EORTC Late Effects Working Group. Overview of late effects normal tissues (LENT) scoring system.
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Diagnostic Imaging↗
Biomedical subjects
L F Fajardo
Publications and source records attributed to L F Fajardo.
Characterization and applications of the disc angiogenesis system.
A model to study microvascular proliferation, the Disc Angiogenesis System (DAS), consists of a synthetic foam disc implanted subcutaneously in experimental animals. After a period of growth, usually 7 to 21 days, the disc is removed. Planar sections are used to measure and characterize the growth. Microvessels grow centripetally into the disc, together with fibroblasts. Concentric growth zones have been defined by light and electron microscopy. Moderate growth occurs spontaneously and is accelerated by angiogenic stimulants placed in the center of the disc. Morphometric analyses have shown that vessel growth is directly proportional to total fibrovascular growth, so the former can be quantified by procedures measuring the latter. These include manual projection of sections and computer-assisted digital image analysis, which is recommended for routine use. The proliferation of endothelial and other cells is determined by incorporation of tritiated thymidine, using scintillation counting and autoradiography. Using the DAS, well-established angiogenic agents such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and prostaglandin E1 were found to increase proliferation of endothelial cells (EC) and microvessels. Heparin augmented the effect of bFGF. When used by itself heparin increased angiogenesis but not EC proliferation, in keeping with in vitro observations indicating that it stimulates migration but not proliferation of EC. Locally applied hyperthermia and ionizing radiation decreased angiogenesis, even when applied after the angiogenic stimulus. Systemic prostaglandin synthetase inhibitors antagonized the angiogenic effects of bFGF and EGF, in accordance with a postulated role of prostaglandins in the transduction of proliferative signals in microvascular EC. The DAS is easy to assemble and implant in small animals, including mice, which tolerate it well. Hence multiple discs can be used for each time or dose point, which allows reproducible measurements of vascular growth and increases statistical accuracy. Another advantage of the system is the capability of discriminating between proliferation and migration of EC and fibroblasts. The DAS can be used to test putative agonists or antagonists of angiogenesis. More generally, the DAS provides a model of wound healing, either uncomplicated or complicated by inflammation, and of angiogenic responses to solid tumors.
Animals↗
Dual role of tumor necrosis factor-alpha in angiogenesis.
The role of tumor necrosis factor-alpha (TNF; cachectin) in angiogenesis has been controversial. In vitro TNF inhibits proliferation of endothelial cells (EC) whereas in the cornea it appears to stimulate vessel growth. The authors tested TNF in their recently developed disc angiogenesis system (DAS), designed to measure the proliferation of microvessels. The DAS, implanted subcutaneously in mice, was either fitted with a central pellet containing mouse recombinant TNF (mrTNF), or exposed to mrTNF delivered subcutaneously by an osmotic minipump. Low doses of mrTNF (0.01-1 ng) induced angiogenesis, which was maximum at 0.1 ng, whereas high doses (1, and 5 micrograms) inhibited it. Subcutaneous mrTNF delivered at the (high) rate of 15-60 ng/hr for 14 days inhibited angiogenesis. These results indicate bimodal, dose-dependent opposing effects and explain some of the in vitro versus in vivo paradoxical results. TNF (native or exogenous) may have opposing effects on microvessels of neoplasms and inflammatory reactions, depending on its local tissue concentrations.
Animals↗
Cultured proliferating rat mammary epithelial cells.
Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occurred as the LA7 cells slowly died from the radiation. By labeling the cultures with 3H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of approximately 1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to approximately 19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco's modified Eagle's medium:Ham's F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no outgrowths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads.
Animals↗
Tumor necrosis factor in human disease.
The Scientific Board of the California Medical Association presents the following inventory of items of progress in pathology. Each item, in the judgment of a panel of knowledgeable physicians, has recently become reasonably firmly established, both as to scientific fact and important clinical significance. The items are presented in simple epitome and an authoritative reference, both to the item itself and to the subject as a whole, is generally given for those who may be unfamiliar with a particular item. The purpose is to assist busy practitioners, students, researchers, or scholars to stay abreast of these items of progress in pathology that have recently achieved a substantial degree of authoritative acceptance, whether in their own field of special interest or another. The items of progress listed below were selected by the Advisory Panel to the Section on Pathology of the California Medical Association, and the summaries were prepared under its direction.
Biomarkers, Tumor↗
Mechanism of antitumor activity of tumor necrosis factor alpha with hyperthermia in a tumor necrosis factor alpha-resistant tumor.
Cells from a radiation-induced fibrosarcoma (RIF-1) are exceedingly resistant to tumor necrosis factor alpha (TNF-alpha) in vitro. We tested whether the addition of mild hyperthermia (42.5 degrees C, 30 minutes) could enhance TNF-alpha activity against RIF-1 tumors growing in syngeneic hosts (C3H mice). TNF-alpha was administered intratumorally. Tumor cell killing essentially was not measurable following TNF-alpha, hyperthermia, or a combination of the two. Single-modality treatments also had no effect on tumor growth delay or on the x-ray dose (given 24 hours after the primary treatment) required to sterilize 50% of the tumors. The combination of TNF-alpha and hyperthermia, however, resulted in a marked increase in tumor doubling time and a highly significant reduction in the x-ray dose required to sterilize the tumors. Syngeneic lymph nodal lymphocytes and blood leukocytes did not appear to mediate the action of TNF-alpha on RIF-1 cells in vitro. Necrosis and hemorrhage were the most prominent histopathological alterations in the treated tumors. Electron microscopic studies 6 hours after therapy showed increased damage to capillary endothelial cells and accumulation of neutrophils in the capillaries of tumors treated with TNF-alpha with or without heat, suggesting that neutrophils may mediate the endothelial cell injury. These observations indicate a greater than additive tumoricidal effect of TNF-alpha with hyperthermia. Furthermore, they support the concept that the interaction between the two agents damages the vasculature, compromising the microcirculation and ultimately causing ischemic tumor necrosis.
Animals↗
Effects of X irradiation on angiogenesis.
We have evaluated the effect of X irradiation on the mesenchymal tissue growth (blood capillaries and stromal cells) in an angiogenesis system in the mouse. This was accomplished by implanting a polyvinyl alcohol sponge disc in the subcutis of the thorax, and quantifying the extent of growth reduction of capillaries and stromal cells following graded doses of X rays. The sponge disc contained a centrally located pellet impregnated with 20 micrograms of epidermal growth factor and coated with a thin film of slow-releasing compound. Total growth of vessels and fibroblasts was determined by morphometric analysis of histologic sections. The incorporation of [3H]TdR was measured during a 24-h period. A dose-response relationship was observed when X irradiation was given on Day 11 after implantation, with the disc removed on Day 20. A single dose of 15 Gy reduced both the rate of incorporation of [3H]TdR and the total growth area. These and previous observations point to endothelial cells as important targets of ionizing radiation in the stroma, especially during the period of active proliferation of these cells, induced by growth factors.
Animals↗
The complexity of endothelial cells. A review.
This review of the biology peculiar to endothelial cells (ECs) is based mainly on data available within the last decade. The functions described refer to angiogenesis, coagulation, and EC-platelet interaction, inflammation and immune response, synthesis of stromal components, vascular tone regulation, and miscellaneous metabolic activities. Emphasis is placed on the marked variability of ECs from tissue to tissue and from one species to another. This heterogeneity is evident morphologically, functionally, and in the response of ECs to injury.
Animals↗
Morphologic patterns of radiation injury.
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Humans↗
The unique physiology of endothelial cells and its implications in radiobiology.
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Animals↗
Hyperthermia inhibits angiogenesis.
Since in vitro studies have demonstrated that capillary endothelial cells are thermosensitive, experiments were performed to determine the (in vivo) heat sensitivity of blood capillaries and their endothelial cells. Angiogenesis discs were implanted subcutaneously in mice, and vascular growth was stimulated by slow release of epidermal growth factor placed in the center of each disc. After 5 days of growth the discs were subjected to radiofrequency-induced hyperthermia. Heat exposures were 41, 42, 43, and 44 degrees C for 30 min. Control discs were sham treated. Seven days after heating the discs were extracted and paraffin embedded. Centripetal (radial) vessel growth was measured in magnified medial planar sections. An inverse relationship was demonstrated between vessel growth and exposure temperature. The extent of the fibroblastic growth was also inversely proportional to temperature. Thus, at least in this system, the microvasculature shows dose-dependent damage by hyperthermia, consistent with preceding in vitro observations. This inhibition of angiogenesis may result from endothelial cell killing, interference with cell replication, inhibition of cell migration, or a combination of these mechanisms.
Animals↗
The disc angiogenesis system.
A new system for the study of angiogenesis in vivo has been devised. It consists of a small disc of polyvinyl alcohol foam, covered on both flat sides by Millipore filters, leaving only the edge as the area for cell penetration into the disc. Angiogenic agonists or antagonists, as well as other substances to be studied, are placed in the center of the disc. The slow release of these substances is maintained by a film of ethylene-vinyl acetate co-polymer, or by the use of agarose. The disc is implanted subcutaneously in the host animal through a distant skin incision. In mice, the optimal times for examination of the discs are 7 to 12 days after implantation for discs containing angiogenic stimulants and 12 to 20 days for those without stimulants. After a period of growth is completed, the disc is removed, fixed, and embedded in paraffin or methacrylate. Medial plane sections, stained by a variety of methods, are used to observe and measure the growth of vessels and stroma into the disc. Whether stimulated or not, this growth is centripetal and can be easily quantitated by simple morphometric technics. This system has already been used in mice, to study the proliferation of vessels and fibroblasts into discs devoid of, or containing angiogenic stimulants (epidermal growth factor, acidic fibroblastic growth factor). We have also utilized the discs to demonstrate the inhibition of vessel growth by hyperthermia. Examples of these applications are presented. The disc angiogenesis system is easy to prepare, inexpensive, and well tolerated, at least by mice. Its simplicity and reproducibility make it suitable for a wide range of applications beyond those described here.
Angiogenesis Inducing Agents↗
Vascular lesions following radiation.
The special radiation sensitivity of the vascular system is mainly linked to that of endothelial cells, which are perhaps the most radiation-vulnerable elements of mesenchymal tissues. Within the vascular tree, radiation injures most often capillaries, sinusoids, and small arteries, in that order. Lesions of veins are observed less often, but in certain tissues the veins are regularly damaged (e.g., intestine) or are the most affected structures (i.e., liver). Large arteries do suffer the least; however, when significant damage does occur in an elastic artery (e.g., thrombosis or rupture), it tends to be clinically significant and even fatal. Although not always demonstrable in human tissues, radiation vasculopathy generally is dose and time dependent. Like other radiation-induced lesions, the morphology in the vessels is not specific, but it is characteristic enough to be often recognizable. Vascular injury, especially by therapeutic radiation is not just a morphologic marker. It is a mediator of tissue damage; perhaps the most consistent pathogenetic mechanism in delayed radiation injury.
Animals↗
Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria.
Tumor necrosis factor, or cachectin (TNF-alpha), a protein with a wide range of biological activities, is produced mainly by macrophages and may be important in inflammatory processes. The role of TNF-alpha in the pathogenesis of cerebral malaria was investigated in a murine model. Most CBA mice infected with Plasmodium berghei anka die between days 6 and 14 with acute neurological manifestations unrelated to the level of parasitemia, whereas mice of some other strains have malaria of the same severity that ends in death after 3 to 4 weeks without neurological manifestations. The activity of serum TNF-alpha was considerably increased in CBA/Ca mice with cerebral malaria but not in Plasmodium berghei-infected mice that did not develop this complication. One injection of rabbit antibody to TNF-alpha on day 4 or 7 fully protected infected mice from cerebral malaria without modifying the parasitemia, whereas immunoglobulins from normal rabbit had no effect. In mice with cerebral malaria, the cerebral vessels showed focal accumulations of packed macrophages often containing infected erythrocytes; this lesion was not seen in mice treated with antibody to TNF-alpha or in untreated mice without cerebral malaria. These findings indicate that TNF-alpha has an important role in the pathogenesis of cerebral malaria in this murine model and suggest that local accumulation and activation of macrophages may lead to the predominance of lesions in the central nervous system.
Animals↗
Von Willebrand factor levels do not predict or diagnose radiation pneumonitis.
In search for an index of endothelial injury that would provide an early diagnosis of radiation pneumonitis, we investigated the plasma levels of von Willebrand Factor (Factor VIII Related Antigen, FVIII:RAg) in 14 patients undergoing pulmonary irradiation. This study was based on observations indicating that damage to the endothelium-rich pulmonary parenchyma may produce alterations in the synthesis, storage or release of FVIII:RAg, detectable in plasma. There was no correlation between FVIII:RAg levels and radiation pneumonitis, radiation dose, volume of irradiated lung, tumor burden, or time-interval between exposure and sampling. The heterogeneity of the neoplasms and the inconstant effects of radiation in the tumor vasculature are among several variables that may explain this lack of correlation. The plasma levels of FVIII:RAg cannot be used to diagnose or predict radiation pneumonitis.
Adolescent↗
Ionizing radiation and neoplasia.
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Animals↗
Correlation of the dermal microvasculature morphology with the epidermal and the endothelial population changes produced by single X ray fractions of 1649, 2231 and 2619 rad in swine.
The role of the dermal microvasculature in the production of skin changes has not been specified. The healing moist reaction observed between 21 and 36 days following X ray exposures of 1649, 2231, and 2619 rad results from the loss and repopulation of the epidermal basal cells. The second breakdown occurring between 36 and 70 days results from the loss of endothelial cells in vessels of the papillary plexus. The epidermal population in swine skin decreases linearly to a nadir at 23 days and returns to control levels (and beyond) exponentially at 28-32 days. During this period, the endothelial cell density remains at control levels. At 28-36 days there is an abrupt decrease to 50% of control levels following administration of 1649 rad and a further decrease to 0 after 2231 and 2619 rad by 70 days. The qualitative morphology of the microvasculature remains unchanged until 32-36 days following irradiation. At that time there is increased endothelial cell pyknosis and loss of endothelial cells. A decrease in the vascular lumen cross-section profile density occurs by 43-49 days. This is accompanied by a progressive vascular dilatation. Interstitial edema becomes marked, with an inflammatory infiltrate of varying amount. Micro thrombi are found after 2619 rad, but are nearly absent after 1649 rad.
Animals↗
Safety of 125iodine and 192iridium implants to the canine carotid artery: preliminary report.
Thirty-nine healthy dogs underwent a simulated radical neck dissection followed by implantation of either 125Iodine (125I) or 192Iridium (192Ir) in various dose regimes randomized prospectively from 3,000 to 30,000 rad. Bilateral selective carotid angiography was performed immediately postoperatively and at six months and one year. No significant effects occurred to the animals who received 15,000 rad 125I or 6,000 rad 192Ir. In the higher dosed animals the 125I treated group fared better than the 192Ir treated group, probably due to the lower dose rate delivery. All surviving animals will be maintained an additional year to determine the late effects of brachytherapy irradiation to the carotid artery.
Animals↗