Dose- and LET-dependent changes in mouse skin contracture up to a year after either single dose or fractionated doses of carbon ion or gamma rays.

Time dependence of relative biological effectiveness (RBE) of carbon ions for skin damage was investigated to answer the question of whether the flat distribution of biological doses within a Spread-Out Bragg peak (SOBP) which is designed based on in vitro cell kill could also be flat for in vivo late responding tissue. Two spots of Indian ink intracutaneously injected into the legs of C3H mice were measured by calipers. An equieffective dose to produce 30% skin contraction was calculated from a dose-response curve and used to calculate the RBE of carbon ion beams. We discovered skin contraction progressed after irradiation and then reached a stable/slow progression phase. Equieffective doses decreased with time and the decrease was most prominent for gamma rays and least prominent for 100 keV/µm carbon ions. Survival parameter of alpha but not beta in the linear-quadratic model is closely related to the RBE of carbon ions. Biological doses within the SOBP increased with time but their distribution was still flat up to 1 year after irradiation. The outcomes of skin contraction studies suggest that (i) despite the higher RBE for skin contracture after carbon ions compared to gamma rays, gamma rays can result in a more severe late effect of skin contracture. This is due to the carbon effect saturating at a lower dose than gamma rays, and (ii) the biological dose distribution throughout the SOBP remains approximately the same even one year after exposure.

A Potential Renewed Use of Very Heavy Ions for Therapy: Neon Minibeam Radiation Therapy.

(1) Background: among all types of radiation, very heavy ions, such as Neon (Ne) or Argon (Ar), are the optimum candidates for hypoxic tumor treatments due to their reduced oxygen enhancement effect. However, their pioneering clinical use in the 1970s was halted due to severe side effects. The aim of this work was to provide a first proof that the combination of very heavy ions with minibeam radiation therapy leads to a minimization of toxicities and, thus, opening the door for a renewed use of heavy ions for therapy; (2) Methods: mouse legs were irradiated with either Ne MBRT or Ne broad beams at the same average dose. Skin toxicity was scored for a period of four weeks. Histopathology evaluations were carried out at the end of the study; (3) Results: a significant difference in toxicity was observed between the two irradiated groups. While severe da-mage, including necrosis, was observed in the broad beam group, only light to mild erythema was present in the MBRT group; (4) Conclusion: Ne MBRT is significantly better tolerated than conventional broad beam irradiations.

Closed Fracture Diagnosed by Bedside Ultrasonography During Hemodialysis: A Report of Seven Cases and Relevant Clinical Characteristics.

BACKGROUND: Patients undergoing dialysis have a high incidence of fracture, and early diagnosis is important. We report seven cases of closed rib or upper-limb fractures diagnosed by bedside ultrasonography during maintenance hemodialysis sessions and describe relevant clinical characteristics. CASE PRESENTATION: We identified seven patients who were injured by falls in their homes. No injuries occurred on the day of dialysis. Five of the 7 patients did not visit the emergency room. All patients complained of persistent unexplained pain during a regular hemodialysis session. Ultrasonography (US) was performed during dialysis sessions, without any reports of pain. Before US evaluation, the sensitivity of radiography for diagnosis of fracture was 25%, while the sensitivity of US was 100%. Compared with other patients in our clinic, these patients were significantly older and had lower serum albumin concentrations and lower hemodialysis efficiency as determined by Kt/V. They also had a higher incidence of diabetes and a greater need for vasopressors during dialysis. These findings were consistent with the results of previous studies of the characteristics of fractures in dialysis patients. However, blood levels of creatinine, corrected calcium, phosphate, intact parathyroid hormone, and hemoglobin, as well as bone density and blood pressure, after the previous dialysis session were not different. CONCLUSIONS: To our knowledge, this is the first report of closed fracture of superficial bone diagnosed by bedside ultrasonography during a hemodialysis session. Ultrasonography is especially useful for diagnosis in these cases because it is noninvasive and highly accurate. Doctors should determine the differential diagnosis for closed fracture in patients undergoing dialysis, especially in those who are older, have diabetes, and are malnourished, and in those with recent contusions and persistent pain.

The FGF1/CPP-C chimera protein protects against intestinal adverse effects of C-ion radiotherapy without exacerbating pancreatic carcinoma.

BACKGROUND AND PURPOSE: Carbon ion (C-ion) beams are concentrated to irradiate pancreatic carcinoma in the upper abdomen; however, this radiotherapy potentially causes adverse reactions in the gastrointestinal tract. FGF1 is a candidate radioprotector for radiation-induced intestinal damage, but may promote the malignancy of pancreatic cancer. An FGF1/CPP-C chimeric protein was created to enhance the intracellular signaling mode of FGF1 instead of FGFR signaling. The present study investigated the effects of FGF1/CPP-C on the intestinal adverse reactions of C-ion radiotherapy as well as its influence on the malignancy of pancreatic cancer. MATERIALS AND METHODS: FGF1/CPP-C was administered intraperitoneally to BALB/c mice without heparin 12 h before total body irradiation (TBI) with low-LET C-ion (17 keV/µm) at 6-8 Gy. Several radioprotective effects were examined in the jejunum. The invasion and migration of the human pancreatic carcinoma cell lines MIAPaCa-2 and PANC-1 were assessed using Boyden chambers after cultures with FGF1/CPP-C. RESULTS: The FGF1/CPP-C treatment promoted crypt survival after C-ion irradiation at 7-8 Gy significantly more than the FGF1 treatment. FGF1/CPP-C also inhibited C-ion radiotherapy-induced apoptosis and reduced γH2AX foci in crypt cells more than FGF1. However, FGF1/CPP-C inhibited the downstream signaling pathways of FGFRs and suppressed the activation of cell-cycle regulatory molecules in the intestine until 4 h after TBI. Furthermore, IEC6 cells were arrested in G2M after cultures with FGF1/CPP-C or FGF1, suggesting that DNA repair after irradiation is promoted by FGF1/CPP-C-induced G2M arrest. In contrast, FGF1/CPP-C appeared to be internalized into MIAPaCa-2 and PANC-1 cells more efficiently than FGF1. Therefore, FGF1/CPP-C reduced the in vitro proliferation, invasion, and migration of MIAPaCa-2 and PANC-1 cells significantly more than FGF1 through the cellular internalization of FGF1. CONCLUSION: These results suggest that the intracellular signaling mode of FGF1/CPP-C attenuates the intestinal adverse effects of C-ion radiotherapy without enhancing the malignancy of pancreatic carcinoma.

Antimetastatic Effects of Carbon-Ion Beams on Malignant Melanomas.

The goal of this work was to clarify the effect of carbon-ion beams on reduction of the metastatic potential of malignant melanoma using in vitro and in vivo techniques. We utilized a 290 MeV/u carbon beam with a 6-cm spread-out Bragg-peak (SOBP), 137Cs γ rays or 200 kVp X rays for irradiation, and in vitro murine melanoma B16/BL6 cells that were implanted into C57BL/6J mice. The metastatic abilities (migration, invasion and adhesion) were suppressed by carbon ion treatment at all doses that were tested, whereas invasion and migration tended to increase after X-ray irradiation at low dose. Biological effects of carbon ions increased with linear energy transfer (LET) for both cell killing and metastatic abilities, although the effects were more pronounced for migration and invasion. mRNA expression of E-cadherin was significantly downregulated with low-dose photon exposures, but increased with dose or LET. Expression of Mel-CAM and L1-CAM was upregulated after low-dose photon exposure, but decreased with dose, especially after carbon-ion treatment. Conversely, these molecules showed a reversal in expression changes, especially after low-dose photon exposure. Cell-cell adhesion may be an important contributor to the antimetastatic effect of carbon ion treatment. The number of lung metastases after local tumor irradiation significantly decreased with increased dose and LET, with carbon ions being more effective than γ rays. Integrating dose-response curves to examine the relationship between cell killing and lung metastasis clearly showed that carbon ions inhibit lung metastasis more efficiently than photons at the iso-effective level of cell killing. Thus, carbon ions were more effective than photon beams, not only at killing tumor cells, but also at inhibiting metastatic spread caused by tumor cells that survived irradiation.

Utility of Ultrasonography of the Median Nerve With a High-Frequency Probe for the Diagnosis of Dialysis-Related Carpal Tunnel Syndrome.

This cross-sectional study aimed to determine the utility of ultrasonography with improved resolution using a high-frequency probe for dialysis-related carpal tunnel syndrome (CTS). This study targeted 125 hemodialysis patients at our hospital. A 12 MHz probe was placed on the carpal tunnel area to identify the median nerve. The compression rate of the nerve was calculated by measuring the smallest diameter of the compressed nerve and largest diameter of the unaffected part. To quantify CTS symptoms, we determined the presence of Tinel's sign, measured pinch strength, and used questionnaires to assess numbness and pain. The association of these clinical data with the compression rate was examined. Mean compression rate was 12.1 ± 1.1%. The compression rate cutoff value for those positive with Tinel's sign was 25%, (sensitivity and specificity were 0.80 and 0.91, respectively), and that for those with a history of CTS surgery was 25% (sensitivity and specificity were 0.67 and 0.89, respectively). Multiple regression analysis identified duration of dialysis, ß2-microglobulin(ß2-Mg) concentration, positivity for Tinel's sign, history of CTS surgery, and pinch strength as independent compression rate determinants. Notably, compression rates were significantly higher in patients with a ≥4-year duration of dialysis and a ß2-Mg level of 20 mg/L or more. The compression rate of the median nerve measured by an improved ultrasound device significantly correlated with clinical symptoms, medical history, and serological features associated with dialysis-related CTS. Because ultrasonography is non-invasive, the examination might be a simple method especially for early diagnosis of dialysis-related CTS.

Determination of the relative biological effectiveness and oxygen enhancement ratio for micronuclei formation using high-LET radiation in solid tumor cells: An in vitro and in vivo study.

We determined the relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) of micronuclei (MN) formation in clamped (hypoxic) and non-clamped (normoxic) solid tumors in mice legs following exposure to X-rays and heavy ions. Single-cell suspensions (aerobic) of non-irradiated tumors were prepared in parallel and used directly to determine the radiation response for aerobic cells. Squamous cell carcinoma (SCCVII) cells were transplanted into the right hind legs of syngeneic C3H/He male mice. Irradiation doses with either X-rays or heavy ions at a dose-averaged LET (linear energy transfer) of 14-192keV/µm were delivered to 5-mm diameter tumors and aerobic single-cells in sample-tubes. After irradiation, the tumors were excised and trypsinized to observe MN in single-cells. The single-cell suspensions were used for MN formation assays. The RBE values increased with increasing LET. The maximum RBE values for the three different oxygen conditions; hypoxic tumor, normoxic tumor, and aerobic cells, were 8.18, 5.30, and 3.76 at an LET of 192keV/µm, respectively. After X-irradiation, the OERh/n values (hypoxic tumor/normoxic tumor) were lower than the OERh/a (hypoxic tumor/aerobic cells), and were 1.73 and 2.58, respectively. We found that the OER for the in vivo studies were smaller in comparison to that for the in vitro studies. Both of the OER values at 192keV/µm were small in comparison to those of the X-ray irradiated samples. The OERh/n and OERh/a values at 192keV/µm were 1.12 and 1.19, respectively. Our results suggest that high LET radiation has a large biological effect even if a solid tumor includes substantial numbers of hypoxic cells. To conclude, we found that the RBE values under each oxygen state for non-MN fraction increased with increasing LET and that the OER values for both tumors in vivo and cells in vitro decreased with increasing LET.

Evaluation of therapeutic gain for fractionated carbon-ion radiotherapy using the tumor growth delay and crypt survival assays.

BACKGROUND AND PURPOSE: The aim of the study was to evaluate the therapeutic gain of carbon ion (C-ion) radiotherapy using a mouse model. MATERIALS AND METHODS: Transplanted fibrosarcoma (NFSa) growing in C3H/He mice and murine small intestine were irradiated with 290 MeV/nucleon C-ion beams (C-ions) in 1-12 fractions separated by 4h. The cell killing efficiencies of C-ions were measured using jejunum crypt survival and tumor growth delay (TGD) assays. RESULTS: The equieffect dose for crypt survival and TGD increased with increasing number of fractions after X-rays and 20 keV/µm C-ions, whereas TGD after 77 keV/µm C-ions rather decreased. Crypts showed stronger LET-dependent increase in α terms than the tumor while ß terms less depended on LET irrespective of tissues. Therapeutic gain factor, i.e., a ratio of tumor RBE over crypt RBE, of 77 keV/µm C-ions was more than unity at any doses while that of 20 keV/µm C-ions increased with an increase in dose per fraction. CONCLUSIONS: These specific data imply that use of large dose per fraction would be suitable for C-ion radiotherapy irrespective of LET from the point of view of therapeutic gain, though small dose per fraction by high-LET radiation decreases total dose for tumor.

Designing a ridge filter based on a mouse foot skin reaction to spread out Bragg-peaks for carbon-ion radiotherapy.

BACKGROUND AND PURPOSE: Carbon-ion radiotherapy uses spread-out Bragg peaks (SOBP) to produce uniform biological effects within a target volume. The relative biological effectiveness is determined by the in vitro cell kill after a single dose is employed to design the SOBP. A question remains as to whether biological effects for in vivo tissues after fractionated doses are also uniform within the SOBP. MATERIAL AND METHODS: Mouse foot skin was irradiated with fractionated doses of carbon ions at various linear energy transfer (LET) values. A new ridge filter was designed based on alpha and beta values for each LET to cause moderate skin reaction, and was studied concerning its uniformity. RESULTS: The reciprocal total doses of intermediate-LET carbon ions and of reference gamma rays linearly increased with an increase of a dose per fraction in Fe-plots. As the single total dose of higher LET run off linearity, data obtained from 2 to 6 fractions were used to design a new ridge filter. The physical dose distribution of the new ridge filter was almost identical to, and indistinguishable from, the ridge filter designed based on the in vitro cell kill. CONCLUSIONS: The LET dependence of alpha is a principle of the biological factor to be used for designing spread-out Bragg peaks of carbon-ion radiotherapy.

Tumor induction in mice after local irradiation with single doses of either carbon-ion beams or gamma rays.

PURPOSE: To determine the dose-dependent relative biological effectiveness (RBE) for tumor prevalence in mice receiving single localized doses to their right leg of either carbon ions (15, 45 or 75 keV/µm) or 137Cs gamma rays. METHODS AND MATERIALS: A total of 1647 female C3H mice were irradiated to their hind legs with a localized dose of either reference gamma rays or 15, 45 or 75 keV/µm carbon-ion beams. Irradiated mice were evaluated for tumors twice a month during their three-year life span, and the dimensions of any tumors found were measured with a caliper. The tumor induction frequency was calculated by Kaplan-Meier analysis. RESULTS: The incidence of tumors from 50 Gy of 45 keV/µm carbon ions was marginally higher than those from 50 Gy of gamma rays. However, 60 Gy of 15 keV/µm carbon ions induced significantly fewer tumors than did gamma rays. RBE values of 0.87 + 0.12, 1.29 + 0.08 or 2.06 + 0.39 for lifetime tumorigenesis were calculated for 15, 45 or 75 keV/µm carbon-ion beams, respectively. Fibrosarcoma predominated, with no Linear Energy Transfer (LET)-dependent differences in the tumor histology. Experiments measuring the late effect of leg skin shrinkage suggested that the carcinogenic damage of 15 keV/µm carbon ions would be less than that of gamma rays. CONCLUSIONS: We conclude that patients receiving radiation doses to their normal tissues would face less risk of secondary tumor induction by carbon ions of intermediate LET values compared to equivalent doses of photons.

Nd:YAG Laser Treatment for Keloids and Hypertrophic Scars: An Analysis of 102 Cases.

BACKGROUND: The present retrospective cohort study was performed to determine the efficacy of contact-mode 1064 nm neodymium-yttrium-aluminum-garnet (Nd:YAG) laser laser for keloids and hypertrophic scars. The indication and limitations of this modality are discussed. METHODS: The cohort consisted of 102 consecutive Japanese patients (23 males and 79 females) with keloids and hypertrophic scars for more than 1 year. They were treated every 3-4 weeks for 1 year with a long-pulsed 1064 nm Nd:YAG laser (Cutera, Brisbane, Calif.) in contact mode. Thirty-eight patients had hypertrophic scars and 64 had keloids. The scars were evaluated before the treatment commenced and 1 month after the last session by using the Japan Scar Workshop Scar Scale 2011. Recurrence was assessed at 6 months after the termination of treatment. RESULTS: The average total Japan Scar Workshop score of the keloid and hypertrophic scar region groups dropped significantly after 1 year of treatment compared with before treatment (all P < 0.05). None of the hypertrophic scars or keloids deteriorated. However, 3 of the 34 anterior chest keloids (8.8%) did not respond. The following recurrence rates were observed 6 months after stopping laser treatment: 1 of the abdomen hypertrophic scars (4%), 18 of the anterior chest keloids (52.9%), 5 of the upper arm keloids (35.7%), and 4 of the scapula keloids (25%). CONCLUSIONS: Hypertrophic scars responded significantly better to 1064 nm Nd:YAG laser treatment than keloids. However, keloid recurrence occurred when there was remaining redness and induration, even if only a small part of the scar was affected.

Evaluation of SCCVII tumor cell survival in clamped and non-clamped solid tumors exposed to carbon-ion beams in comparison to X-rays.

The aim of this study was to measure the RBE (relative biological effectiveness) and OER (oxygen enhancement ratio) for survival of cells within implanted solid tumors following exposure to 290MeV/nucleon carbon-ion beams or X-rays. Squamous cell carcinoma cells (SCCVII) were transplanted into the right hind legs of syngeneic C3H male mice. Irradiation with either carbon-ion beams with a 6-cm spread-out Bragg peak (SOBP, at 46 and 80keV/µm) or X-rays was delivered to 5-mm or less diameter tumors. We defined three different oxygen statuses of the irradiated cells. Hypoxic and normoxic conditions in tumors were produced by clamping or not clamping the leg to avoid blood flow. Furthermore, single-cell suspensions were prepared from non-irradiated tumors and directly used to determine the radiation response of aerobic cells. Single-cell suspensions (aerobic condition) were fully air-saturated. Single-cell suspensions were prepared from excised and trypsinized tumors, and were used for in vivo-in vitro colony formation assays to obtain cell survival curves. The RBE values increased with increasing LET in SOBP beams. The maximum RBE values in three different oxygen conditions; hypoxic tumor, normoxic tumor and aerobic cells, were 2.16, 1.76 and 1.66 at an LET of 80keV/µm, respectively. After X-ray irradiation the OERh/n values (hypoxic tumor/normoxic tumor) were lower than the OERh/a (hypoxic tumor/aerobic cells), and were 1.87±0.13 and 2.52±0.11, respectively. The OER values of carbon-ion irradiated samples were small in comparison to those of X-ray irradiated samples. However, no significant changes of the OER at proximal and distal positions within the SOBP carbon-ion beams were observed. To conclude, we found that the RBE values for cell survival increased with increasing LET and that the OER values changed little with increasing LET within the SOBP carbon-ion beams.

Quantitative 17O imaging towards oxygen consumption study in tumor bearing mice at 7 T.

(17)O magnetic resonance imaging (MRI) using a conventional pulse sequence was explored as a method of quantitative imaging towards regional oxygen consumption rate measurement for tumor evaluation in mice. At 7 T, fast imaging with steady state (FISP) was the best among gradient echo, fast spin echo and FISP for the purpose. The distribution of natural abundance H2(17)O in mice was visualized under spatial resolution of 2.5 × 2.5mm(2) by FISP in 10 min. The signal intensity by FISP showed a linear relationship with (17)O quantity both in phantom and mice. Following the injection of 5% (17)O enriched saline, (17)O re-distribution was monitored in temporal resolution down to 5 sec with an image quality sufficient to distinguish each organ. The image of labeled water produced from inhaled (17)O2 gas was also obtained. The present method provides quantitative (17)O images under sufficient temporal and spatial resolution for the evaluation of oxygen consumption rate in each organ. Experiments using various model compounds of R-OH type clarified that the signal contribution of body constituents other than water in the present in vivo(17)O FISP image was negligible.

Nd:YAG Laser Treatment of Keloids and Hypertrophic Scars.

Pathological cutaneous scars such as keloids and hypertrophic scars (HSs) are characterized by a diffuse redness that is caused by the overgrowth of capillary vessels due to chronic inflammation. Our group has been using long-pulsed, 1064-nm Nd:YAG laser in noncontact mode with low fluence and a submillisecond pulse duration to treat keloids and hypertrophic scars since 2006 with satisfactory results. The present study examined the efficacy of this approach in 22 Japanese patients with keloids (n = 16) or hypertrophic scars (n = 6) who were treated every 3 to 4 weeks. Treatment settings were as follows: 5 mm spot size diameter; 14 J/cm(2) energy density; 300 µs exposure time per pulse; and 10 Hz repetition rate. The responses of the pathological scars to the treatment were assessed by measuring their erythema, hypertrophy, hardness, itching, and pain or tenderness. Moreover, skin samples from 3 volunteer patients were subjected to histological evaluation and 5 patients underwent thermography during therapy. The average total scar assessment score dropped from 9.86 to 6.34. Hematoxylin and eosin staining and Elastica Masson-Goldner staining showed that laser treatment structurally changed the tissue collagen. This influence reached a depth of 0.5 to 1 mm. Electron microscopy revealed plasma protein leakage, proteoglycan particles, and a change in the collagen fiber fascicles. Further analyses revealed that noncontact mode Nd:YAG laser treatment is highly effective for keloids and hypertrophic scars regardless of patient age, the origin and multiplicity of scarring, the location of the scar(s), or the tension on the scar.

Clinical applications of basic research that shows reducing skin tension could prevent and treat abnormal scarring: the importance of fascial/subcutaneous tensile reduction sutures and flap surgery for keloid and hypertrophic scar reconstruction.

We use evidence-based algorithms to treat abnormal scarring, including keloids and hypertrophic scars (HSs). This involves a multimodal approach that employs traditional methods such as surgical removal, postoperative radiotherapy, corticosteroid injection, laser, and silicone gel sheets. As a result, the rate of abnormal scarring recurrence has decreased dramatically over the past 10 years. However, several problems remain to be solved. First, despite the optimization of a radiotherapy protocol, over 10% of cases who are treated with surgery and postoperative radiotherapy still recur in our facility. Second, the treatment options for cases with huge keloids are very limited. To address these problems, we performed basic research on the mechanisms that drive the formation of keloids and HSs. Extrapolation of these research observations to the clinic has led to the development of two treatment strategies that have reduced the rate of abnormal scar recurrence further and provided a means to remove large scars. Our finite element analysis of the mechanical force distribution around keloids revealed high skin tension at the keloid edges and lower tension in the keloid center. Moreover, when a sophisticated servo-controlled device was used to stretch wounded murine dorsal skin, it was observed that the stretched samples exhibited upregulated epidermal proliferation and angiogenesis, which are also observed in keloids and HSs. Real-time RT-PCR also revealed that growth factors and neuropeptides are more strongly expressed in cyclically stretched skin than in statically stretched skin. These findings support the well-established notion that mechanical forces on the skin strongly influence the cellular behavior that leads to scarring. These observations led us to focus on the importance of reducing skin tension when keloids/HSs are surgically removed to prevent their recurrence. Clinical trials revealed that subcutaneous/fascial tensile reduction sutures, which apply minimal tension on the dermis, are more effective in reducing recurrence than the three-layered sutures used by plastic surgeons. Moreover, we have found that by using skin flaps (e.g., perforator flaps and propeller flaps), which release tension on the wound, in combination with postoperative radiotherapy, huge keloids can be successfully treated.

Apparent absence of a proton beam dose rate effect and possible differences in RBE between Bragg peak and plateau.

PURPOSE: Respiration-gated irradiation for a moving target requires a longer time to deliver single fraction in proton radiotherapy (PRT). Ultrahigh dose rate (UDR) proton beam, which is 10-100 times higher than that is used in current clinical practice, has been investigated to deliver daily dose in single breath hold duration. The purpose of this study is to investigate the survival curve and relative biological effectiveness (RBE) of such an ultrahigh dose rate proton beam and their linear energy transfer (LET) dependence. METHODS: HSG cells were irradiated by a spatially and temporally uniform proton beam at two different dose rates: 8 Gy/min (CDR, clinical dose rate) and 325 Gy/min (UDR, ultrahigh dose rate) at the Bragg peak and 1.75 (CDR) and 114 Gy/min (UDR) at the plateau. To study LET dependence, the cells were positioned at the Bragg peak, where the absorbed dose-averaged LET was 3.19 keV/microm, and at the plateau, where it was 0.56 keV/microm. After the cell exposure and colony assay, the measured data were fitted by the linear quadratic (LQ) model and the survival curves and RBE at 10% survival were compared. RESULTS: No significant difference was observed in the survival curves between the two proton dose rates. The ratio of the RBE for CDR/UDR was 0.98 +/- 0.04 at the Bragg peak and 0.96 +/- 0.06 at the plateau. On the other hand, Bragg peak/plateau RBE ratio was 1.15 +/- 0.05 for UDR and 1.18 +/- 0.07 for CDR. CONCLUSIONS: Present RBE can be consistently used in treatment planning of PRT using ultrahigh dose rate radiation. Because a significant increase in RBE toward the Bragg peak was observed for both UDR and CDR, further evaluation of RBE enhancement toward the Bragg peak and beyond is required.

Influence of manipulating hypoxia in solid tumors on the radiation dose-rate effect in vivo, with reference to that in the quiescent cell population.

PURPOSE: The aim of this study was to clarify the effect of manipulating intratumor hypoxia on radiosensitivity under reduced dose-rate (RDR) irradiation. MATERIALS AND METHODS: Tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. They received gamma-rays or accelerated carbon-ion beams at high dose-rate (HDR) or RDR with or without tumor clamping to induce hypoxia. Some mice without clamping received nicotinamide, an acute hypoxia-releasing agent or misonidazole, a hypoxic cell radio-sensitizer before irradiation. The responses of quiescent (Q) and total (= P + Q) cells were assessed by the micronucleus frequency using immunofluorescence staining for BrdU. RESULTS: The clearer decrease in radiosensitivity in Q than total cells after RDR gamma-ray irradiation was suppressed with carbon-ion beams, especially with a higher linear energy transfer value. Repressing the decrease in the radiosensitivity under RDR irradiation through keeping tumors hypoxic during irradiation and enhancing the decrease in the radiosensitivity by nicotinamide were clearer with gamma-rays and in total cells than with carbon-ion beams and in Q cells, respectively. Inhibiting the decrease in the radiosensitivity by misonidazole was clearer with gamma-rays and in Q cells than with carbon-ion beams and in total cells, respectively. CONCLUSION: Manipulating hypoxia during RDR as well as HDR irradiation influences tumor radiosensitivity, especially with gamma-rays.

Microscope-assisted reduction clitoroplasty used to treat two patients with clitoromegaly.

We report 2 cases of clitoromegaly, 1 in a patient with true hermaphroditism, and the other in a patient with adrenogenital syndrome. Both were treated surgically with reduction clitoroplasty. There are 3 different clitoroplasty procedures: clitorectomy, clitoral recession, and reduction clitoroplasty. Reduction clitoroplasty with preservation of the neurovascular bundle is considered superior in terms of formation of the external genitals and sensation. However, the disadvantages are that detachment of the neurovascular bundle from the clitoral shaft is difficult and that there is a high possibility of sensory and blood flow disorders in the clitoris. In an attempt to achieve safe and reliable surgical manipulation, we used a surgical microscope (OPMI 6-SDFC, Carl Zeiss Surgical GmbH, magnification x8) to detach the neurovascular bundle from the clitoral shaft in our 2 patients. Our impression is that our efforts were extremely effective. Furthermore, our experience leads us to believe that the procedure for neurovascular bundle detachment required in reduction clitoroplasty is not particularly difficult if performed with a surgical microscope by a plastic surgeon who regularly performs microsurgery. Because the procedure can be performed simply and safely, we believe that reduction clitoroplasty with preservation of the neurovascular bundle is the best overall of the 3 clitoroplasty procedures.

Therapeutic angiogenesis by autologous bone marrow cell implantation together with allogeneic cultured dermal substitute for intractable ulcers in critical limb ischaemia.

Therapeutic angiogenesis by autologous bone marrow cell implantation improves blood supply in patients with critical limb ischaemia. In addition, allogeneic cultured dermal substitute is effective for intractable ulcers. The present study determined the effectiveness of bone marrow cell implantation combined with allogeneic cultured dermal substitute in treating severely ischaemic ulcers. We treated eight consecutive patients with severely ischaemic ulcers using this procedure. Stromal cells aspirated from bone marrow were processed to obtain suspensions of mononuclear cells, platelets and endothelial progenitor cells and immediately injected intramuscularly into the lower leg and around the wound, on which allogeneic cultured dermal substitute was applied and changed weekly. Skin ulcers were subsequently closed by skin grafting, if necessary. Angiogenesis was confirmed by postoperative analyses such as ankle-brachial pressure index, angiography, thermography and (99m)Technetium-Tetrofosmin perfusion scintigraphy. Above- or below-knee amputation was avoided in all patients and wounds were completely closed in six of them. These results indicate that this combined therapy effectively treated ischaemic ulcers. Since the incidence of this condition might increase in the future, this therapeutic approach should play an important role in the preservation of ischaemic limbs.

[11C]Gefitinib ([11c]Iressa): radiosynthesis, in vitro uptake, and in vivo imaging of intact murine fibrosarcoma.

OBJECTIVE: Gefitinib (N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(morpholin-4-yl)propoxy]quinazolin-4-amine, Iressa) is an approved anticancer drug. In this study, we labeled gefitinib with carbon-11 and evaluated [(11)C]gefitinib to explore its specific binding in intact fibrosarcoma (NFSa)-bearing mice. METHODS: [(11)C]Gefitinib was synthesized by the reaction of desmethyl precursor (1) with [(11)C]CH(3)I. In vitro uptake of [(11)C]gefitinib into NFSa, human-A431 epidermoid carcinoma, and Jurkat T cells was determined. Positron emission tomography (PET) imaging using [(11)C]gefitinib was performed for NFSa-bearing mice. RESULTS: [(11)C]Gefitinib accumulated into NFSa cells with 2.1 uptake ratio (UR)/mg protein in cells. Addition of nonradioactive gefitinib decreased uptake in a concentration-dependent manner. [(11)C]Gefitinib also had high uptake (2.6 UR/mg protein) into epidermal growth factor receptor/tyrosine kinase (EGFR/TK)-rich A431 cells but low uptake (0.2 UR/mg protein) into EGFR/TK-poor Jurkat cells. In vivo distribution study on NFSa-bearing mice by the dissection method revealed that [(11)C]gefitinib specifically accumulated into the tumor. The ratio of radioactivity in tumors to that in blood and muscle as two comparative regions increased from 0.4 to 6.0 and from 0.6 to 5.0 during this experiment (0-60 min), respectively. PET for NFSa-bearing mice produced a clear tumor image, although high radioactivity was distributed throughout the body. Treatment with nonradioactive gefitinib (100 mg/kg) decreased uptake in the tumor. In vivo metabolite analysis demonstrated that [(11)C]gefitinib was stable in the tumor, liver, kidney, and blood. CONCLUSION: These results demonstrated the promising potential of [(11)C]gefitinib to serve as a PET ligand for in vivo imaging of NFSa-bearing mice.