PECAM-1 modulates liver damage induced by synergistic effects of TNF-α and irradiation.

The mechanisms of radiation-induced liver damage are poorly understood. We investigated if tumour necrosis factor (TNF)-α acts synergistically with irradiation, and how its activity is influenced by platelet endothelial cell adhesion molecule-1 (PECAM-1). We studied murine models of selective single-dose (25 Gy) liver irradiation with and without TNF-α application (2 µg/mouse; i.p.). In serum of wild-type (wt)-mice, irradiation induced a mild increase in hepatic damage marker aspartate aminotransferase (AST) in comparison to sham-irradiated controls. AST levels further increased in mice treated with both irradiation and TNF-α. Accordingly, elevated numbers of leucocytes and increased expression of the macrophage marker CD68 were observed in the liver of these mice. In parallel to hepatic damage, a consecutive decrease in expression of hepatic PECAM-1 was found in mice that received radiation or TNF-α treatment alone. The combination of radiation and TNF-α induced an additional significant decline of PECAM-1. Furthermore, increased expression of hepatic lipocalin-2 (LCN-2), a hepatoprotective protein, was detected at mRNA and protein levels after irradiation or TNF-α treatment alone and the combination of both. Signal transducer and activator of transcription-3 (STAT-3) seems to be involved in the signalling cascade. To study the involvement of PECAM-1 in hepatic damage more deeply, the liver of both wt- and PECAM-1-knock-out-mice were selectively irradiated (25 Gy). Thereby, ko-mice showed higher liver damage as revealed by elevated AST levels, but also increased hepatoprotective LCN-2 expression. Our studies show that TNF-α has a pivotal role in radiation-induced hepatic damage. It acts in concert with irradiation and its activity is modulated by PECAM-1, which mediates pro- and anti-inflammatory signalling.

Role of PECAM-1 in radiation-induced liver inflammation.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is known to play an important role in hepatic inflammation. Therefore, we investigated the role of PECAM-1 in wild-type (WT) and knock-out (KO)-mice after single-dose liver irradiation (25 Gy). Both, at mRNA and protein level, a time-dependent decrease in hepatic PECAM-1, corresponding to an increase in intercellular cell adhesion molecule-1 (ICAM-1) (6 hrs) was detected in WT-mice after irradiation. Immunohistologically, an increased number of neutrophil granulocytes (NG) (but not of mononuclear phagocytes) was observed in the liver of WT and PECAM-1-KO mice at 6 hrs after irradiation. The number of recruited NG was higher and prolonged until 24 hrs in KO compared to WT-mice. Correspondingly, a significant induction of hepatic tumour necrosis factor (TNF)-α and CXC-chemokines (KC/CXCL1 interleukin-8/CXCL8) was detected together with an elevation of serum liver transaminases (6-24 hrs) in WT and KO-mice. Likewise, phosphorylation of signal transducer and activator of transcription-3 (STAT-3) was observed in both animal groups after irradiation. The level of all investigated proteins as well as of the liver transaminases was significantly higher in KO than WT-mice. In the cell-line U937, irradiation led to a reduction in PECAM-1 in parallel to an increased ICAM-1 expression. TNF-α-blockage by anti-TNF-α prevented this change in both proteins in cell culture. Radiation-induced stress conditions induce a transient accumulation of granulocytes within the liver by down-regulation/absence of PECAM-1. It suggests that reduction/lack in PECAM-1 may lead to greater and prolonged inflammation which can be prevented by anti-TNFα.

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects.

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small "scars" on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of "provisional clot"-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.

Single-dose gamma-irradiation induces up-regulation of chemokine gene expression and recruitment of granulocytes into the portal area but not into other regions of rat hepatic tissue.

Liver damage is a serious clinical complication of gamma-irradiation. We therefore exposed rats to single-dose gamma-irradiation (25 Gy) that was focused on the liver. Three to six hours after irradiation, an increased number of neutrophils (but not mononuclear phagocytes) was observed by immunohistochemistry to be attached to portal vessels between and around the portal (myo)fibroblasts (smooth muscle actin and Thy-1(+) cells). MCP-1/CCL2 staining was also detected in the portal vessel walls, including some cells of the portal area. CC-chemokine (MCP-1/CCL2 and MCP-3/CCL7) and CXC-chemokine (KC/CXCL1, MIP-2/CXCL2, and LIX/CXCL5) gene expression was significantly induced in total RNA from irradiated livers. In laser capture microdissected samples, an early (1 to 3 hours) up-regulation of CCL2, CXCL1, CXCL8, and CXCR2 gene expression was detected in the portal area but not in the parenchyma; with the exception of CXCL1 gene expression. In addition, treatment with an antibody against MCP-1/CCL2 before irradiation led to an increase in gene expression of interferon-gamma and IP-10/CXCL10 in liver tissue without influencing the recruitment of granulocytes. Indeed, the CCL2, CXCL1, CXCL2, and CXCL5 genes were strongly expressed and further up-regulated in liver (myo)fibroblasts after irradiation (8 Gy). Taken together, these results suggest that gamma-irradiation of the liver induces a transient accumulation of granulocytes within the portal area and that (myo)fibroblasts of the portal vessels may be one of the major sources of the chemokines involved in neutrophil recruitment. Moreover, inhibition of more than one chemokine (eg, CXCL1 and CXCL8) may be necessary to reduce leukocytes recruitment.

Comparison of the micronucleus and chromosome aberration techniques for the documentation of cytogenetic damage in radiochemotherapy-treated patients with rectal cancer.

PURPOSE: The goal of the interdisciplinary Clinical Research Unit KFO179 (Biological Basis of Individual Tumor Response in Patients with Rectal Cancer) is to develop an individual Response and Toxicity Score for patients with locally advanced rectal cancer treated with neoadjuvant radiochemotherapy. The aim of the present study was to find a reliable and sensitive method with easy scoring criteria and high numbers of cell counts in a short period of time in order to analyze DNA damage in peripheral blood lymphocytes. Thus, the cytokinesis-block micronucleus (CBMN) assay and the chromosome aberration technique (CAT) were tested. MATERIALS AND METHODS: Peripheral blood lymphocytes obtained from 22 patients with rectal cancer before (0 Gy), during (21.6 Gy), and after (50.4 Gy) radiochemotherapy were stimulated in vitro by phytohemagglutinin (PHA); the cultures were then processed for the CBMN assay and the CAT to compare the two methods. RESULTS: A significant increase of chromosomal damage was observed in the course of radiochemotherapy parallel to increasing radiation doses, but independent of the chemotherapy applied. The equivalence of both methods was shown by Westlake's equivalence test. CONCLUSION: The results show that the CBMN assay and the CAT are equivalent. For further investigations, we prefer the CBMN assay, because it is simpler through easy scoring criteria, allows high numbers of cell counts in less time, is reliable, sensitive, and has higher statistical power. In the future, we plan to integrate cytogenetic damage during radiochemotherapy into the planned Response and Toxicity Score within our interdisciplinary Clinical Research Unit.

High-grade acute organ toxicity during preoperative radiochemotherapy as positive predictor for complete histopathologic tumor regression in multimodal treatment of locally advanced rectal cancer.

PURPOSE: To test for a possible correlation between high-grade acute organ toxicity during preoperative radiochemotherapy and complete tumor regression after total mesorectal excision in multimodal treatment of locally advanced rectal cancer. PATIENTS AND METHODS: From 2001 to 2008, 120 patients were treated. Preoperative treatment consisted of normofractionated radiotherapy at a total dose of 50.4 Gy, and either two cycles of 5-fluorouracil (5-FU) or two cycles of 5-FU and oxaliplatin. Toxicity during treatment was monitored weekly, and any toxicity CTC (Common Toxicity Criteria) >or= grade 2 of enteritis, proctitis or cystitis was assessed as high-grade organ toxicity for later analysis. Complete histopathologic tumor regression (TRG4) was defined as the absence of any viable tumor cells. RESULTS: A significant coherency between high-grade acute organ toxicity and complete histopathologic tumor regression was found, which was independent of other factors like the preoperative chemotherapy schedule. The probability of patients with acute organ toxicity >or= grade 2 to achieve TRG4 after neoadjuvant treatment was more than three times higher than for patients without toxicity (odds ratio: 3.29, 95% confidence interval: [1.01, 10.96]). CONCLUSION: Acute organ toxicity during preoperative radiochemotherapy in rectal cancer could be an early predictor of treatment response in terms of complete tumor regression. Its possible impact on local control and survival is under further prospective evaluation by the authors' working group.

Gold markers for tumor localization and target volume delineation in radiotherapy for rectal cancer.

BACKGROUND AND PURPOSE: In locally advanced rectal cancer, neoadjuvant radiochemotherapy is indicated. To improve target volume definition for radiotherapy planning, the potential of implanted gold markers in the tumor region was evaluated. PATIENTS AND METHODS: In nine consecutive patients, two to three gold markers were implanted in the tumor region during rigid rectoscopy. Computed tomography scans were performed during treatment planning. All electronic portal imaging devices (EPIDs) recorded during treatment series were analyzed. All patients underwent complete tumor resection with meticulous histopathologic examination. RESULTS: The gold markers could easily be implanted into the mesorectal tissue at the caudal tumor border without any complications. They were helpful in identifying the inferior border of the planning target volume in order to spare normal tissue (in particular anal structures). No significant shift of the markers was found during the course of therapy. Marker matching of the EPIDs did not improve patient positioning in comparison to bone structure matching. The former position of at least one marker could be identified in all patients during histopathologic examination. CONCLUSION: The use of gold marker enables a more precise definition of the target volume for radiotherapy in patients with rectal cancer. This could eventually allow a better protection of anal structures of patients with a tumor localization > or = 5 cm cranial of the anal sphincter. The implantation of the gold markers improved communication between the surgeon, the radiooncologist and the pathologist resulting in intensified exchange of relevant informations.

The impact of prostate volume changes during external-beam irradiation in consequence of HDR brachytherapy in prostate cancer treatment.

PURPOSE: To evaluate prostate volume changes during external-beam irradiation in consequence of high-dose-rate (HDR) brachytherapy in prostate cancer treatment. PATIENTS AND METHODS: 20 patients who underwent radiotherapy for prostate cancer were included in this prospective evaluation. All patients had a computed tomography (CT) scan for planning of the external-beam irradiation and additional scans after each HDR brachytherapy. For the planning target volume (PTV), a safety margin of 10 mm was added to the clinical target volume (CTV) in each direction. The prostate volume measured in the planning CT was compared with the prostate volumes measured after HDR brachytherapy and, subsequently, the change of prostate volume was calculated. Volume changes which resulted in differences of the prostate radius of > 5 mm for the CTV were defined as a reason for a new treatment-planning procedure for the patient. RESULTS: Taking all patients together, prostate volumes before HDR, 1 day and 4-6 days after the first HDR treatment, as well as 1 day and 4-6 days after the second HDR treatment were in median 37.7 cm(3), 37.6 cm(3), 38.2 cm(3), 39.3 cm(3), and 40.5 cm(3), respectively. In none of the patient, a volume change resulted in a change of the prostate radius of > 5 mm for the CTV. Prerequisite for this calculation was the simplification of the complex prostate geometry to a sphere. No new treatment-planning procedure was necessary during external-beam radiotherapy. CONCLUSION: HDR brachytherapy does change the prostate volume. Under the condition of a 10-mm safety margin in each direction added to the CTV for the PTV, no new treatment-planning procedure was necessary after HDR brachytherapy. There is no need for CT scans at regular intervals during external-beam radiotherapy.

Effect of irradiation on gene expression of rat liver adhesion molecules: in vivo and in vitro studies.

BACKGROUND AND PURPOSE: Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro. MATERIAL AND METHODS: Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology. RESULTS: Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), beta1-integrin, beta2-integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, beta1-integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), beta2-integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-)alpha, interleukin-(IL-)1beta, or IL-6 plus TNF-alpha led to an upregulation of P-selectin, ICAM-1 and VCAM-1. CONCLUSION: The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of inflammation in the irradiated rat liver.

Noninvasive imaging of liver repopulation following hepatocyte transplantation.

Near infrared fluorescence (NIRF) optical imaging is a technique particularly powerful when studying in vivo processes at the molecular level in preclinical animal models. We recently demonstrated liver irradiation under the additional stimulus of partial hepatectomy as being an effective primer in the rat liver repopulation model based on hepatocyte transplantation. The purpose of this study was to assess optical imaging and the feasibility of donor cell expansion tracking in vivo using a fluorescent probe. Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with irradiation. Four days later, a partial hepatectomy was performed and wild-type (DPPIV+) hepatocytes were transplanted into recipient livers via the spleen. Repopulation by transplanted DPPIV+ hepatocytes was detected in vivo with Cy5.5-conjugated DPPIV antibody using the eXplore Optix System (GE HealthCare). Results were compared with nontransplanted control animals and transplanted animals receiving nonspecific antibody. Optical imaging detected Cy5.5-specific fluorescence in the liver region of the transplanted animals, increasing in intensity with time, representing extensive host liver repopulation within 16 weeks following transplantation. A general pattern of donor cell multiplication emerged, with an initially accelerating growth curve and later plateau phase. In contrast, no specific fluorescence was detected in the control groups. Comparison with ex vivo immunofluorescence staining of liver sections confirmed the optical imaging results. Optical imaging constitutes a potent method of assessing the longitudinal kinetics of liver repopulation in the rat transplantation model. Our results provide a basis for the future development of clinical protocols for suitable fluorescent dyes and imaging technologies.

Effect of tumour necrosis factor-alpha and irradiation alone or in combination on the viability of hepatocellular and biliary adenocarcinoma cell lines in vitro.

BACKGROUND: Tumour necrosis factor alpha (TNF-alpha) may exhibit antitumoral activity and can influence the reaction of both tumour and normal tissue to radiation. AIMS: To test the effect of TNF-alpha and/or irradiation on hepatocellular (HepG2, Hep3B, Sk-Hep1, HuH7) and cholangiocellular (Sk-chA1, Mz-chA1) tumour cell lines. METHODS: Colony formation, apoptosis analysis and trypan blue exclusion were used to assess cell viability. Doses of radiation (2-25 Gy) and TNF-alpha (100-50,000 U) as well as their respective sequencing were varied (24 and 12 h before and 6 h after). The expression of TNF-alpha and TNF receptors 1/2 was determined using real-time polymerase chain reaction and IkappaBalpha protein expression was detected by Western blot. RESULTS: Sole irradiation induced a reduction in colony formation in all cell lines and sole TNF-alpha in HepG2 and Sk-chA1 cells only. No difference in apoptosis induction after TNF-alpha or irradiation was observed. Cellular death induced by the combination of TNF-alpha and radiation was not superior to the use of any of the two agents alone. All cell lines revealed that radiation induced upregulation of TNF-alpha whereas the extent of TNF receptor-specific transcription did not change. Furthermore, radiation-induced changes in IkappaBalpha expression were not detectable. CONCLUSIONS: Our data suggest that both TNF-alpha and radiation may be treatment options for hepatocellular and cholangiocellular carcinomas. Because TNF-alpha and radiation do not interact in terms of radiosensitization, anti-TNF-alpha treatment may have the potential to protect against hepatocellular injury after abdominal irradiation. However, further in vivo studies are needed to confirm that anti-TNF-alpha treatment does not compromise tumour control and actually attenuates radiation-induced liver injury.

In vivo dosimetry in the urethra using alanine/ESR during (192)Ir HDR brachytherapy of prostate cancer--a phantom study.

A phantom study for dosimetry in the urethra using alanine/ESR during (192)Ir HDR brachytherapy of prostate cancer is presented. The measurement method of the secondary standard of the Physikalisch-Technische Bundesanstalt had to be slightly modified in order to be able to measure inside a Foley catheter. The absorbed dose to water response of the alanine dosimetry system to (192)Ir was determined with a reproducibility of 1.8% relative to (60)Co. The resulting uncertainty for measurements inside the urethra was estimated to be 3.6%, excluding the uncertainty of the dose rate constant Lambda. The applied dose calculated by a treatment planning system is compared to the measured dose for a small series of (192)Ir HDR irradiations in a gel phantom. The differences between the measured and applied dose are well within the limits of uncertainty. Therefore, the method is considered to be suitable for measurements in vivo.

In vivo alanine/electron spin resonance (ESR) dosimetry in radiotherapy of prostate cancer: a feasibility study.

PURPOSE: We have developed a device to evaluate the potential of alanine/electron spin resonance (ESR) dosimetry for quality assurance in 3D conformal radiotherapy for prostate cancer. It consists of a rectal balloon carrying eight alanine dosimeter probes and two metal markers to document the exact position of the balloon. We measured the effects of an air-filled rectal balloon on the dose at the rectal wall and compared these results with the applied dose distribution of the treatment planning system. MATERIALS AND METHODS: During 10 fractions with 2.0 Gy per fraction, the accumulated doses were measured in 3 patients. The results of the ESR measurements were compared to the applied doses. RESULTS: It was possible to insert the device without clinical complications and without additional rectal discomfort for the patients. The measurements of the dose accumulated at the anterior and the posterior rectal wall agreed with the applied dose within a mean deviation of 1.5% (overestimation of the dose) and 3.5% (underestimation of the dose), respectively. However, clinically significant differences between applied and measured rectal doses were seen in a patient with a hip prosthesis. In this case, the dose at the anterior rectal wall was overestimated by the TPS by about 11% and the dose at the posterior rectal wall was underestimated by approximately 7%. CONCLUSION: The method presented in this study is useful for quality control of irradiations in vivo.

An easy irradiation technique (partial half-beam) to reduce renal dose in radiotherapy of cervical cancer including paraaortic lymph nodes.

PURPOSE: : For radiation treatment of patients with cervical cancer and a high risk for paraaortic lymph node involvement, an easy three-dimensional (3-D) conformal irradiation technique (partial half-beam [PHB]) for protection of organs at risk, especially of renal tissue, was developed. PATIENTS AND METHODS: : In five consecutive female patients a computed tomography scan was performed. Dose-volume histograms of the renal tissue and other organs at risk were analyzed for PHB, three other 3-D conformal techniques, and an intensitymodulated radiotherapy (IMRT) technique. RESULTS: : The PHB technique reduced the renal volume and volumes of other organs at risk exposed to radiation doses when comparing all patients to the other 3-D conformal techniques. With use of the IMRT technique more renal tissue volume received very low radiation doses (

Effect of radiation on gene expression of rat liver chemokines: in vivo and in vitro studies.

The aim of the study was to analyze the effect of a single irradiation on chemokine gene expression in the rat liver and in isolated rat hepatocytes. RNA extracted from livers and from hepatocytes within the first 48 h after irradiation was analyzed by real-time PCR and the Northern blot assay. The chemokine concentrations in the serum of irradiated rats were measured quantitatively by ELISA. A significant radiation-induced increase of CINC1, IP10, MCP1, MIP3alpha, MIP3beta, MIG and ITAC gene expression could be detected at the RNA level in the liver. CINC1, MCP1 and IP10 serum levels were significantly increased. In rat hepatocytes in vitro, only MIP3alpha showed a radiation-induced increase in expression, while CINC1, IP10, MIP3beta, MIG, MIP1alpha, ITAC and SDF1 RNA levels were significantly down-regulated. However, incubation of irradiated hepatocytes in vitro with either TNF-alpha, IL1beta, or IL6 plus TNF-alpha led to up-regulation of MCP1, IP10 and MCP1 or CINC1 and MIP3beta, respectively. Irradiation of the liver induces up-regulation of the genes of the main proinflammatory chemokines, probably through the action of locally synthesized proinflammatory cytokines. The reason for the lack of liver inflammation in this model has still to be clarified.

Irradiation as preparative regimen for hepatocyte transplantation causes prolonged cell cycle block.

PURPOSE: Hepatocyte transplantation following liver irradiation (IR) and partial hepatectomy (PH) leads to extensive liver repopulation. We investigated the changes in the liver induced by IR explaining the loss of reproductive integrity in endogenous hepatocytes. MATERIALS AND METHODS: Right lobules of rat liver underwent external beam IR (25 Gy). A second group was subjected to additional 33% PH of the untreated left liver lobule. Liver specimens and controls were analyzed for DNA damage, apoptosis, proliferation and cell cycle related genes (1 hour to up to 12 weeks). RESULTS: Double strand breaks (phosphorylated histone H2AX) induced by IR rapidly declined within hours and were no longer detectable after 4 days. No significant apoptosis was noted and steady mRNA levels (B-cell lymphoma 2-associated X protein (BAX), caspase 3 and 9) were in line with the lack of DNA fragmentation. However, gene expression of p53 and p21 in irradiated liver tissue increased. Transcripts of cyclin D1, proliferating cell nuclear antigen (PCNA), and cyclin B augmented progressively, whereas cyclin E was only affected moderately. Following PH, irradiated livers displayed persistently high protein levels of p21 and cyclin D1. However, cell divisions were infrequent, as reflected by low PCNA levels up to four weeks. CONCLUSION: IR leads to a major arrest in the G(1)/S phase and to a lesser extent in the G(2)/M transition of the cell cycle, resulting in reduced regenerative response following PH. The persistent block of at least four weeks may promote preferential proliferation of transplanted hepatocytes in this milieu.

A special device (double-hole belly board) and optimal radiation technique to reduce testicular radiation exposure in radiotherapy of rectal cancer.

PURPOSE: Patients with rectal cancer are treated in prone position on a belly board to reduce the volume of irradiated small bowel. With this technique the testes obtain radiation doses, which often result in partial or complete impairment of the spermatogenesis and a dose-dependent decrease of testosterone levels. We developed a double-hole belly board (DHBB) and evaluated its potential to reduce testicular dose. METHODS AND MATERIALS: In nine consecutive male patients (3 very low tumor localisations [inguinal RT], 3 low [RT perineum], 3 high [lower border ischial tuberosities]) CT scans were performed on a conventional single-hole belly board (SHBB) and on a DHBB. Dose-volume histograms of the testes were analysed for both belly boards and for different treatment techniques (3-field and 4-field). RESULTS: To reduce testicular dose in high tumors, positioning on DHBB was most effective (V(1.5Gy) 20-30% vs. 60% for SHBB, V(4Gy) 7% vs. 35%). In low tumors, a 3-field technique reduced high testicular doses (V(14Gy) 0-6% vs. 28-34% for 4-fields). In very low tumors a combination of DHBB and 3-fields led to a decrease of high dose exposure (V(33Gy) 0% vs. 24-78%). CONCLUSION: In male patients with rectal cancer the use of a DHBB and a 3-field technique is recommended to reduce testicular radiation exposure.

External-beam radiotherapy as preparative regimen for hepatocyte transplantation after partial hepatectomy.

PURPOSE: The transplantation of donor hepatocytes is considered a promising option to correct chronic liver failure through repopulation of the diseased organ. This study describes a novel selective external-beam irradiation technique as a preparative regimen for hepatocyte transplantation. METHODS AND MATERIALS: Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with external-beam single-dose irradiation (25 Gy) delivered to two thirds of the liver. Four days later, a one-third partial hepatectomy (PH) was performed to resect the untreated liver section, and 15 million wild-type (DPPIV+) hepatocytes were transplanted via the spleen into the recipient livers. The degree of donor-cell integration and growth was studied 8 h, 3 days, and 5 and 12 weeks after transplantation. RESULTS: Transplanted hepatocytes integrated rapidly into the irradiated liver and proliferated as clusters, finally repopulating the host liver to approximately 20% hepatocyte mass. After 12 weeks, donor cells and their numerous descendents were fully integrated and expressed functional markers to the same extent as host hepatocytes. CONCLUSIONS: We demonstrate that external-beam liver irradiation is sufficient to achieve partial repopulation of the host liver after hepatocyte transplantation, under the additional stimulus of one-third PH. The method described has potentially good prospects for its application in a clinically viable form of treatment.

Identification of genes responsive to gamma radiation in rat hepatocytes and rat liver by cDNA array gene expression analysis.

The mechanisms underlying hepatocellular damage after irradiation are obscure. We identified genes induced by radiation in isolated rat hepatocytes in vitro by cDNA array gene expression analysis and then screened in vivo experiments with those same genes using real-time PCR and Western blotting. Hepatocytes were irradiated and cDNA array analyses were performed 6 h after irradiation. The mRNA of differentially expressed genes was quantitatively analyzed by real-time PCR. cDNA array analyses showed an up-regulation of 10 genes in hepatocytes 6 h after irradiation; this was confirmed by real-time PCR. In vivo, rat livers were irradiated selectively. Treated and sham-irradiated controls were killed humanely 1, 3, 6, 12, 24 and 48 h after irradiation. Liver RNA was analyzed by real-time PCR; expression of in vivo altered genes was also analyzed at the protein level by Western blotting. Up-regulation was confirmed for three of the in vitro altered genes (multidrug resistance protein, proteasome component C3, eukaryotic translation initiation factor 2). Histologically, livers from irradiated animals were characterized by steatosis of hepatocytes. Thus we identified genes that may be involved in liver steatosis after irradiation. The methods shown in this work should help to further clarify the consequences of radiation exposure in the liver.

Irradiation leads to susceptibility of hepatocytes to TNF-alpha mediated apoptosis.

BACKGROUND AND PURPOSE: The pathogenesis of radiation-induced-liver-disease (RILD) is still unknown. We tested the hypothesis that irradiated liver macrophages influence the viability of radiation stressed hepatocytes. PATIENTS AND METHODS: Hepatocytes and liver macrophages were isolated from rat liver, cultured and irradiated with doses of 2, 8, and 25 Gy. Cell viability was measured by trypan blue exclusion, and by annexin V/propidium iodide staining. TNF-alpha in the supernatants from liver macrophage cell culture was quantitatively detected by ELISA. TNF-alpha mRNA from liver macrophages was measured by real time PCR. RESULTS: Irradiation had no influence on cell viability. Apoptosis of irradiated hepatocytes was detected 24h after replacing 50% of medium with supernatants of irradiated liver macrophages 6 h after irradiation (32.0+/-5.8% compared to solely irradiated cells (12+/-2.9%, P=0.02)). In supernatants of hepatocytes, no TNF-alpha secretion could be measured. A radiation dependent increase was found in supernatants of liver macrophages. Addition of anti-TNF-alpha-antibodies to the supernatants of irradiated liver macrophages reduced apoptosis (20+/-0.9%). Incubation of irradiated hepatocytes with purified recombinant TNF-alpha increased apoptosis in irradiated hepatocytes. This effect could be abrogated by additional administration of TNF-alpha-antibodies. CONCLUSIONS: Irradiation leads to susceptibility of hepatocytes to TNF-alpha mediated apoptosis. Liver macrophages may be one of the sources of TNF-alpha in case of liver-irradiation. This cell-cell-interaction may be an important initial step towards RILD and liver fibrosis.