Performing SELEX experiments in silico.

Due to the sequence-dependent nature of the elasticity of DNA, many protein-DNA complexes and other systems in which DNA molecules must be deformed have preferences for the type of DNA sequence they interact with. SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments and similar sequence selection experiments have been used extensively to examine the (indirect readout) sequence preferences of, e.g., nucleosomes (protein spools around which DNA is wound for compactification) and DNA rings. We show how recently developed computational and theoretical tools can be used to emulate such experiments in silico. Opening up this possibility comes with several benefits. First, it allows us a better understanding of our models and systems, specifically about the roles played by the simulation temperature and the selection pressure on the sequences. Second, it allows us to compare the predictions made by the model of choice with experimental results. We find agreement on important features between predictions of the rigid base-pair model and experimental results for DNA rings and interesting differences that point out open questions in the field. Finally, our simulations allow application of the SELEX methodology to systems that are experimentally difficult to realize because they come with high energetic costs and are therefore unlikely to form spontaneously, such as very short or overwound DNA rings.

The anatomy of the thoracic spinal canal investigated with magnetic resonance imaging (MRI).

BACKGROUND AND OBJECTIVES: Anesthesiologists are reluctant to consider higher levels for spinal anesthesia, largely due to direct threats to the spinal cord. The goal of this study is to investigate, with magnetic resonance imaging (MRI), the distances between the relevant structures of the spinal canal (spinal cord, thecal tissue, etc.) to determine modal anatomical positions for neuraxial anesthesia. METHOD: A group of 19 patients were imaged with an MRI scanner in supine position. Medial sagittal slices of the thoracic and lumbar spine were measured for the relative distances between anatomical structures, including epidural space, dura, and spinal cord. RESULTS: The posterior dura - spinal cord distance is significantly greater in the middle thoracic region than at upper and lower thoracic levels (e.g. T6 9.5 +/- 1.8 mm, T12 3.7 +/- 1.2 mm, p < 0.001, T1 4.7 +/- 1.7 mm, p < 0.001). There is variation in modal distances between the structures important for neuraxial anesthesia, at different levels of the spinal canal. CONCLUSIONS: The spinal cord tends to follow the straightest line through the imposed geometry of the spine. Considering the necessary angle of entry of the needle at mid-thoracic levels, there is relatively (more than at upper thoracic and lumbar levels) substantial separation of cord and surrounding thecal tissue. Anesthesiologists perform spinal blockades up to the L2-L3 interspace, but avoid higher levels for fear of neurological damage. The information that there is substantially more space in the dorsal subarachnoid space at thoracic level, might lead to potential applications in regional anesthesia. In contrast, the cauda equina sits more dorsally in the lumbar region.

Effects of hyperthermia on the central nervous system: what was learnt from animal studies?

Animal studies show that nervous tissue is sensitive to heat. Although inter-species variations may play a role, the data indicate that the maximum heat dose without obvious complications after localized hyperthermia in regions of the central nervous system (CNS) lies in the range of 40-60 min at 42-42.5 degrees C or 10-30 min at 43 degrees C. Expression of thermotolerance after a 'conditioning' heat dose was clearly observed in the spinal cord of rodents and the thermotolerance ratio's (ratio between heat doses with and without conditioning required to obtain a certain defined effect) were high, approximately 2. The thermotolerant state of CNS is shown to protect also against other types of injury as well: pre-treatment of rats with hyperthermia protected against spinal cord ischemic injury. During the rather long period required for temperature elevation which is inherent to WBH, some degree of thermotolerance may develop. The correlation between thermotolerance and hsp70 induction in CNS is obvious. Heat, at least if applied shortly after X-rays, enhances the response of nervous tissue to radiation. Data on the combined effects of X-ray irradiation and hyperthermia on rodent spinal cord clearly show that the radiation response can be enhanced with a factor of 1.1-1.3. There are no clear experimental data indicating an increase in adverse effects specific to the CNS after localized or whole body hyperthermia as a result of combined treatment with chemotherapy.

Histopathology of ventricles, coronary arteries and mast cell accumulation in transverse and longitudinal sections of the rat heart after irradiation.

The aim of the study was to compare cross sections with longitudinal sections in histopathological examination of the rat heart after irradiation, to find the most optimal method for the detection of cardiac radiation injuries. For this purpose, rats were irradiated locally on the heart with a single dose of 0 or 20 Gy. At different time points after irradiation, hearts were perfused and cut into longitudinal or cross sections. In both sections, several histopathological changes were scored on a graded scale between 0 and 3. Mast cells, which are thought to play a role in tissue remodelling, were counted. After 20 Gy, frequently occurring lesions were most severe in the upper half of the ventricles and the septum. These lesions could only be detected when using longitudinal sections, resulting in a higher total histopathological score than the examination of a single cross section. From 3 months onwards, changes in coronary arteries of irradiated hearts included endothelial cell loss, a loss of smooth muscle cells and fibrosis in media and adventitia. Up to 1 month after irradiation, mast cell densities of the left and right ventricles were decreased after 15 and 20 Gy, compared to time-matched controls, followed by increases from 3 months onwards. In the left ventricle, mast cell densities correlated with myocardial degeneration and fibre loss. The results of this study show that the usage of a single longitudinal section in the histopathological examination of the irradiated rat heart leads to the recognition of more severe injuries, including myocardial degeneration and fibrosis, in ventricular tissue than the usage of a single midventricular cross section. Morphological changes observed in coronary arteries of irradiated hearts might lead to a decreased compliance of the coronary artery wall. Further investigation is needed to determine the role of mast cells in cardiac tissue remodelling after irradiation.

Irradiation of mechanically-injured human arterial endothelial cells leads to increased gene expression and secretion of inflammatory and growth promoting cytokines.

Radiation therapy is applied to inhibit neointima formation after percutaneous transluminal coronary angioplasty (PTCA). In this study, we evaluated the effect of irradiation on re-endothelialisation of circular denuded tracks made in post-confluent cultures of arterial endothelial cells (ECs) and on cellular factors involved in this process. Image analysis and time-lapse microcinematography revealed cell migration into denuded areas starting 4h after injury. Fifty percent coverage was achieved at 14.8 +/- 2.0 h. Using competitive PCR and flow cytometry techniques, no significant changes in mRNA expression of interleukin-1beta (IL-1beta), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B) and tissue factor (TF), and surface molecule expression of anti-intercellular adhesion molecule-1 (ICAM-1), anti-vascular cell adhesion molecule-1 (VCAM-1), anti-platelet/endothelial cell adhesion molecule-1 (PECAM-1), MHC-1, TF and Fas were observed. However, injury did significantly (P < 0.05) elevate the release of IL-8 and FGF-2 protein in the cell culture supernatant, as assessed by ELISA. Radiation (15Gy) given immediately after injury did not affect the kinetics of re-endothelialisation up to 48 h, in spite of the fact that no cell divisions were observed. Thereafter cell density decreased and cultures deteriorated. Compared to cultures exposed to injury alone, radiation induced significant (P < 0.05) increases in mRNA levels of IL-8 (1.35 +/- 0.10-fold increase at 4h), FGF-2 (1.62 +/- 0.10-fold at 4h; 1.76 +/- 0.33-fold at 24h) and IL-1beta (2.76 +/- 0.40-fold at 24h), whereas mRNA levels of TGF-beta1, PDGF-A and PDGF-B increased about 1.2-fold. IL-8 and FGF-2 protein concentrations in the media were higher than those observed in non-irradiated injured cell cultures; however, this difference was not significant. Radiation induced a 2.3 +/- 0.3-fold increase (P < 0.05) in Fas surface expression only. In conclusion, irradiation of mechanically-injured human EC leads to increased gene expression and protein secretion of inflammatory and growth promoting cytokines.

Effects of hyperthermia on the peripheral nervous system: a review.

The present paper overviews the current knowledge about effects of hyperthermia at temperatures used in clinical oncology on the peripheral nervous system. From the experimental studies it may be concluded that the heat sensitivity of the nerve is determined by the sensitivity of the nerve vasculature. These studies show that in order to avoid induction of severe neuropathy, application of heat to the peripheral nerves should not be in excess of doses of 30 min at 44 degrees C or equivalent. Using modern equipment for application of loco-regional hyperthermia the incidence of even mild neurological complications is very low. In hyperthermic isolated limb perfusion (HILP) neurotoxicity is an often-mentioned side effect, this is in spite of the fact that in all studies a relatively mild hyperthermic temperature is used that, based on the experimental studies, should be well tolerated by the nerves and other normal tissues in the limbs. It seems that the neurotoxicity observed after HILP results from thermal enhancement of drug toxicity, very probably combined with effects of a high tourniquet pressure that is used to isolate the blood flow in the leg. Whole body hyperthermia (WBH), using anesthesia and appropriate monitoring to avoid cardiovascular stress is at present considered a safe procedure. Still in the recent past cases of neuropathy after treatment have been described. When chemotherapy, and notably cisplatin, is administered before or during hyperthermia there are several clinical and experimental observations that indicate a limited tolerance of the peripheral nervous tissue in such case. Also previous radiotherapy may limit the tolerance of nerves to hyperthermia, notably when radiation is applied with a large field size. Experimental studies show that combined treatment with radiation and heat leads to enhancement of effects of radiation (enhancement ratio approximately 1.5 at 60 min at 44 degrees C). A clear contraindication for the application of hyperthermia in patients is the presence of a neurodegenerative disease, such as multiple sclerosis. Vigilance is also required in the treatment of diabetic patients with hyperthermia, this based on experimental animal studies, but so far no clear clinical data are available.

Effects of multiple doses of ionizing radiation on cytokine expression in rat and human cells.

PURPOSE: To determine the effect of daily fractionated irradiation on the expression of growth factors and cytokines in different cardiac and vascular cell types. MATERIALS AND METHODS: Cell cultures of rat cardiac myocytes, fibroblasts, a rat cardiac microvascular endothelial cell line and human artery endothelial cells were irradiated with doses of 2 Gy, given daily during 5 consecutive days. Twenty-four hours after each fraction, gene expression was determined by competitive or semiquantitative polymerase chain reaction. Protein secretion into culture media was determined by enzyme-linked immunoabsorbant assay. RESULTS: Of all investigated mRNA levels, transforming growth factor (TGF)-ss1 and fibroblast growth factor (FGF)-2 were slightly upregulated in the rat cardiac endothelial cell line after irradiation. TGF-ss1 protein secretion by these cells was slightly, but non-significantly, elevated. Interleukin 1ss protein levels in myocyte culture media were decreased in control cultures at days 3 and 4 compared with day 2. No significant changes were observed in expression of FGF-2 in either of the four cell types. Moreover, no changes were observed in gene expression of platelet-derived growth factors A, B and interleukin 8 in the human artery endothelial cells. CONCLUSIONS: Fractionated irradiation leads to minor changes in the expression of specific cytokines in cardiac myocytes, fibroblasts and endothelial cells.

Separation of neonatal rat ventricular myocytes and non-myocytes by centrifugal elutriation.

The preparation of pure cardiac myocyte cultures from neonatal rats is hampered by the presence of non-myocytes, which can proliferate during culturing, thereby causing a progressive decrease in the proportion of myocytes. In order to obtain myocyte cell suspensions of high purity, a method based on centrifugal elutriation was developed. Cardiac cells, isolated from neonatal rat heart ventricles, were subjected to elutriation using flow rates that increased step-wise from 20 to 80 ml/min. The cell fraction obtained at 80 ml/min consisted of 68-90% myocytes. Still, upon culturing, the remaining non-myocytes proliferate, causing the proportion of myocytes to decrease to 60 +/- 2% at day 5. A second elutriation protocol was developed in which myocytes and non-myocytes were separated after a period of co-culturing for 4-5 days. By this approach a fibroblast-rich cell fraction (87 +/- 5%) and a myocyte-rich cell fraction (82 +/- 6%) were obtained. In conclusion, centrifugal elutriation creates the opportunity to separate neonatal rat myocytes from non-myocytes, either freshly isolated or after a period of culturing. Particularly, cell separation after a period of culturing ventricular cells offers an advantage to analyse the experimental effects on myocytes and non-myocytes separately.

Radiation-induced changes in gene expression and distribution of atrial natriuretic peptide (ANP) in different anatomical regions of the rat heart.

PURPOSE: To examine the effects of whole-heart irradiation on gene expression and distribution of atrial natriuretic peptide (ANP) in atrial appendages and left ventricles of the rat heart. MATERIAL AND METHODS: Female Sprague-Dawley rats were irradiated with a single dose of 0, 15 or 20 Gy locally to the heart. At intervals up to 16 months post-irradiation, the localization of ANP was examined using immunohistochemical techniques. Absolute mRNA concentrations were determined using the competitive PCR assay. RESULTS: Histological and immunohistochemical studies demonstrated that whole-heart irradiation caused a reduction of atrial ANP, which is due to a substantial loss of ANP-producing atrial myocytes and accumulation of collagen (replacement fibrosis). On the other hand, ANP became detectable in the subendocardium of the irradiated left ventricles. Positive staining of ANP was mainly found in the proximity of injured areas consisting of degeneration of myocytes and fibrosis. At the transcriptional level, reduction of atrial ANP expression at 1 month post-irradiation was followed by recovery at 3 months. Thereafter, ANP mRNA concentrations followed the mRNA pattern of controls and even appeared to increase at 16 months. In the left ventricle, dose-dependent and progressive elevation of ANP gene expression could be observed during the observation period and reached a 20-fold increase as compared with sham-irradiated age-matched controls. CONCLUSIONS: The distribution of ANP after local irradiation of the rat heart depends on the severity of the pathological/structural changes (i.e. myocyte degeneration and fibrosis). In radiation-induced heart disease, elevated ANP expression in the left ventricle is most probably involved in the observed chronic elevation of plasma ANP levels.

Effects of ionizing radiation on gene expression in cultured rat heart cells.

PURPOSE: To determine the in vitro effect of ionizing radiation on TGF-beta1, FGF-2, IL-1beta, atrial natriuretic peptide (ANP) and procollagen types I and III gene expression in three different cell types of rat heart. MATERIALS AND METHODS: Primary cell cultures of myocytes and fibroblasts and cultures of a rat heart endothelial cell line (RHEC) were irradiated with single doses of 2.0, 8.5 or 15 Gy. At different time-points after irradiation (4-336 h), gene expression was analysed using a competitive PCR technique. RESULTS: Irradiation of cultured rat heart cells may lead to temporary changes in expression of the genes studied. Analysis of the radiation response of cultured myocytes, cardiac fibroblasts and rat heart endothelial cells reveals different responses with regard to (1) the dose necessary to evoke changes in mRNA expression, (2) the level of and (3) the duration of the 'induced' response. The changes observed were small and between parallel experiments the onset and time-course of the induced gene expression varied between 4 and 48 h. The average expression of TGF-beta1 mRNA between 4 and 48 h was significantly elevated in endothelial cells after a dose of 2.0 Gy, in fibroblasts after a dose of 8.5 Gy and in myocytes after a dose of 15 Gy. Down-regulation of TGF-beta1 mRNA in myocytes was observed after a dose of 8.5 Gy. FGF-2 and procollagen type-I mRNAs were significantly elevated in fibroblasts after a dose of 2.0 Gy. For all three cell types, no effect of dose on the timing or size of the gene expression was observed. CONCLUSIONS: Although irradiation of cultured heart cells influences expression of genes involved in tissue remodelling, the observed differences were too small and too restricted in time and dose to explain the exact role of these cell types in processes leading to radiation-induced cardiac fibrosis.

Muscle wasting after radiotherapy in young and adult rats.

The impact of late muscular injury after radiotherapy on the well being of patients, particularly those treated at a relatively young age, is often underestimated. To extend the present knowledge, a detailed comparative study of the radiation response of skeletal muscles was performed. The hind limb of rats was irradiated at the age of 30-60 days (young), at approximately 200 days (adult) or at the age of >360 days (old), with single doses ranging from 15-60 Gy. Muscular wet weight, volume and tibia length was followed up to 12 months post-treatment. Radiation treatment resulted in muscular wasting, which was more severe at higher doses. The radiation response depended strongly on the age of the animal at the time of treatment. Radiation induced muscle wasting and stunted tibia growth were more prominent in young animals than in adult or in old rats. ED50 values based on normalized wet weight were 12.5 (11.5-13.6) Gy for young animals and 22.5 (21.8-23.3) Gy for adult animals. The gastrocnemius muscle of young animals appears to be more radiosensitive than the gastrocnemius muscle of adult or old animals. Furthermore, in young animals, severe wasting of the muscles may already occur after relative low radiation doses (single dose of 15 Gy).

Circulating atrial natriuretic peptide plasma levels as a marker for cardiac damage after radiotherapy.

PURPOSE: To investigate whether plasma concentrations of atrial natriuretic peptide (ANP) could be used to identify patients with radiation mediated cardiac dysfunction. MATERIALS AND METHODS: Circulating levels of ANP were measured in patients who have been irradiated on a large part of the heart (50-80%; Hodgkin's disease) or smaller part of the heart (20-30%; primary breast cancer). C-terminal ANP was determined by radioimmunoassay (RIA) using a commercial kit. RESULTS: In this study ANP plasma levels of 121 patients (Hodgkin's disease, 73 patients; breast cancer, 48 patients) and 67 controls were examined. ANP plasma levels of both Hodgkin patients (28.8+/-2.2, P=0.003) and breast cancer patients (20.4+/-2.8 ng/l, P=0.01) were significantly elevated when compared to age-matched controls (13.5+/-1.2 ng/l). Both for the Hodgkin (R=0.42, P=0.05) and breast cancer group (R=0.50, P=0.09) a positive relation between ANP plasma values and age was found. However, no clear relation between ANP plasma levels and time post treatment could be demonstrated. Patients with clinical symptoms of cardiovascular disease (n=25) had significantly higher ANP plasma levels (P<0.001) compared to patients in the same treatment group without evidence of cardiac disease (50.2+/-7.5 vs. 23.3+/-1.3 ng/l, P<0.001, and 38.2+/-12.4 vs. 16.3+/-1.6 ng/l, P<0.001, for Hodgkin's disease and breast cancer, respectively). Eight patients suffered from essential hypertension (n=8), whereas the remaining group of 17 patients showed a variety of cardiac disorders (i.e. myocardial infarction, decreasing ventricular function, and atrial fibrillations). In 11 patients cardiac problems were manifest either before or within a few years after mediastinal therapy. In two patients treated for Hodgkin's disease, and in four patients treated for breast cancer cardiac problems became manifest a long time (>10 years) after radiotherapy. Probably in this group of patients cardiac problems are related to the therapy. CONCLUSIONS: The present study indicates that ANP plasma levels could be used to identify patients with radiation induced cardiac dysfunction.

Structural changes in the auricles of the rat heart after local ionizing irradiation.

BACKGROUND AND PURPOSE: Irradiation of the heart may lead to late cardiovascular complications and depending on the dose to cardiac-related death. There is increasing evidence that left atrial appendages play an important role in left ventricular filling especially in cardiac disease. The aim of the present study was to investigate the radiation response of the atria of the rat heart (auricles in particular) at morphological, histological and transcriptional level. MATERIAL AND METHODS: Sprague-Dawley rats were irradiated with a single dose locally on the heart (0-22.5 Gy). End-diastolic diameters of left auricles were measured during evaluation of cardiac function. Histopathological evaluations were performed at various time points up to 16 months post irradiation. Changes in mRNA expression of procollagen types I and III and pro-fibrogenic cytokines (TGF-beta1 and IL-1beta) were investigated using competitive PCR. RESULTS: Irradiation leads to a dose-dependent decrease in end-diastolic diameter of the left auricles. This decrease was observed at 4 months post-irradiation, where no gross damage of the ventricle has been reported. Histologically, epicardial fibrosis was found already 1 month post irradiation, and the frequency/severity of the structural changes appeared to be dose-dependent and progressive with time post irradiation. At 9 months, fibrosis was observed in all three layers (epicardium, myocardium and endocardium) of both auricles. On the level of gene expression, increases in procollagen types I and III were observed at 12 and 3 months post irradiation, respectively. Increases in IL-1beta and TGF-beta1, cytokines known to influence collagen deposition at different levels, preceded the upregulation of procollagen mRNA. CONCLUSIONS: Auricles of the rat heart show a marked pathological response to ionizing radiation, characterized by generalized accumulation of collagen (fibrosis) and a reduction of end-diastolic diameter. The reduction of auricular volume and loss of elasticity will negatively contribute to the pump function of the irradiated ventricle.

The influence of the oncogene N-ras on cell cycle delay in a human melanoma cell line with reduced radioresistance.

In a previous study we found that transfection of a human melanoma cell line with the oncogene N-ras led to increased radiosensitivity as measured by clonogenic assays. Since a shift in radiosensitivity is often correlated with altered G2/M delay, we investigated whether this was also the case in this oncogene containing melanoma cell line (IGRras). A human melanoma cell line, stably transfected with mutated N-ras, and its parental cell line transfected with the neomycin phosphotransferase gene only (IGRneo), were irradiated with 5 Gy and cell cycle distribution was measured at hourly time intervals by DNA staining with propidium iodide. Next, the effect of ionising radiation on the duration of the S-phase was determined by pulse labelling cells with BrdUrd before irradiation. Both cell lines showed a radiation induced G2/M delay, which was most prolonged for the ras transfected cell line. After 5 Gy, the S-phase duration was unaltered, although the shape of the relative movement (RM) curves was slightly different. No G1 delay was observed in either cell line. Ras transfection in a melanoma cell line leads to prolonged G2/M delay after radiotherapy. This prolongation is associated with increased radiosensitivity and not with radioresistance. These data throw doubt on the use of oncogene expression or G2/M delay as predictors of radiosensitivity.

Myocardial enzyme activities in plasma after whole-heart irradiation in rats.

Plasma levels of myocardial enzymes present after local heart irradiation were studied in a rat model. The purpose was to investigate whether, within days after irradiation, these enzyme levels change to such an extent that they may be helpful in assessing the severity of cardiac damage after radiotherapy. Therefore, activities of creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) were determined in the plasma and left ventricular myocardium of rats following local heart irradiation with a single dose of 20 Gy. A dose of 20 Gy is known to cause irreversible cardiac damage and to reduce survival times of the animals. Cardiac enzyme assays were performed directly after and twice daily for up to 2 weeks after radiation. Plasma CK, LDH, AST and alpha-HBDH levels were increased between 2 h and 24 h after irradiation. Plasma ALT levels remained unchanged. Myocardial enzyme levels, measured between 24 h and 16 days after radiation, did not differ between irradiated and control animals, although acute (first 12 h) reductions were observed in the irradiated group. The elevated enzyme levels in plasma appeared to correlate with the acutely reduced myocardial enzyme levels. Although irradiation with a dose of 20 Gy induced acute rises of cardiac enzyme levels in plasma, it is doubtful that fractionated radiation, as applied clinically for treatment of solid tumors, will induce plasma enzyme elevations that are large enough to indicate the extent of cardiac damage occurring acutely or chronically.

Changes in transforming growth factor-beta (TGF-beta 1), procollagen types I and II mRNA in the rat heart after irradiation.

PURPOSE: To study alteration in gene transcription (transforming growth factor-beta 1 and procollagen types I and III) involved in radiation-induced cardiac damage. MATERIALS AND METHODS: Female Sprague-Dawley rats were irradiated with a single dose of 0, 15, 20 or 25 Gy locally on the heart. At intervals up to 16 months after irradiation, absolute amounts of mRNA were quantified using a (semi-nested) competitive PCR assay. All values were normalized to equal input cDNA with respect to their GAPDH content. RESULTS: After irradiation, left ventricular TGF-beta 1 mRNA levels increased sharply. This response was bi-phasic with peaks at days 1 and 12 (maximum 6-fold baseline), then returning to control levels by 1 month. After 20 Gy, a persistent elevation was observed from 6 months, but this elevation was less profound (approximately 1.5-fold baseline) when compared with the early response (1-12 days). Absolute mRNA levels of procollagen type I hardly changed during the first 6 months, but thereafter these levels increased progressively until the end of observation. An age-related increase in procollagen I was also observed. Procollagen type III mRNA levels were increased between days 1 and 12, returned to control values and remained low up to 6 months, then mRNA levels rose again with increasing time post-treatment. CONCLUSION: The difference in time-course between TGF-beta 1 and procollagen mRNA expression after local heart irradiation and ageing strongly suggest that the late up-regulation of both procollagen types in the left ventricle occurs without TGF-beta 1 over-expression.

Detection and quantitative measurement of transforming growth factor-beta1 (TGF-beta1) gene expression using a semi-nested competitive PCR assay.

A polymerase chain reaction (PCR) technique was optimized for detection and quantification of very low concentrations (down to a few molecules) of transforming growth factor beta1 (TGF-beta1) mRNA. The strategy involved a combination of a competitive PCR assay and a semi-nested PCR. In the present study, the semi-nested PCR technique was tested in several rat organs containing different concentrations of target mRNA. A control fragment for TGF-beta1 was used to correct for differences in amplification of various cDNA samples. TGF-beta1 mRNA levels were also corrected according to the abundance of the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same samples. The differences of sensitivity among the standard (one-step) and semi-nested (two-step) competitive PCR assays for the detection of TGF-beta1 are discussed. In conclusion, the semi-nested PCR protocol provides greatly enhanced sensitivity over standard PCR analysis. It is a reproducible and very specific method for quantification of only a few molecules of TGF-beta1 mRNA in a background of non-target molecules.

Changes in circulating atrial natriuretic peptide in relation to the cardiac status of Rhesus monkeys after total-body irradiation.

PURPOSE: In order to determine the presence of cardiac damage associated with total-body irradiation (TBI), both echocardiographic parameters and circulating levels of atrial natriuretic peptide (ANP) were measured in three different age-cohorts of Rhesus monkeys (Macaca mulatta) previously treated with TBI without additional chemotherapy, at post irradiation intervals up to 30 years, at the former TNO/Radiobiological Institute at Rijswijk. MATERIALS AND METHODS: Standard echocardiographic techniques were used to measure cardiac dimensions and left ventricular function in situ. Plasma-ANP concentration was measured by radioimmunoassay (RIA). After necropsy, tissue samples from the heart were taken for histological analysis. RESULTS: Plasma-ANP levels of animals which received TBI were significantly (P = 0.0005) elevated when compared to age-matched controls (66.4 +/- 8.4 vs. 33.1 +/- 5.7 ng/l). Moreover, a positive correlation (P = 0.032) between plasma-ANP values and time post treatment was found in the TBI group. TBI affected cardiac dimensions; however, no significant differences in cardiac functional parameters were observed between the different treatment groups. Necropsy reports demonstrated slight but consistent cardiovascular damage in several animals treated with TBI, in terms of increased incidence of mild epicardial and coronary arterial wall fibrosis, compared to age-matched controls. CONCLUSIONS: The concentration of plasma-ANP proved to be an important parameter for subclinical cardiac damage. In humans, serial determinations of plasma ANP in individual patients might provide relevant information about the cardiac status after TBI.

Additive effect of concomitant multiple low-dose doxorubicin and thoracic irradiation on ex vivo cardiac performance of the rat heart.

The effects of doxorubicin alone or combined with local heart irradiation on ex vivo cardiac performance were studied in Sprague-Dawley rats. Rats were treated with doxorubicin either administered as a single bolus injection or administered weekly during a period of 10 weeks. In "combined" experiments, local heart irradiation with a single dose of 15 Gy was given prior to drug administration. Evaluation of cardiac performance was performed 14 weeks after initiation of treatment. At drug doses that were tolerated by the rat, single injections with doxorubicin (sd-DXR; up to a dose of 5 mg/kg) did not lead to a change in cardiac performance whereas multiple injections with low-dose doxorubicin (mld-DXR; up to a cumulative dose of 20 mg/kg) led to a dose-dependent decrease in cardiac function. Extracardial toxicity as a result of mld-DXR (cumulative dose < or =15 mg/kg) was mild when compared to the toxicities observed after sd-DXR (5.0 and 7.5 mg/kg). When administration of mld-doxorubicin was preceded by 15 Gy, cardiac performance further decreased. The present data indicate that the interaction between doxorubicin and local heart irradiation with a dose of 15 Gy is additive, when the treatments are given concomitantly. Irradiation did not lead to an increase of DXR-mediated extracardial toxicities. The isolated working rat heart preparation offers a reliable method to evaluate the effects of doxorubicin and new anthracycline analogue on the heart.

Comparison of in vivo cardiac function with ex vivo cardiac performance of the rat heart after thoracic irradiation.

The aim of the study was to compare in vivo cardiac function with ex vivo cardiac performance after local heart irradiation in the same rat. Left ventricular ejection fraction (LVEF) was measured in vivo by radionuclide ventriculography in Sprague-Dawley rats up to 16 months after a single dose of 20 Gy. Four days after in vivo measurements, cardiac performance was determined ex vivo, using the isolated working rat heart preparation. After irradiation, cardiac performance measured ex vivo deteriorated more rapidly than the in vivo measured LVEF. Within 4 months post-treatment, ex vivo cardiac output and stroke volume started to decrease and declined continuously throughout the observation period of 16 months. The reduction in stroke volume was already significant (p < 0.04) at 4 months post-treatment, whereas the decline in cardiac output was significant (p < 0.05) at 12 months post-treatment. In vivo, no change in LVEF was observed during the first 12 months post-treatment. Thereafter, LVEF decreased rapidly from 65 +/- 2% to 46 +/- 8% (p < 0.01), at 16 months post-treatment. Up to 12 months post-irradiation, LVEF was not correlated to ex vivo cardiac output. At 16 months post-treatment, when clinical symptoms of heart failure become evident, a positive relation between both parameters was found. The lack of correlation between the in vivo and ex vivo measurements of cardiac function during the first 12 months post-treatment might be explained by the involvement of compensatory mechanisms being operative in vivo to maintain sufficient cardiac output.