R0 resection following chemo (radio)therapy improves survival of primary inoperable pancreatic cancer patients. Interim results of the German randomized CONKO-007± trial.

PURPOSE: Chemotherapy with or without radiotherapy is the standard in patients with initially nonmetastatic unresectable pancreatic cancer. Additional surgery is in discussion. The CONKO-007 multicenter randomized trial examines the value of radiotherapy. Our interim analysis showed a significant effect of surgery, which may be relevant to clinical practice. METHODS: One hundred eighty patients received induction chemotherapy (gemcitabine or FOLFIRINOX). Patients without tumor progression were randomized to either chemotherapy alone or to concurrent chemoradiotherapy. At the end of therapy, a panel of five independent pancreatic surgeons judged the resectability of the tumor. RESULTS: Following induction chemotherapy, 126/180 patients (70.0%) were randomized to further treatment. Following study treatment, 36/126 patients (28.5%) underwent surgery; (R0: 25/126 [19.8%]; R1/R2/Rx [n = 11/126; 6.1%]). Disease-free survival (DFS) and overall survival (OS) were significantly better for patients with R0 resected tumors (median DFS and OS: 16.6 months and 26.5 months, respectively) than for nonoperated patients (median DFS and OS: 11.9 months and 16.5 months, respectively; p = 0.003). In the 25 patients with R0 resected tumors before treatment, only 6/113 (5.3%) of the recommendations of the panel surgeons recommended R0 resectability, compared with 17/48 (35.4%) after treatment (p < 0.001). CONCLUSION: Tumor resectability of pancreatic cancer staged as unresectable at primary diagnosis should be reassessed after neoadjuvant treatment. The patient should undergo surgery if a resectability is reached, as this significantly improves their prognosis.

Time management in radiation oncology: evaluation of time, attendance of medical staff, and resources during radiotherapy for prostate cancer: the DEGRO-QUIRO trial.

PURPOSE: In order to evaluate resource requirements, the German Society of Radiation Oncology (DEGRO) recorded the times needed for core procedures in the radio-oncological treatment of various cancer types within the scope of its QUIRO trial. The present study investigated the personnel and infrastructural resources required in radiotherapy of prostate cancer. METHODS: The investigation was carried out in the setting of definitive radiotherapy of prostate cancer patients between July and October 2008 at two radiotherapy centers, both with well-trained staff and modern technical facilities at their disposal. Personnel attendance times and room occupancy times required for core procedures (modules) were each measured prospectively by two independently trained observers using time measurements differentiated on the basis of professional group (physician, physicist, and technician), 3D conformal (3D-cRT), and intensity-modulated radiotherapy (IMRT). RESULTS: Total time requirements of 983 min for 3D-cRT and 1485 min for step-and-shoot IMRT were measured for the technician (in terms of professional group) in all modules recorded and over the entire course of radiotherapy for prostate cancer (72-76 Gy). Times needed for the medical specialist/physician were 255 min (3D-cRT) and 271 min (IMRT), times of the physicist were 181 min (3D-cRT) and 213 min (IMRT). The difference in time was significant, although variations in time spans occurred primarily as a result of various problems during patient treatment. CONCLUSION: This investigation has permitted, for the first time, a realistic estimation of average personnel and infrastructural requirements for core procedures in quality-assured definitive radiotherapy of prostate cancer. The increased time needed for IMRT applies to the step-and-shoot procedure with verification measurements for each irradiation planning.

Radiobiological mechanisms in inflammatory diseases of low-dose radiation therapy.

PURPOSE: Whereas X-irradiation with high doses is established to exert pro-inflammatory effects, low-dose radiotherapy (LD-RT) with single fractions below 1.0 Gy and a total dose below 12 Gy is clinically well known to exert anti-inflammatory and analgesic effects on several inflammatory diseases and painful degenerative disorders. Experimental studies to confirm the effectiveness, the empirical dose and fractionation schemes, and the underlying radiobiological mechanisms are still fragmentary. METHOD: The anti-inflammatory efficiency of LD-RT was confirmed in several experimental in vitro and in vivo models. RESULTS: In vitro studies revealed a variety of mechanisms related to the anti-inflammatory effect, in particular the modulation of cytokine and adhesion molecule expression on activated endothelial cells and leukocytes, and of nitric oxide (NO) production and oxidative burst in activated macrophages and native granulocytes. CONCLUSION: Inflammatory diseases are the result of complex and pathologically unbalanced multicellular interactions. It is, therefore, reasonable to assume that further molecular pathways and cellular components contribute to the anti-inflammatory effect of LD-RT. This review discusses data and models revealing aspects of the mechanisms underlying the anti-inflammation induced by low doses of X-irradiation and may serve as a basis for systematic analyses, necessary to optimize LD-RT in clinical practice.

Radiotherapy for prevention of disease progression in early-stage Dupuytren's contracture: initial and long-term results.

PURPOSE: Radiotherapy (RT) was given to prevent disease progression in early-stage Dupuytren's contracture. Initial response, long-term outcome, and treatment toxicity were evaluated. METHODS: Between 1982 and 1993, 96 patients (142 hands) received orthovoltage RT, which consisted of two courses with daily fractionation of 5 x 3 Gy (total dose 30 Gy) separated by a 6-week interval. The extent of disease was staged according to the classification of Tubiana et al.. Initial evaluation was performed 3 months after completion of RT; long-term outcome was analyzed at last follow-up (i.e., between February and April 1994). The mean follow-up was 6 +/- 2 (range 1-12) years. Fifty-seven patients with a minimum follow-up of 5 (median 7.5; mean 9.5-12) years were separately evaluated for long-term outcome (i.e., prevention of disease progression). Acute and late treatment toxicity was assessed using the Radiation Therapy Oncology Group/EORTC criteria. RESULTS: According to stage, 130 cases (92%) remained stable at 3 months follow-up, 10 improved (7%), and 2 progressed (1%). An objective reduction of symptomatic cords and nodules was achieved in 107 cases (75%) at 3 months follow-up. Moreover, 87% of the patients reported a subjective relief of symptoms. In long-term follow-up, only 16 of 142 cases (11%) had progressed according to stage. In the group with minimum follow-up 5 years (n = 57), 44 patients (77%) experienced no disease progression, whereas 13 progressed (23%) inside [8 cases (14%)] or outside [5 cases (9%)] of the RT field. Most failures could have been avoided with appropriate choice of larger safety margins included in the treated portals; however, the failures outside were still amenable for another RT course. CONCLUSION: Radiotherapy is effective to prevent disease progression for early-stage Dupuytren's contracture. Thus, it helps to avoid an otherwise necessary surgical procedure which is performed in adavanced stages of Dupuytren's contracture.

Preoperative versus postoperative radiotherapy for prevention of heterotopic ossification (HO): first results of a randomized trial in high-risk patients.

PURPOSE: In vivo data support the effectiveness of pre- and postoperative radiotherapy in suppressing the development of heterotopic ossification after hip surgery. In June 1992 a prospectively randomized trial was initiated to assess the comparative efficacy of pre- vs. postoperative prophylactic radiotherapy in patients with high risk to develop heterotopic ossification after hip surgery. METHODS AND MATERIAL: Between June 1992 and September 1993 a total of 84 eligible patients with high risk profile for the development of heterotopic ossification were entered in the study. They were randomized to receive radiotherapy either preoperatively (< 4 h before surgery) or according to a "standard protocol" postoperatively (< 72 h after surgery). A single 7 Gy fraction was administered to the preoperative group, while the postoperative group received a previously tested scheme of five fractions of 3.5 Gy (total dose 17.5 Gy). The treatment portal encompassed the soft tissues between the periacetabular region of the pelvis and the intertrochanteric portion of the femur. Important patient variables (age, sex, prior surgery) and predisposing risk factors were equally distributed between both treatment arms. X rays of the irradiated hips were obtained prior and immediately after surgery as well as at 6 months after surgery. The modified Brooker grading was used to score the extent of heterotopic ossification. The Harris score was applied to judge the overall functional status of the hip. If the Brooker grade and Harris score decreased from the immediate postoperative or preoperative status respectively to the follow-up situation, the case was considered as a "treatment failure." RESULTS: At a minimum 6 months follow-up after hip surgery 44 patients were available for evaluation. Effective prophylaxis was achieved in 41 (93%) hips. Two "radiological failures" were observed in the preoperative group and one in the postoperative group. Neither the pre- nor the postoperative interval affected the prophylactic efficacy. There were no increased intra- and postoperative complications seen in the preoperative group. The interval of partial strain (50% body weight) to the operated hip was longer in the preoperative group (19 days +/- 27) as compared to the postoperative group (8 days +/- 13), however the interval to full strain (100% body weight) was equal in both groups. The functional status (Harris Score change) of the operated hip decreased only in two (5%) patients ("functional failures"). The overall change was better in the postoperative group (42.7 +/- 17.1) as compared to the preoperative group (34.3 +/- 13.7) (p = 0.08, NS) as well as with regard to the criteria "limp" (p = 0.05) and "use of walking support" (p = 0.10, NS). In in all other aspects no differences were observed between both treatment arms. Therefore, the preliminary results for preoperative radiotherapy are similar to historical results obtained with postoperative radiotherapy regimens. CONCLUSION: Preoperative radiotherapy of the operative site applied within 4 h prior to elective hip surgery and total hip arthroplasty appears to be equally effective to currently accepted postoperative radiotherapy regimens in prevention of clinically significant heterotopic ossification about the hip. Improved patient comfort, ease of treatment management, and avoidance of possible postoperative complications associated with moving and positioning the patient in the immediate postoperative period are the major advantages of the preoperative radiotherapy concept.

Palliative radiotherapy for recurrent and metastatic malignant melanoma: prognostic factors for tumor response and long-term outcome: a 20-year experience.

PURPOSE: Radiotherapy is used as a "last resort" for patients with advanced cutaneous malignant melanoma. We have analyzed our 20-year clinical experience with respect to different endpoints and prognostic factors in patients with locally advanced, recurrent, or metastatic malignant melanoma. METHODS: From 1977 to 1995, 2,917 consecutive patients were entered in the melanoma registry of our hospital. Radiotherapy was indicated in 121 patients (56 females, 65 males) for palliative reasons in advanced malignant melanoma stages UICC IIB/III/IV. The histology of the primary lesion was nodular in 51 patients, superficial spreading in 35, acral-lentiginous in 8, and lentigo maligna melanoma in 4 patients. Eleven patients had primary or recurrent lesions which were either not eligible for surgery or had residual disease (R2) after resection of a primary or recurrent lesion (UICC IIB); 57 patients had lymph node (n = 33) or in-transit metastases (n = 24) (UICC III), and 53 had distant organ metastases (7 M1a; 46 M1b) (UICC IV). Time from first diagnosis to on-study radiotherapy averaged 19 (median: 18; range: 3-186) months. In most cases, conventional RT was applied with 2-6 Gy single fractions up to a median total radiation dose of 48 (mean: 45; range: 20-66) Gy. RESULTS: At 3 months follow-up, complete response (CR) was achieved in 7 (64%) and overall response [complete (CR) and partial response (PR)] in all (100%) UICC IIB patients, in 25 (44%) and 44 (77%) of 57 UICC III patients, and in 9 (17%) and 26 (49%) of 53 UICC IV patients. Tumor progression during radiotherapy occurred in 25 (21%) patients. Patients with CR survived longer (median: 40 months) than those without CR (median 10 months) (p < 0.01). At last follow-up (Dec 31, 1996), 26 patients were still alive: 6 (55%) UICC IIB, 17 (30%) UICC III, and 3 (6%) UICC IV patients (p < 0.01). Univariate analysis revealed the following prognostic factors for complete response and long-term survival: UICC stage (p < 0.001), primary location in the head and neck region, total radiation dose above 40 Gy (all p < 0.05), while age, gender, and histology had no impact. In multivariate analysis, UICC stage was the only independent prognostic factor (p < 0.001). CONCLUSION: External beam radiotherapy can provide long-term local control and effective palliation in malignant melanoma UICC stages IIB-IV. The current UICC staging system is an excellent prognostic factor for initial and long-term tumor response in metastatic melanoma. Therefore, prospective randomized trials using external radiotherapy with or without adjuvant therapy for advanced malignant melanoma are justified.

Prevention of heterotopic ossification about the hip: final results of two randomized trials in 410 patients using either preoperative or postoperative radiation therapy.

PURPOSE: Experimental and clinical data support effectiveness of perioperative radiotherapy to prevent heterotopic ossification after hip surgery or trauma. Since 1987, two prospectively randomized trials were performed in patients with high-risk factors to develop heterotopic ossification: the first (HOP 1) to assess the prophylactic efficacy of postoperative low vs. medium dose radiotherapy, and the second (HOP 2) to assess the prophylactic efficacy of pre vs. postoperative radiotherapy. METHODS AND MATERIAL: 410 patients with high risk to develop heterotopic ossifications about the hip following hip surgery were recruited. Between June 1987 and June 1992, 249 patients were randomized in HOP 1 to postoperative "low dose" (5 x 2 Gy; total: 10 Gy) or "medium dose" (5 x 3.5 Gy; total: 17.5 Gy) radiotherapy. Between July 1992 and December 1995, 161 patients were randomized in HOP 2 to either 1 x 7 Gy preoperatively (< or = 4 h before surgery) or 5 x 3.5 Gy (total: 17.5 Gy) postoperatively (< or = 96 h after surgery). With exception of age and type of implant (cemented vs. uncemented prosthesis) all confounding patient variables (gender, prior surgery) and predisposing risk factors were similarly distributed between both trials and treatment arms. Portals encompassed the periacetabular and intertrochanteric soft tissues. Radiographs were obtained prior and immediately after surgery and at least 6 months after surgery to assess the extent of ectopic bone formation about the hip. Modified Brooker grading was used to score the extent of heterotopic ossification. Harris scoring was applied to evaluate the functional hip status. If the scores decreased from immediate post or preoperative status, respectively, to the last follow-up, radiological or functional failures were assumed. RESULTS: Effective prophylaxis was achieved in 227 (91%) hips of HOP 1 and in 142 (88%) of HOP 2. In HOP 1, 15 (11%) radiological failures were observed in the low-dose group compared to 7 (6%) in the medium dose group (p > 0.05). In HOP 2, 4 (5%) radiological failures were observed in the postoperative and 11 (19%) in the preoperative group (p < 0.05). Subgroup analysis of the preoperative group revealed that the highest failure rate occurred in patients with prophylactic radiotherapy prior to removal of ipsilateral Brooker Grade III and IV ossification (39%) (p < 0.001), while all other patients in the preoperative group had a failure rate that was comparable to postoperative treatment groups. In multivariate logistic regression analysis the number of high-risk factors for development of heterotopic ossification (p = 0.03) and the time to RT initiation (p = 0.05) were independent prognostic factors in the HOP 1 study. For the HOP 2 study, the multivariate logistic regression analysis revealed the number of high-risk factors for development of heterotopic ossification (p = 0.003), the preoperative HO grade (p = 0.001) and the RT dose concept (p = 0.05) as independent prognostic factors. Other factors including type of implant (cemented vs. uncemented) did not affect the prophylactic efficacy of radiotherapy. There were no increased intra- and postoperative complications seen in the preoperative group, and no long-term complications were observed in both HOP studies. For functional failures (decrease of Harris score) no statistically prognostic factors were found. There were less functional failures in HOP 1 (18 = 7%) than in HOP 2 (23 = 14%, but this difference was not statistically significant. Only patients with high Brooker Grade III and IV at last FU achieved a lower Harris score than those with low Brooker Grade 0, I and II (p < 0.05). CONCLUSION: With the exception of a small subgroup of patients with ipsilateral high Brooker Grade III and IV, pre- and postoperative radiotherapy are equally effective to prevent heterotopic ossification about the hip after hip surgery and total hip arthroplasty. Fractionated medium dose radiotherapy resulted in the low

Epicondylopathia humeri (EPH) and peritendinitis humeroscapularis (PHS): evaluation of radiation therapy long-term results and literature review.

BACKGROUND: The effectiveness of radiotherapy (RT) for degenerative inflammatory disorders has been clinically documented in historical studies, but long-term follow-up and assessment with objective criteria are still not available. PATIENTS AND METHODS: From 1986 to 1991, 200 consecutive patients with symptomatic epicondylopathia humeri (EPH, n = 104) and peritendinitis humeroscapularis (PHS, n = 96) were referred to our clinic. All patients were refractory to conventional therapy prior to irradiation. One hundred fifty-six patients with 192 sites (due to bilateral symptoms) received a full treatment course and were available for long-term follow-up, i.e. 83 patients with 93 elbows and 73 patients with 89 shoulders. The treatment response was evaluated with regard to pain symptoms grouped into five categories (pain at strain, pain at night, persistent pain during daytime, pain at rest and morning stiffness) and four grades (none, mild, moderate and severe) and with regard to established orthopedic scores (Morrey score and Constant and Murley score). The analysis was performed before and 6 weeks after RT and at last follow-up. All joints received two RT series applied in three weekly fractions (EPH, 6 x 1 Gy (total 12 Gy); PHS, 6 x 0.5 Gy (total 6 Gy)). The second RT series started 6 weeks after the first RT series. The minimum follow-up was 1 year for both groups and the mean follow-up reached 4 years (range 1-8 years). RESULTS: Fifty elbows (43 patients) and 44 shoulders (39 patients) achieved complete pain relief in all pain categories; 24 elbows and 28 shoulders substantially improved, i.e. had only minor symptoms. Thus, 74 elbows and 72 shoulders responded to RT. Nineteen elbows (17 patients) had surgery after RT due to persisting symptoms or subjective dissatisfaction; 17 shoulders (12 patients) were non-responders and five of those were operated on; seven elbows and one shoulder were completely free of pain after surgery. The mean Morrey score improved by 18 points (from 78 to 96) and the mean Constant and Murley score improved by 48 points (from 18 to 66). Two cases worsened according to the Morrey score and one case worsened according to the Constant and Murley score. Bi- and multivariate analysis revealed two factors with negative prognostic value on treatment outcome, i.e. EPH, long symptom interval prior to RT and long-term immobilization with plaster (P < 0.05) and PHS, long symptom interval prior to RT and lack of pain intensification during the first RT course (P < 0.05) were poor prognostic factors. CONCLUSION: RT is highly effective for refractory EPH and PHS. Structured pain scores and quantitative orthopedic scores are important for evaluation. Prognostic factors for outcome can be established. Due to minimal side effects and low costs, RT represents an excellent treatment compared to conventional methods of treatment and surgery in the chronic disease.

Low-dose radiotherapy selectively reduces adhesion of peripheral blood mononuclear cells to endothelium in vitro.

BACKGROUND AND PURPOSE: The anti-inflammatory effect of low-dose radiotherapy (LD-RT) still is not understood. The adhesion of leukocytes to endothelial cells (EC) of the vessel wall is the initial event of tissue invasion, and thus, crucially contributes to the regulation of inflammation. We investigated the influence of LD-RT on the adhesion process in vitro. MATERIALS AND METHODS: Isolated peripheral-blood-mononuclear-cells (PBMC) were incubated with an activated murine endothelioma cell-line under shear conditions at 4 degrees C after irradiation with single doses between 0.1 and 10.0 Gy. Adherent cells were counted microscopically and compared to a non-irradiated control. In parallel, viability and expression of adhesion molecules, especially of L-selectin, and lineage-specific markers on the cell surface were determined by dye exclusion and cytofluorometry, respectively. Modulation of adhesion by soluble L-selectin was tested in the adhesion assay. RESULTS: Radiation doses of 0.1-0.5 Gy reduced the adhesion of viable PBMC to EC in vitro by 70% of the control level 4 h after irradiation. Leukocytes showed a marked reduction of L-selectin expression after LD-RT. Soluble L-selectin can inhibit the adhesion of PBMC to EC. CONCLUSION: The anti-inflammatory effect of LD-RT might, in part, be due to the reduction in the adhesion of PBMC to EC. This reduction in adhesion might be a consequence of the reduced expression of L-selectin on the surface of PBMC, and the inhibition of adherence by soluble L-selectin shed by PBMC in vitro.

In vitro apoptosis in peripheral blood mononuclear cells induced by low-dose radiotherapy displays a discontinuous dose-dependence.

PURPOSE: Cells undergoing apoptosis contribute to the regulation of activated mononuclear cells (Voll et al. 1997). Low-dose radiotherapy (LD-RT) is known to improve inflammatory symptoms, but the mechanism of action is still unclear. The aim of this study was to investigate the rate of apoptosis of peripheral blood mononuclear cells (PBMC) induced by LD-RT within the therapeutic dose range of anti-inflammatory RT. MATERIALS AND METHODS: PBMC were isolated from venous blood of ten healthy volunteers and were irradiated with single doses between 0.1 and 3.0 Gy. Apoptotic nuclei were detected by flow cytometry after propidium iodide (PI) triton staining, and apoptotic cells were detected by annexin V/PI staining and cell scatter analysis. Since apoptotic cells display increased cytoplasmatic granularity and concomitant reduced cell size, they can be distinguished from viable cells in forward/side scatter (FSC/SSC) histograms. Apoptotic PBMC were further subtyped by double staining with annexin V and directly labelled monoclonal antibodies recognizing the lineage-specific surface markers CD4, CD8, and CD19, respectively. The apoptosis rate of irradiated cells was analysed in a time and dose dependent fashion and was compared to a sham-irradiated control. RESULTS: After irradiation, a dose-dependent increase in apoptosis was observed, with a discontinuity (plateau or peak) between 0.3Gy and 0.7Gy in 9/10 donors (90%) and 59/80 samples (74%). 8/10 donors (80%) and 38/80 samples (47%) showed not only a discontinuous increase with a plateau but a relative maximum of apoptosis peaking within the dose range of 0.3 Gy and up to 0.7 Gy. CONCLUSION: LD-RT induces a relative maximum of apoptosis in PBMC in the does range between 0.3 Gy and 0.7 Gy. This may contribute to its anti-inflammatory effect observed clinically.

Anti-inflammatory effect of low-dose X-irradiation and the involvement of a TGF-beta1-induced down-regulation of leukocyte/endothelial cell adhesion.

PURPOSE: Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, but the underlying radiobiological and immunological mechanisms remain elusive. In recent studies, we observed a reduced adhesion of peripheral blood mononuclear cells (PBMC) to endothelial cells (EC) after LD-RT (0.3-0.7 Gy). This shows that this treatment affects the initial steps of the inflammatory response. To explore the role of inflammatory mediators in this process, we investigated the expression of Transforming growth factor beta(1) (TGF-beta(1)) and Interleukin 6 (IL-6) after LD-RT. MATERIALS AND METHODS: EC were grown to subconfluence and irradiated with single-dose LD-RT. Twenty-hours after irradiation, EC were treated with IL-1beta for 4 h and then incubated with peripheral blood mononuclear cells (PBMC). Adherent PBMC were counted when using light microscopy. Expression of the cytokines TGF-beta(1) and IL-6 was measured by ELISA, and mRNA levels were analysed by the RNAse-protection assay (RPA). Surface expression of E-selectin was quantified by flow cytometry. RESULTS: A relative minimum of adhesion was observed after LD-RT between 0.3 and 0.7 Gy. This was paralleled by an expression maximum of TGF-beta(1) and IL-6, as shown by protein and mRNA levels, respectively. Neutralization of TGF-beta(1) by monoclonal antibodies, but not of IL-6, increased PBMC adhesion to EC nearly to control levels. In addition, fluorescence activated cell sorter (FACS) analysis of irradiated EC demonstrated a down-regulation of E-selectin in the same dose range. CONCLUSION: Low-dose X-irradiation between 0.3 and 0.7 Gy induced a relative maximum of TGF-beta(1) production by stimulated EC. This results in a down-regulation of leukocyte/PBMC adhesion and may contribute to the anti-inflammatory effect of LD-RT.

UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotese-mediated shedding.

PURPOSE: L-selectin (CD62L) is a prerequisite for leucocyte adhesion to endothelial cells of blood vessels and consequently for transmigration. Its expression on the cell surface therefore regulates the ability of lymphocytes to enter lymph nodes, to re-enter blood vessels or to invade tissues at sites of inflammation. The aim of this study was to determine the expression of CD62L on apoptotic lymphocytes after UVB irradiation. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of normal healthy volunteers. Cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for activation. Apoptosis in peripheral T-cells and Jurkat cells was induced by irradiation with UVB (120 mJ/cm2). In addition, T-cells or Jurkat cells were cultured for the indicated time with anti-Fas antibody CH11. The CH11-induced apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk. For detection of apoptosis, cells were analysed by cytofluorometry for morphological changes typical for apoptosis. The reliability of the apoptotic cell gate was confirmed by staining with FITC-labelled annexin-V in the presence ofpropidium iodide (PI). For FACS analysis of CD62L expression on the cell-surface immunofluorescence was performed using FITC-conjugated anti-CD62L and PE-conjugated anti-CD3 antibodies. Soluble CD62L (sCD62L) in the cell supernatants was measured by standard ELISA technique. Assays were performed in the presence and absence of metalloprotease inhibitor KB8301. RESULTS: PBMC from healthy volunteers undergoing apoptosis following UVB irradiation selectively shed CD62L, whereas the expression of the lineage-specific marker CD3 showed only minor changes. Shedding was blocked by the hydroxamic acid-based metalloprotease inhibitor KB8301. When Jurkat cells were treated with the caspase inhibitor zVAD-fmk, anti-CD95 antibodies did not induce apoptosis, and the expression of CD62L remained unaltered. CONCLUSION: UVB or ionizing radiation induce apoptosis in lymphocytes. The loss of CD62L is associated with apoptosis and will influence lymphocyte trafficking and, by excluding them from CD62L-mediated adhesion and tissue invasion, might contribute to the regulation of inflammation.

Apoptotic UV-irradiated lymphocytes undergo protease mediated shedding of L-selectin in vitro.

Adhesion between circulating lymphocytes and endothelial cells of the vessel wall depends on the expression of selectins and is the first step of tissue invasion which characterises inflammation. UV-B is well known to induce apoptosis in lymphocytes. We show that induction of apoptosis by any procedure leads to a metalloprotease mediated shedding of L-selectin from the surface of T-lymphocytes. Together with the previously published immunosuppressive action of apoptotic cells, this may contribute to the clinical effect of UV-B application, especially in photopheresis.

Experimental model for transplantation of a modified free myocutaneous gracilis flap to an irradiated neck region in rats.

In 102 Wistar rats (male, weight 300-500 g), a modified free myocutaneous gracilis flap was obtained from the groin and transplanted to the neck. To create a pre-irradiated transplant bed, a local area of the neck was irradiated preoperatively with 30 Gy (fractionation: 3 x 10 Gy) in 30 animals, and with 50 Gy (fractionation: 5 x 10 Gy) in a further 30 animals. The interval between preoperative irradiation and transplantation was 4 weeks. Forty-two animals received no such preoperative radiation. The evaluation of healing and the success of the transplanted flap was based on a clinical assessment, carried out on postoperative days 1 7. Testing for significant differences was done nonparametrically using the Kruskal-Wallis test. The survival rate in the nonirradiated animals was 86%. In contrast, the healing of the free flaps in the pre-irradiated transplant bed was significantly lower (P=0.003) 76%, after irradiation with 30 Gy and 50% after 50 Gy. The significant difference (P=0.020) in survival rates after irradiation with 30 and 50 Gy was evidence for the dependence of successful healing on the preoperative radiation dose. Transplantation of the free myocutaneous gracilis flap to a previously irradiated transplant bed in the region of the neck is a suitable model for investigating the healing of free transplants to irradiated tissue. The success rate observed in non-irradiated transplant beds is comparable to that seen with other flap models in rats.

Histomorphometric analysis of irradiated recipient vessels and transplant vessels of free flaps in patients undergoing reconstruction after ablative surgery.

The aim of the study was to investigate, histomorphometrically, quantitative and qualitative changes in irradiated neck recipient vessels and transplant vessels used for microsurgical anastomoses in free flaps in patients undergoing preoperative radiotherapy and chemotherapy. In 55 patients receiving 42 radial forearm flaps, 6 latissimus dorsi flaps, 6 osteomyocutaneous fibula grafts and 1 lateral arm flap, a total of 220 vessels were obtained from neck recipient vessels and transplant vessels during anastomosis. Three groups were formed: Group 1 (16 patients) treated with no radiotherapy or chemotherapy; Group 2 (20 patients) treated with preoperative irradiation (40-50 Gy) and chemotherapy (800 mg/m2 5-FU and 20 mg/m2 cisplatin) 1.5 months prior to surgery; Group 3 (19 patients) treated with radiotherapy (60-70 Gy) (median interval 78.7 months; IQR 31.3 months) prior to surgery. From each of the 220 vessel specimens, 3 sections each were histomorphometrically investigated, both qualitatively and quantitatively. To evaluate these changes as a function of age, radiation dose and chemotherapy, a statistical analysis was performed using analysis of covariance and chi-square tests. In Group 3, qualitative changes (intima dehiscence, hyalinosis) were found in recipient arteries significantly more frequently (25%, P=0.009) than in Groups 1 and 2. For Group 3 recipient arteries, histomorphometry revealed a significant decrease in the ratio of media area/total vessel area (median 0.53, IQR 0.10) in comparison with Group 1 (P= 0.02) (median 0.60, IQR 0.29) and Group 2 (P=0.046) (median 0.59, IQR 0.10). No significant differences were found between the vessels of Groups 1 and 2 (P= 0.48). Age and chemotherapy did not appear to have a significant influence on vessel changes in this study.

Modulation of inflammatory immune reactions by low-dose ionizing radiation: molecular mechanisms and clinical application.

During the last decade, a multitude of experimental evidence has accumulated showing that low-dose radiation therapy (single dose 0.5-1 Gy) functionally modulates a variety of inflammatory processes and cellular compounds including endothelial (EC), mononuclear (PBMC) and polymorphonuclear (PMN) cells, respectively. These modulations comprise a hampered leukocyte adhesion to EC, induction of apoptosis, a reduced activity of the inducible nitric oxide synthase, and a lowered oxidative burst in macrophages. Moreover, irradiation with a single dose between 0.5-0.7 Gy has been shown to induce the expression of X-chromosome linked inhibitor of apoptosis and transforming growth factor beta 1, to reduce the expression of E-selectin and L-selectin from EC and PBMC, and to hamper secretion of Interleukin-1, or chemokine CCL20 from macrophages and PMN. Notably, a common feature of most of these responses is that they display discontinuous or biphasic dose dependencies, shared with "non-targeted" effects of low-dose irradiation exposure like the bystander response and hyper-radiosensitivity. Thus, the purpose of the present review is to discuss recent developments in the understanding of low-dose irradiation immune modulating properties with special emphasis on discontinuous dose response relationships.

Radio-immunological mechanisms of anti-inflammatory treatment: is there a way from the past into the future?

The anti-inflammatory efficiency of low-dose radiotherapy (LD-RT) for degenerative joint disorders was demonstrated over decades but had no explanation on a cellular or molecular level. As inflammatory diseases are the results of complex and pathologically unbalanced cellular and molecular interactions more recent in-vivo and in-vitro data will be discussed for possible explanation of the mechanism underlying ant-inflammatory LD-RT.2.

Whole body low dose irradiation improves the course of beginning polyarthritis in human TNF-transgenic mice.

Rheumatoid arthritis (RA) displays a chronic inflammatory joint disease, accompanied by symmetric polyarthritis (PA) which evokes synovial inflammation, cartilage damage, and bone erosion. Patients with RA are routinely treated by immunosuppressive drugs. The therapy of inflammatory diseases and degenerative disorders with Low-dose radiotherapy (LD-RT) (single doses from 0.3 to 1.0 Gy) represents a low cost therapy with low toxicity, and is able to substitute at least in part treatment with drugs. The efficiency of LD-RT has already been proven in several animal models of inducible arthritis. In the present study we used a human TNF transgenic mouse model to examine the effects of LD-RT on PA. We observed a significant temporal improvement of the clinical progression of disease when mice were irradiated at the beginning of the disease. These data emphasize the role of LD-RT in clinical settings to treat patients with chronic and degenerative disorders and diseases.