[Biochemical markers of bone turnover. New aspect. Bone metabolic markers available in daily practice].

Changes of bone remodeling markers reflect bone growth and bone turnover. Information on bone metabolism can be attained by blood and urine laboratory tests. Recently developed bone specific markers are categorized by bone remodeling process, i.e. bone formation and resorption. The formation markers include bone-specific alkaline phosphatase (BAP), osteocalcin (OC), undercarboxylated osteocalcin (ucOC), procollagene type I C- and N-terminal peptides (P1CP and P1NP). Bone resorption markers include deoxypyridinoline, collagen I C- and N-terminal telopeptides (CTX and NTX) , and tartrate resistent acid phosphatase (TRACP) isoform 5b. These laboratory tests offer lots of advantages for the diagnosis of bone metabolic disorders and for the evaluation of clinical states of primary osteoporosis and other metabolic skeletal diseases.

[Bone quality changes in diabetes].

Diabetes-related bone fragility has recently drawn many researchers' attention. Diabetes would affect bone remodeling by various mechanisms, including deficiency of insulin actions, increased accumulation of advanced glycation end products and microangiopathy. The combination of poor bone quality of microstructure and nanoarchitecture (type I collagen and non-collageous proteins) would reduce bone strength. Bone mineral density is the best predictor for fractures of primary osteoporosis, but presumably not for type 2 diabetes. Quality changes of diabetic bone, therefore, should be more thoroughly studied.

[Newly developed drugs for osteoporosis in overseas and their future roles for the therapy].

Bisphosphonates are widely used, though gastrointestinal tolerance is a problem on daily administration. Intermittent regimen, from once weekly to once yearly, is now available in overseas and can overcome GI adverse events. New generation of anti-resorptive agents (anti-RANKL antibody and a new SERM, bazedoxifene) are promising and will be soon available for the treatment of osteoporosis. Anabolic agents such as teriparatide and strontium ranelate have marked effects on BMD and reduction on fracture risk. While none of these options is suitable for everyone, the range of future available therapies does mean that most patients can find an intervention that is effective and acceptable.

[Parathyroid and bone. Evidence and perspective of parathyroid therapy for patients with osteoporosis].

Parathyroid hormone (PTH) is a new management option for patients with osteoporosis. As an anabolic agent that affects bone remodeling and modeling, a novel approach to reducing fracture risk could be considered for patients with severe conditions. A number of trials have shown that increases in spine and hip bone mineral density (BMD), and reduction of fracture risk in postmenopausal women. Although the combination of PTH and alendronate does not seem to be additive, PTH followed by alendronate would yield maximum increase in BMD. Treatment with PTH can change the course of osteoporosis by directly stimulating formation of new bone, and its application should be explored in daily clinical practice.

[Osteocalcin and bone].

Osteocalcin (OC) is a product of osteoblasts and accumulated in the extracellular matrix of bone. It has been recognized that serum OC is a marker of osteoblast activity, and the levels reflect the rate of bone formation. The present assay system was developed to assess the major circulating forms of intact and the large N-terminal fragments. OC binds to the crystal of hydroxyapatite, at least partly, through gamma-carboxylation of three residues. Increased rate of immature undercarboxylated osteocalcin, therefore, might display risks for osteoporotic fractures in clinical studies. However, at present, measurement of OC does not substitute for bone mass measurement and only provide limited values to evaluate the conditions of patients with primary osteoporosis.

Repair of skin damage during fractionated irradiation with gamma rays and low-LET carbon ions.

In clinical use of carbon-ion beams, a deep-seated tumor is irradiated with a Spread-Out Bragg peak (SOBP) with a high-LET feature, whereas surface skin is irradiated with an entrance plateau, the LET of which is lower than that of the peak. The repair kinetics of murine skin damage caused by an entrance plateau of carbon ions was compared with that caused by photons using a scheme of daily fractionated doses followed by a top-up dose. Right hind legs received local irradiations with either 20 keV/microm carbon ions or gamma rays. The skin reaction of the irradiated legs was scored every other day up to Day 35 using a scoring scale that consisted of 10 steps, ranging from 0.5 to 5.0. An isoeffect dose to produce a skin reaction score of 3.0 was used to obtain a total dose and a top-up dose for each fractionation. Dependence on a preceding dose and on the time interval of a top-up dose was examined using gamma rays. For fractionated gamma rays, the total dose linearly increased while the top-up dose linearly decreased with an increase in the number of fractions. The magnitude of damage repair depended on the size of dose per fraction, and was larger for 5.2 Gy than 12.5 Gy. The total dose of carbon ions with 5.2 Gy per fraction did not change till 2 fractions, but abruptly increased at the 3rd fraction. Factors such as rapid repopulation, induced repair and cell cycle synchronization are possible explanations for the abrupt increase. As an abrupt increase/decrease of normal tissue damage could be caused by changing the number of fractions in carbon-ion radiotherapy, we conclude that, unlike photon therapy, skin damage should be carefully studied when the number of fractions is changed in new clinical trials.

[Management of osteoporosis in patients with inhaled glucocorticoids].

Inhaled glucocorticoids are the standard of therapy in asthma and are commonly prescribed for chronic obstructive pulmonary disease. Accumulating evidence suggests that the effect of inhaled glucocorticoids on bone is not small, especially in patients taking moderate or high doses for long periods of time. The risk of adverse events is likely to differ between inhaled glucocorticoids. Inhaled glucocorticoids should be used widely, since they reduce the need of oral corticosteroids and improve respiratory function, but that they need to be managed carefully to minimize the risk of fracture with long-term use. This article described the effects of inhaled glucocorticoids on bone and fracture risk.

Repair kinetics of DNA-DSB induced by X-rays or carbon ions under oxic and hypoxic conditions.

We studied the relation between initial DNA double-strand breaks (DNA-DSB) and the rejoining kinetics of the strand breaks, as well as the OER (oxygen enhancement ratio) after low- and high-LET (linear energy transfer) radiations. CHO cells were exposed to 200 kVp X-rays or 80 keV/microm carbon ions under oxic and hypoxic conditions. DNA-DSB in the cells were analyzed by a static-field gel electrophoresis (SFGE). The kinetics of the rejoining could be described by a sum of fast and slow components. The initial released DNA after X-ray irradiation was higher for cells irradiated under an oxic condition than that under a hypoxic condition. The OER of DNA-DSB after X-ray irradiation was 5.7. This value decreased rapidly to be 3.4 with the fast component by 15 minutes. On the other hand, the OER of DNA-DSB after carbon ion irradiation was 2.2, and this value was not changed by rejoining incubation. The OER values for cell killing were 2.8 and 1.8 after X-ray and carbon ion irradiations, respectively. These values matched to the OER for DNA-DSB with complete rejoining. We conclude that the rejoining of DNA-DSB is an important factor in the mechanism of the oxygen effect.

Biological gain of carbon-ion radiotherapy for the early response of tumor growth delay and against early response of skin reaction in mice.

The biological effectiveness of carbon ions relative to gamma rays (RBE) was compared between the tumor growth delay and an early skin reaction of syngeneic mice. The RBE was larger for a tumor than skin when irradiated with large doses of high-LET (linear energy transfer) carbon ions. The intra-track damage (a term of a linear quadratic model) of a tumor and skin increased equally with an increase of the LET, while the inter-track damage (beta term) of skin alone increased with the LET. These data provide evidence that high-LET radiotherapy could achieve therapeutic gain by minimizing the difference in response to fractionated irradiation between the tumor and normal tissue.

Radioprotective activities of beer administration for radiation-induced acute toxicity in mice.

BACKGROUND AND PURPOSE: We previously found that drinking beer reduces chromosome aberrations in blood lymphocytes that were collected and irradiated in vitro. In this study, we investigated the radioprotective activities of beer-administration for bone marrow and intestine in mice. METHODS: C3H/He female mice received an oral administration of beer, ethanol or saline at a dose of 1 ml/mouse 30 min before whole body irradiation with 137Cs gamma rays or LET 50 keV/microm carbon ions. Radioprotective activities were estimated using a LD(50/30) (The dose required to kill 50% of the mice within 30 days) and a microcolony technique for intestine. RESULTS: The LD(50/30) for the beer-administered mice was significantly increased in comparison with saline administered mice. The LD(50/30) of gamma-ray was 7.8 Gy (p < 0.05), 7.6 Gy and 7.3 Gy for beer-, ethanol- and saline-administered group, respectively. The LD(50/30) of carbon ions was 6.6 Gy (p < 0.05), 6.2 Gy and 5.9 Gy for the beer-, ethanol- and saline-administered groups, respectively. The crypt survivals that were semi-logarithmically plotted against dose were well fitted to a linear regression line. The dose reduction factor (DRF) (D10) of beer- and ethanol-administered mice for gamma rays was 1.09 and 1.08, respectively. The DRF (D10) of beer- and ethanol-administered mice for carbon ions was 1.08 and 1.07, respectively. CONCLUSIONS: The radioprotection by beer-administration is due to not only OH radical-scavenge action by the ethanol contained in beer.

Differences in pO2 peaks of a murine fibrosarcoma between carbon-ion and X-ray irradiation.

We measured and compared the oxygen partial pressure (pO(2)) profiles in experimental tumors after irradiation with carbon ions and with X-rays. The NFSa fibrosarcomas grown in the hind legs of C3H male mice received isoeffect single doses of carbon ions or X-rays. Coaxial oxygen microelectrodes of high spatial resolution were inserted into the tumor with 20 microm steps by a computerized micromanipulator. The number of pO(2) peaks that reached 15 mmHg were at least 0.45 per 3,000 microm in unirradiated tumors and significantly increased to 1.55 per 3,000 microm as early as day 1 of carbon-ion irradiation (p < 0.001). The tumors that received X-ray irradiation also significantly increased pO(2) peaks, but as late as day 3. The time course of pO(2) peak appearance in the present study coincides with a previous report where reoxygenation was measured by paired growth delay assay. The pO(2) peaks appeared selectively in peripheral regions of X-ray irradiated tumors, but they appeared rather homogeneously in the tumor after carbon-ion irradiation. It is concluded that carbon-ion irradiation reoxygenated the NFSa fibrosarcomas earlier in time and deeper in space than the X-ray irradiation did.

Relative biological effectiveness of 290 MeV/u carbon ions for the growth delay of a radioresistant murine fibrosarcoma.

The relative biological effectiveness (RBE) for animal tumors treated with fractionated doses of 290 MeV/u carbon ions was studied. The growth delay of NFSa fibrosarcoma in mice was investigated following various daily doses given with carbon ions or those given with cesium gamma-rays, and the RBE was determined. Animal tumors were irradiated with carbon ions of various LET (linear energy transfer) in a 6-cm SOBP (spread-out Bragg peak), and the isoeffect doses; i.e. the dose necessary to induce a tumor growth delay of 15 days were studied. The iso-effect dose for carbon ions of 14 and 20 keV/microm increased with an increase in the number of fractions up to 4 fractions. The increase in the isoeffect dose with the fraction number was small for carbon ions of 44 keV/microm, and was not observed for 74 keV/microm. The alpha and beta values of the linear-quadratic model for the radiation dose-cell survival relationship were calculated by the Fe-plot analysis method. The alpha values increased linearly with an increase in the LET, while the beta values were independent of the LET. The alpha/beta ratio was 129 +/- 10 Gy for gamma-rays, and increased with an increase in the LET, reaching 475 +/- 168 Gy for 74 keV/microm carbon ions. The RBE for carbon ions relative to Cs-137 gamma-rays increased with the LET. The RBE values for 14 and 20 keV/microm carbon ions were 1.4 and independent of the number of fractions, while those for 44 and 74 keV/microm increased from 1.8 to 2.3 and from 2.4 to 3.0, respectively, when the number of fractions increased from 1 to 4. Increasing the number of fractions further from 4 to 6 was not associated with an increase in the RBE. These results together with our earlier study on the skin reaction support the use of an RBE of 3.0 in clinical trials of 80 keV/microm carbon beams. The RBE values for low doses of carbon beams were also considered.

IL-27 suppresses RANKL expression in CD4+ T cells in part through STAT3.

The receptor activator of NF-κB ligand (RANKL), which is expressed by not only osteoblasts but also activated T cells, plays an important role in bone-destructive diseases such as rheumatoid arthritis. IL-27, a member of the IL-6/IL-12 family cytokines, activates STAT1 and STAT3, promotes early helper T (Th)1 differentiation and generation of IL-10-producing type 1 regulatory T (Tr1) cells, and suppresses the production of inflammatory cytokines and inhibits Th2 differentiation. In addition, IL-27 was recently demonstrated to not only inhibit Th17 differentiation but also directly act on osteoclast precursor cells and suppress RANKL-mediated osteoclastogenesis through STAT1-dependent inhibition of c-Fos, leading to amelioration of the inflammatory bone destruction. In the present study, we investigated the effect of IL-27 on the expression of RANKL in CD4(+) T cells. We found that IL-27 greatly inhibits cell surface expression of RANKL on naive CD4(+) T cells activated by T cell receptor ligation and secretion of its soluble RANKL as well. The inhibitory effect was mediated in part by STAT3 but not by STAT1 or IL-10. In contrast, in differentiated Th17 cells, IL-27 much less efficiently inhibited the RANKL expression after restimulation. Taken together, these results indicate that IL-27 greatly inhibits primary RANKL expression in CD4(+) T cells, which could contribute to the suppressive effects of IL-27 on the inflammatory bone destruction.

In vitro interaction of high-LET heavy-ion irradiation and chemotherapeutic agents in two cell lines with different radiosensitivities and different p53 status.

BACKGROUND: To investigate the differences between two rat yolk sac tumor cell lines with different radiosensitivities and p53 status in sensitivity to high-LET heavy-ion beam and in sensitizing effect of etoposide or cisplatin in combination with heavy-ion beam. MATERIALS AND METHODS: NMT-1 (wild-type p53 cell) is a parent radiosensitive cell line and NMT-1R (mutant-type p53 cell) is a variant radioresistant cell line. Heavy ion (carbon ion) was accelerated to 290 MeV per nucleon by a heavy-ion medical accelerator in Chiba at National Institute of Radiological Sciences. The effects of carbon ion irradiation and carbon ion irradiation plus chemotherapeutic agents were assessed by clonogenic assay. RESULTS: There was no significant difference between NMT-1 cells and NMT-1R cells in sensitivity to high-LET heavy-ion irradiation. The RBE of carbon beam was larger in mutant-type p53 cells than in wild-type p53 cells. Etoposide showed a supra-additive effect in combination with carbon beam irradiation in NMT-1R cells. Etoposide potentiation in NMT-1R cells was manifested by the decrease in the slope of the radiation dose-response curve. On the other hand, cisplatin had no enhancement of radiosensitivity in either cell lines. CONCLUSION: Our findings suggested that high-LET radiotherapy is expected to be effective for patients carrying radioresistant tumors and mutated p53 tumor cells. Etoposide might be effective for radioresistant tumors in combination with heavy-ion beam irradiation.

Effects of IL-23 and IL-27 on osteoblasts and osteoclasts: inhibitory effects on osteoclast differentiation.

Interleukin (IL)-23 and IL-27 are IL-6/IL-12 family members that play a role in the regulation of T helper 1 cell differentiation. Cytokines are known to be involved in the bone remodeling process, although the effects of IL-23 and IL-27 have not been clarified. In this study, we examined the possible roles of these cytokines on osteoblast phenotypes and osteoclastogenesis. We found that IL-27 induced signal transducers and activators of transcription 3 activation in osteoblasts. However, neither IL-23 nor IL-27 showed any significant effects on alkaline phosphatase activity, receptor activator of nuclear factor kappaB ligand (RANKL) expression, mRNA expression such as alkaline phosphatase type I procollagen, or the proliferation of osteoblasts. Osteoclastogenesis from bone marrow cells induced by soluble RANKL was partially inhibited by IL-23 and IL-27 with reduced multinucleated cell numbers, but these interleukins did not affect the proliferation of osteoclast progenitor cells. These results indicate that IL-23 and IL-27 could partly modify cell fusion or the survival of multinucleated osteoclasts. On the other hand, partially purified T cells, which are activated by 2 microg/ml anti-CD3 antibody, completely inhibited osteoclastogenesis by M-CSF/RANKL. On using T cells activated with 0.2 microg/ml anti-CD3 antibody, in which osteoclastogenesis was partially inhibited, the interleukins had additive effects for inhibiting osteoclastogenesis. Although the consequences of phosphorylated signals in osteoblasts have not been identified, IL-23 and IL-27, partly and indirectly through activated T cells, inhibited osteoclastogenesis, indicating that these interleukins may protect against bone destructive autoimmune disorders.