption rates between tumour lines may also be involved.

Differences in ICAM-1 and TNF-alpha expression between large single fraction and fractionated irradiation in mouse brain.

PURPOSE: To elucidate the brain molecular response to irradiation. The expression of the intercellular adhesion molecule (ICAM-1) and tumour necrosis factor-alpha (TNF-alpha) in the mouse brain was compared after single-dose and fractionated whole-brain irradiation. MATERIALS AND METHODS: Mice received a single dose of 2, 10 or 20 Gy or a fractionated dose (2 Gy day(-1)) of 10, 20 or 40 Gy. ICAM-1, and TNF-alpha mRNA expression were quantified by the highly sensitive real-time polymerase chain reaction technique. Expression of ICAM-1 protein was quantified by dual-labelled monoclonal antibody assay. RESULTS: After a 20-Gy single dose, there was an increase in ICAM-1 and TNF-alpha mRNA levels (14- and 11-fold, respectively) as well as a significant increase in the level of ICAM-1 protein (p=0.0243). The expression of ICAM-1 and TNF-alpha mRNA increased at the end of the 40-Gy fractionated regimen (3.55- and 2.30-fold, respectively). CONCLUSIONS: The molecular response of the brain to single-dose irradiation was rapid, while its response to fractionated irradiation was slow. This finding is consistent with clinical observations and could be of use when designing strategies to mitigate radiation sequelae.

The role of wall shear stress in microvascular network adaptation.

The possible role of wall shear stress in microvascular network adaptation was investigated by computer simulation in planar polygonal model networks. Adaptation of vessel diameter to local wall shear stress resulted in false geometry and excessive rarefaction of the networks. Adaptation to mean wall shear stress decreased the range of shear stress, decreased the total power dissipation, and prevented network rarefaction. A measure of mean wall shear stress may serve as a control signal for the adaptation of vessel diameter to blood flow in microvascular networks.

Computer simulation of cerebral microhemodynamics.

Microvascular network hemodynamics was simulated by computer in an anatomically reconstructed cerebral microvascular network. A video microscope system was used for three-dimensional mapping of the vessel network in the rat brain cortex. The complete topology, length and mean diameter of the microvessels were determined. The distribution of blood flow and red cell flux in the network was calculated based on vessel resistance estimated from geometrical data and a rheological model of blood. This model described apparent relative blood viscosity as a function of vessel diameter and local discharge hematocrit. The calculations predicted highly heterogeneous cell flux distribution at any feed hematocrit between 10 and 40 percent. The frequency distribution of microvessel hematocrit was bimodal and included values exceeding the feed hematocrit value. A probabilistic simulation of cell transit resulted in transit time distributions which agree with experimental findings. The most probable transit time and capillary path length and 4s and 300 microns, respectively.

A semi-empirical model of apparent blood viscosity as a function of vessel diameter and discharge hematocrit.

A semi-empirical model is developed to describe the dependence of apparent viscosity of blood on vessel diameter (2.7 to 500 microns) and vessel discharge hematocrit (5% to 60%). The blood flow is modeled as a cell-rich core and a cell-free marginal layer in the larger vessels and an axial-train in the smaller vessels. Laminar (Poiseuille) flow is assumed in all cases. An equation is derived in which apparent viscosity is a function of vessel diameter, core viscosity, and width of marginal layer. This is then complemented by empirical equations in which core viscosity varies exponentially with discharge hematocrit while the width of marginal layer varies linearly with discharge hematocrit. The model correlates well with several sets of experimental data and behaves according to the Fahraeus-Lindqvist effect. Predicted apparent viscosity tends to the expected finite value for large vessel diameters. Dependence of apparent viscosity on vessel diameter is realistically smooth in the whole diameter range.

Molecular mimicry in inducing DNA damage between HIV-1 Vpr and the anticancer agent, cisplatin.

The human immunodeficiency virus type 1 (HIV-1) viral protein R (vpr) gene is an evolutionarily conserved gene among the primate lentiviruses. Several functions are attributed to Vpr including the ability to cause cell death, cell cycle arrest, apoptosis and DNA damage. The Vpr domain responsible for DNA damage as well as the mechanism(s) through which Vpr induces this damage is unknown. Using site-directed mutagenesis, we identified the helical domain II within Vpr (aa 37-50) as the region responsible for causing DNA damage. Interestingly, Vpr Delta(37-50) failed to cause cell cycle arrest or apoptosis, to induce Ku70 or Ku80 and to suppress tumor growth, but maintained its capability to activate the HIV-1 LTR, to localize to the nucleus and to promote nonhomologous end-joining. In addition, our cytogenetic data indicated that helical domain II induced chromosomal aberrations, which mimicked those induced by cisplatin, an anticancer agent. This novel molecular mimicry function of Vpr might lead to its potential therapeutic use as a tumor suppressor.

Differentials in child mortality and health care in Pakistan.

PIP: Differentials in child mortality are analyzed by parent's education, health care, and rural-urban residence with data on 7405 currently married women, obtained from the Pakistan Contraceptive Prevalence Survey, 1984-85. The assumption is that educated parents and fathers working in the formal sector have less risks of child mortality than illiterates and those working in the traditional sector; the findings support this assumption. Educated mothers have a greater impact on child mortality risk than educated fathers. Child mortality is highest in families where both parents are illiterates. Mothers, who are housewives or who are working in a family business, have higher child mortality risk than working mothers. Child mortality risk was reduced with a hospital delivery. Father's education and a hospital delivery only reduced risk when women were aged 35 years or less. Urban educated mothers with a hospital delivery had a lower risk of child mortality than mothers with no education. Residence was not a significant factor, when hospital delivery was controlled for younger mothers. Older mothers with a hospital delivery had lower child mortality. Educated mothers with an immunized child had lower child mortality than mothers aged over 25 years. Father's education and immunization showed even lower mortality. Younger educated urban mothers, who immunized their child or infant, had lower child mortality than educated mothers. Rural educated mothers had lower child mortality than rural uneducated mothers, but including immunization as a factor, did not improve child survival. The use of oral rehydration therapy among urban educated mothers also reduced child mortality risk compared with other drug use. The majority of the population was rural (70%) and had the highest child mortality. Not only is child survival reflected in the maldistribution of health facilities, but in lack of access to these facilities, and low levels of literacy, poor sanitation, and low income. Improvement in living conditions in rural areas is recommended; development programs should emphasize access to health and education.^ieng

Mean ages at parities: an indirect estimation.

"The objective of this paper is to describe an indirect approach for estimating the age patterns of occurrence of birth by parity. The main concern here is not of estimating the frequency of occurrence of births (in a period of time) in relation to the population (birth rate) or in relation to the number of females (fertility). Rather, the focus here is on the ages of first, second, and subsequent births, and on their shifts over time. Essentially, the approach is an extension of Hajnal's method for using proportions single to estimate singulate mean age at marriage...." The approach is illustrated using data from the 1975 Pakistan Fertility Survey and the 1990-1991 Pakistan Demographic and Health Survey. A comment by Sultan S. Hashmi is included (pp. 560-1).

Computer simulation of growth of anastomosing microvascular networks.

Stochastic growth of polygonal microvascular networks was simulated on computer by dichotomous terminal branching and bridging (anastomosing with an existing segment). The model was applied to describe microvascular growth into a rectangular plane from the sides when vessels bifurcate in a probabilistic manner. The angle of bifurcation was drawn from a normal distribution, the mean of which was varied between 40 degrees and 80 degrees. The resulting networks contained an average of 88-104 nodes of which 30-38% were due to bridging. Number of nodes, number of branches, number of vascular polygons and a fractal dimension representing the density of nodes were calculated for each simulated network. Capillary density increased when mean angle of bifurcation was increased between 40 degrees and 80 degrees. Distributions of normalized vessel lengths and polygon shapes were compared with those of a mesenteric vascular network. The distributions were not found to be significantly different (p less than 0.05) for most values of the mean angle of bifurcation, matching best for the mean bifurcation angle of 50 degrees. Vascular polygons had an average shape between pentagonal and hexagonal for the mesenteric network as well as for all values of the mean bifurcation angle used in this study.

Dimensions of urban growth in Pakistan.

PIP: The nature and patterns of Pakistani urban growth and implications for the future are discussed. Specific attention is given to the effects of natural increase, in migration, and reclassification or city extension or urban growth. Development expenditures on urban population are also provided. The effects of population pressure on housing, education, health, and employment are also presented. Future growth is anticipated at 55.5 million by 2000 with a constant fertility rate; 36% are expected to live in cities. The maximum increase is expected in Karachi and the urban areas in the provinces of the Punjab and Sindh. The contribution of natural increase to urban growth is estimated at 3.36%, while internal migration is .83% and reclassification or city extension is .11%. The rural-to-urban flow contributes the highest volume to migration flows over urban-to-urban migration. Urban fertility has been stable, and other urban fertility continues to be higher than rural fertility and major urban fertility. Urban infrastructure costs 27% or by 2000 4.7% of the projected gross national products (GNP). Housing growth rates of 3.71% between 1960-80 have not kept pace with population growth of 4.4%, but the shortage of housing has declined faster in urban areas than rural areas where the shortages of housing are greater. Housing conditions in urban areas have not improved. 45% live in pucca kutcha and kutcha housing. 1.3 million housing units are needed in urban areas during 1988-93. There is overcrowding in schools and literacy remains low in rural and urban areas. 2% of the GNP during 1971-85 went to educational funding. 80% of hospital beds and physicians are located in urban areas. There is 1 doctor/1801 persons, 1 bed/610 persons, and 1 hospital/50,000 persons. Only 7% of the 19.4 million urban residents in 1983 had access to potable water, and 48% of 12.12 million people had sewerage and drainage facilities. Agricultural employment is being replaced in urban areas by employment in the informal sector. Out-migration to the Middle East has contributed to a strong economic performance, but recent trends are for return migration. Also affected but not discussed are the effects on urban transportation, water supply, the environment, and town planning.^ieng

Delayed marriages in Pakistan.

PIP: Data from the Migration Module of the Population, Labor Force, and Migration (PLM) Survey of 1979 and from the population censuses of 1961, 1972, and 1981 were examined to explore the impact of modernization, particularly of expansion of education and modern sector employment, urbanization, and migration on proportions never married in various age groups in Pakistan. Table 1 shows a noticeable and substantial increase in proportions never married between 1961 and 1975 and subsequently until 1981. The increase in proportions never married was more pronounced for young females aged 15-29 years than for males in the same age group. The figures for 1972 and 1981 were similar, indicating that increases in the proportions never married occurred more in the 1960s. The singulate mean age at marriage for females was computed to be 18.1 years in 1961, 19.8 years in 1972, and 20.7 years in 1981. Over the 1961-81 period, marital postponement for males was considerably less pronounced. In the age groups above age 30, the proportions never married were lower in 1981 than in 1961 for both males and females. Overall increases in the proportions never married were not as marked in the case of Pakistani males, which may be attributed to the fact that beginning in 1961 male marriage age was already considerably higher than female marriage age -- 23.6 years. Patterns of marriage behavior were expected to vary in the 4 provinces -- Punjab, Sind, NWFP, and Baluchistan -- because of differences in cultural patterns, levels of development, and urbanization. Punjab, the most developed province, contained the bulk of the proportion never married in the 15-19 age group, both in urban and in rural areas. The provincial differential in proportions never married was much greater for females than for males. Punjab had the highest proportions of never married females, followed by the NWFP and Sind, in both urban and rural areas. Urban Punjab had the least differences in average ages at marriage between men and women (4.6 years); in other provinces this difference was more than 5 years. As Table 4 demonstrates, there was a clear pattern of higher proportions never married by each level of education for both males and females in urban and rural areas. The extent of postponement in marriage was more pronounced among younger educated females in urban areas the among rural females. Although the number of persons sampled with 9 or more years of education were on the low side in rural areas, there was a distinct relationship between acquisition of education and marriage postponement there also. Male nuptiality behavior also varied by educational levels.^ieng

Dynamics of birth spacing in Pakistan.

PIP: The data of the Population Labour Force and Migration (PLM) Survey of 1979-1980 was analyzed in order to study spacing between births as well as differentials in urban and rural areas, differentials by age at marriage, urbanization, education, work pattern, and contraceptive use. The median intervals from PLM Survey of 1979-80 and Pakistan Fertility Survey (PFS) of 1975 birth histories were compared by urban and rural residence for each parity in order to analyze trends in birth spacing. Findings indicate only a slight increase in child spacing, the 1st birth interval of urban females has increased consistently by 1 month from 1st parity to 4th parity according to PLM 1979-80. Urban women had shorter spacing of births than rural females in both surveys. In addition, the Pakistan Demographic Survey of 1985-86 showed that the medium of the 1st birth interval declined to 18.09 months compared to the 22.46 months of PLM. Women in urban areas had a higher rate of fertility than women in rural areas where the interval between marriage and 1st birth increased. Urban females also had shorter duration of breastfeeding and tended to have shorter median birth intervals at higher ages at marriage. Longer intervals were attributed to subfecundity or delayed cohabitation. In rural areas women do not space their births deliberately. The medium birth interval of women in urban centers was 4 months shorter than those for rural females with the same education increasing by 7 months in case of no female education. Educated females in major urban areas achieved their family size faster than women residing in other urban and rural areas. Educated urban females with high parity (4+) had longer intervals due to greater contraceptive use and a diminished desire to have more children. In the urban areas of Punjab and Sind longer birth spacing was recorded than in the rural areas but differences disappeared after the 2nd parity. Obviously, women engaged in economic activity want to complete their family size earlier, thus the birth interval is shorter. Among contraceptive users, ever users had shorter median intervals, as this small group was more educated and wanted to complete family size in a short time. There are certain associations between variables, but the impact of changes of birth spacing on marital fertility in inconclusive.^ieng

Health care determinants of child survival in Pakistan.

PIP: The authors examined the relationships between selected individual and household level factors and the survival of children under five years old based upon data in the Pakistan Demographic and Health Survey of 1990-91. Analysis found that maternal age greater than 35 years enhances child survival. Mother's secondary and higher education have a net positive and significant influence upon child survival in urban areas only, with only a weak effect in rural areas; father's education is not a powerful net predictor of child mortality. Among health care factors, breastfeeding and having the child ever immunized are the most beneficial factors for both urban and rural children. Furthermore, household sanitary and living conditions do not appear to significantly influence the survival probability of children under five years old in both urban and rural areas.^ieng

Additional pressure drop at a bifurcation due to the passage of flexible disks in a large scale model.

The flow of red blood cells (RBC) through a microvascular capillary bifurcation was modeled in a large scale system in which rigid circular tubes and bifurcations (diameter = .95 cm) simulated capillaries and capillary bifurcations, flexible disks (undeformed diameter = 0.75 cm) simulated RBC and glycerol simulated plasma. At low Reynolds numbers (0.01 to 0.1), pressure drop was measured in the tubes upstream and downstream from the bifurcation as well as across the bifurcation itself, for various flow splits at the bifurcation while the inflow in the upstream tube was held constant. Pressure gradient across the bifurcation is taken to be the average of the upstream and downstream pressure gradients if the additional pressure drop at the bifurcation due to the partitioning of flow and disks is negligible. For the case of glycerol alone, the ratio of pressure gradient (G) at the bifurcation to the one at the upstream region was always greater than expected and reached 1.14 when the flow in the side branch was zero. With introduction of flexible disks into the system, G at the bifurcation was as much as 10 times the G at the upstream region as disks came in contact with, or close to, the dividing line of the bifurcation and paused momentarily before they entered one or the other side of the bifurcation. The largest G was for even flow split at the bifurcation and the smallest G was for the case where the flow in the side branch was smallest. Therefore, for the range of tube hematocrits (0-30 percent) and flow splits tested here, a significant additional pressure drop at the bifurcation is observed.

A "geographic information systems" based technique for the study of microvascular networks.

An automated system (ANET) has been developed to construct interactive maps of microvascular networks, calculate blood flow parameters, and simulate microvascular network blood flow using the geographic information systems (GIS) technology. ANET enables us to automatically collect and display topological, structural, and functional parameters and simulate blood flow in microvascular networks. The user-definable programming interface was used for the manipulation of drawings and data. Visual enhancement techniques such as color can be used to display useful information within a network. In ANET the network map becomes a graphical interface through which network information is stored and retrieved and simulations of microvascular network blood flow are carried out. We have used ANET to study the effects of ionizing radiation on normal tissue microvascular networks. Our results indicate that while vessel diameters significantly increased with age in control animals they decreased in irradiated animals. The tortuosity of irradiated vessels (16.3+/-1.1 mean+/-standard error of the mean) was significantly different from control vessels (10.0+/-1.3) only at 7 days postirradiation. Average red blood cell transit time was significantly different between control (1.6+/-0.6s) and irradiated (10.7+/-5.7s) microvascular networks at 30 days postirradiation. ANET provides an effective tool for handling the large volume of complex data that is usually obtained in microvascular network studies and for simulating blood flow in microvascular networks.