Effect of vitamin K<Subscript>2</Subscript> and growth hormone on the long bones in hypophysectomized young rats: a bone histomorphometry study

The purpose of the present study was to determine whether vitamin K₂ and growth hormone (GH) had an additive effect on the long bones in hypophysectomized young rats. Forty-eight female Sprague–Dawley rats (6 weeks old) were assigned to the following five groups by the stratified weight randomization method: intact controls, hypophysectomy (HX) alone, HX + vitamin K₂ (30 mg/kg, p.o., daily), HX + GH (0.625 mg/kg, s.c., 5 days a week), and HX + vitamin K₂ + GH. The duration of the experiment was 4 weeks. HX resulted in a reduction of the cancellous bone volume/total tissue volume (BV/TV) at the proximal tibial metaphysis, as well as decreasing the total tissue area and cortical area of the tibial diaphysis. These changes resulted from a decrease of the longitudinal growth rate and the bone formation rate (BFR)/TV of cancellous bone, as well as a decrease of the periosteal BFR/bone surface (BS) and an increase of endocortical bone turnover (indicated by the BFR/BS) in cortical bone. Administration of vitamin K₂ to HX rats did not affect the cancellous BV/TV or the cortical area. On the other hand, GH completely prevented the decrease of total tissue area and cortical area in cortical bone, as well as the decrease of marrow area and endocortical circumference, by increasing the periosteal BFR/BS compared with that in intact controls and reversing the increase of endocortical bone turnover (BFR/BS). However, GH only partly improved the reduction of the cancellous BV/TV, despite an increase of the longitudinal growth rate and BFR/TV compared with those of intact controls. When administered with GH, vitamin K₂ counteracted the reduction of endocortical bone turnover (BFR/BS) and circumference caused by GH treatment, resulting in no significant difference of marrow area from that in untreated HX rats. These results suggest that, despite the lack of an obvious effect on bone parameters, vitamin K₂ normalizes the size of the marrow cavity during development of the bone marrow in young HX rats treated with GH.     

Response of serum carboxylated and undercarboxylated osteocalcin to alendronate monotherapy and combined therapy with vitamin K<Subscript>2</Subscript> in postmenopausal women

Alendronate decreases the risk of femoral neck fracture by suppressing bone turnover, and also decreases the serum total osteocalcin level. A low serum carboxylated osteocalcin level or high undercarboxylated osteocalcin level could be risk factors for femoral neck fracture. Vitamin K mediates the carboxylation of osteocalcin, but the effect of alendronate therapy with or without vitamin K₂ supplementation remains unknown. Forty-eight postmenopausal women were enrolled in a 1-year prospective randomized trial and assigned to alendronate monotherapy (5 mg/day) (group A, n = 26) or vitamin K₂ (45 mg/day) plus alendronate (5 mg/day) (group AK, n = 22). Bone mineral density was measured by dual-energy X-ray absorptiometry at 0 and 12 months; bone turnover parameters were measured at 0, 3, and 12 months. Four patients discontinued alendronate therapy, and we analyzed the remaining 44 patients (23 in group A and 21 in group AK) who completed 1 year of treatment. Alendronate decreased undercarboxylated osteocalcin; carboxylated osteocalcin was not affected. Addition of vitamin K₂ enhanced the decrease of undercarboxylated osteocalcin levels and led to a greater increase of femoral neck bone mineral density. Alendronate monotherapy does not decrease carboxylation of osteocalcin, and combination of vitamin K₂ and alendronate brings further benefits on both osteocalcin carboxylation and BMD of femoral neck in postmenopausal women with osteoporosis.     

Regulation of mineral-to-matrix ratio of lumbar trabecular bone in ovariectomized rats treated with risedronate in combination with or without vitamin K<Subscript>2</Subscript>

The relationship between bone turnover and bone tissue and material properties was examined in ovariectomized (OVX) rats treated with risedronate in combination with or without vitamin K₂. Seventy female rats, 18 weeks of age, were assigned to 7 groups (n = 10): sham-operated + vehicle control; OVX + vehicle control; OVX + risedronate 0.1, 0.5, or 2.5mg/kg/day po; OVX + vitamin K₂ 30mg/kg/day po; OVX + vitamin K₂ (30mg/kg/day) and risedronate (0.5mg/kg/day). Treatments were given daily for 9 months. To assess bone turnover, we measured serum osteocalcin and urinary deoxypyridinoline at 0, 3, and 9 months. To assess vertebral and femoral tissue and material properties, bone mass, bone mineral density (BMD by DXA), trabecular bone structure (vertebra: 3D-CT), cortical bone structure (femur: histomorphometry), biomechanical properties, and mineral properties (mineral-to-matrix and carbonate-to-phosphate ratios by Fourier transform infrared microspectroscopy) were measured ex vivo at 9 months. Ovariectomy increased bone turnover and induced significant loss of bone mass/density, structure, mineral properties (mineral-to-matrix ratio), and strength. Risedronate produced dose-dependent inhibition of the ovariectomy-induced increase in turnover and loss of bone mass/density, structure, mineral-to-matrix ratio, and strength, with a lowest effective dose of 0.1–0.5mg/kg/day. High-dose risedronate (2.5mg/kg/day) did not induce increases in any parameter above that of sham control. Vitamin K₂ had no effects. In the OVX groups, urinary deoxypyridinoline at 3 and 9 months correlated significantly with vertebral BMD, trabecular bone volume, ultimate load, stiffness, and mineral-to-matrix ratio, and with femoral BMD, cortical area, and ultimate load. These results support the concept that changes in bone tissue and material properties can result directly from changes in bone turnover. Different effects among different drugs on material properties, including mineral-to-matrix ratio, may reflect differences in the relative rate and magnitude of osteoclastic bone resorption and osteoblastic primary bone mineralization.     

Combination use of vitamin K<Subscript>2</Subscript> further increases bone volume and ameliorates extremely low turnover bone induced by bisphosphonate therapy in tail-suspension rats

 Bisphosphonate is a potent inhibitor of bone resorption, which results in the increase of bone volume. However, bisphosphonate treatment may lead to extremely low bone turnover and abnormal bone microstructure. In this study, we examined whether the combination of bisphosphonate with vitamin K₂ treatment may have beneficial effects on bone turnover and trabecular microstructure as well as on bone volume loss by using tail-suspension model rats. In these model rats, bone mineral density (BMD) decreased with histological evidence of enhanced bone resorption and suppressed bone formation. By bisphosphonate treatment, BMD was increased compared with that of tail-suspended rats. Osteoclast surface per bone surface (Oc.S/BS) and number of osteoclasts per bone perimeter (N.Oc/B.Pm) were reduced and mineral apposition rate (MAR) decreased, suggesting extreme suppression of bone turnover. However, trabecular structure examined by microfocus CT was apparently abnormal. By contrast, combination of bisphosphonate with vitamin K₂ leads to further increase of bone volume. MAR and BFR as well as Oc.S/BS and N.Oc/B.Pm were increased compared with those of the bisphosphonate-treated group. However, abnormal structure of trabeculae in secondary spongiosa was not completely ameliorated. These data suggested that concomitant use of vitamin K₂ with bisphosphonate excessively ameliorates too much suppression of bone turnover while more efficiently preventing bone volume loss. Received: January 30, 2002 / Accepted: November 6, 2002 RID="*" ID="*"  Present address: Department of Health Sciences, Oita University of Nursing and Health Sciences, Oita, Japan Acknowledgments. This work was supported in part by a Special Grant for Medical Research from Ministry of Post and Telecommunications, Japan (to M.F.), a grant in aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (#13671115 to M.F.), and by a grant from the Research Society for Metabolic Bone Disease (to M.F.). We are grateful to Miss Sachiko Suzuki for technical assistance. Offprint requests to: M. Fukagawa     

Local injection of 1,25-dihydroxyvitamin D<Subscript>3</Subscript> enhanced bone formation for tooth stabilization after experimental tooth movement in rats

The present investigation evaluated the effect of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on alveolar bone formation during tooth movement in rats. Orthodontic elastics were inserted between the maxillary first and second molars on bilateral sides in male rats. 1,25(OH)₂D₃ was injected locally, at the concentration of 10^–10M, once every 3 days in the submucosal palatal area of the root bifurcation of the molar on the right side. Histomorphometric analysis revealed that tooth movement without application of 1,25(OH)₂D₃ decreased the mineral appositional rate (MAR) on the compression area at 7 days. Repeated injections of 1,25(OH)₂D₃ in the orthodontically treated animals distinctly stimulated alveolar bone formation on the mesial side at 14 days. There was a significant increase in MAR associated with elevated osteoblast surface (Ob.S/BS) value on the tension surface. These findings suggest that local application of 1,25(OH)₂D₃ enhances the reestablishment of supporting tissue, especially alveolar bone of teeth, after orthodontic treatment.     

Effects of vitamin K2 and calcitonin on bone resorption model induced by vitamin A in thyroparathyroidectomized rats

Vitamin K₂ is considered to have two different effects: one is to enhance bone formation, and the other is to suppress bone resorption. However, as these effects have not been observed in a single experiment, it is unclear whether bone formation can proceed during a state of accelerating bone resorption. We therefore examined the effects of vitamin K₂ and calcitonin on a vitamin A-induced bone resorption model of thyroparathyroidectomized rats using bone histomorphometry and bone metabolism markers. The seven groups of male Sprague-Dawley rats (6 weeks old) were sham operation of thyroparathyroidectomy (TPTX) (sham group), TPTX (TPTX group), treated with vitamin A (20 mg/kg per day) from 11th to 20th day after TPTX (A group), treated with vitamin A and vitamin K₂ (30 mg/kg per day) or its vehicle from 11th to 20th day after TPTX [K group or K (veh) group], and treated with vitamin A and calcitonin (10IU/kg/ per day) or its vehicle during the same period [CT group or CT (veh) group]. Serum and urine samples were taken for marker determination on days 10, 13, 16, and 19 of TPTX and at death on the 21st day after TPTX. Undecalcified sections (Villanueva bone stain) were made of the left tibiae and decalcified sections [tartrate-resistant acid phosphatase (TRAP) stain] of the right tibiae. In the undecalcified sections, secondary trabeculae were used for histomorphometry, and in the decalcified sections primary and secondary trabeculae were used. Serum Ca of the vitamin A-administered group was significantly higher than that of the TPTX group, but this change was inhibited by vitamin K₂ or calcitonin. Serum alkaline phosphatase (ALP) in the K group was significantly higher than in all the thyroparathyroidectomized groups except the K (veh) group. In the undecalcified sections, although there was no significant difference between any of the groups in bone volume, the K group showed an increase of osteoid surface and mineralizing surface. In the decalcified sections, the K group showed a decrease of TRAP-positive areas compared to the K (veh) group in primary trabeculae. There was no significant difference between the K and K (veh) groups in secondary trabeculae. Results from the CT group were compatible with bone resorption inhibition in both bone metabolism markers and bone histomorphometry. We found that vitamin K₂ enhances bone formation and suppresses bone resorption in areas with a high turnover of bone metabolism. Vitamin K₂ is therefore expected to increase bone content if it is administered over an extended period.     

Comparison of the effects of room air and N<Subscript>2</Subscript>O + O<Subscript>2</Subscript> used for ProSeal LMA cuff inflation on cuff pressure and oropharyngeal structure

This study aimed to evaluate the effects of different inflating gases used for ProSeal LMA (PLMA) cuff inflation on cuff pressure, oropharyngeal structure, and the incidence of sore throat. Eighty patients (American Society of Anesthesiologists; ASA I–II) were randomly divided into two groups. PLMA cuff inflation was achieved with appropriate volumes of 50% N₂O + 50% O₂ in group I and room air in group II, respectively. When the PLMA was removed, oropharyngeal examination was carried out immediately, using a rigid optical telescope. Patients were asked about sore throat symptoms postoperatively. Cuff pressures were significantly lower in group I, except at the initial pressure measurement. Cuff pressure was positively correlated with the length of the operation in group II, and negatively correlated in group I. PLMA cuff inflation with room air led to increased cuff pressure during the operation, possibly due to the diffusion of N₂O into the cuff. We consider that a PLMA cuff inflated with an N₂O-O₂ mixture is convenient, especially in operations in which N₂O has been used.     

Peritoneal and systemic pH during pneumoperitoneum with CO<Subscript>2</Subscript> and helium in a pig model

Background Local peritoneal effects of laparoscopic gases might be important in peritoneal biology during and after laparoscopic surgery. The most commonly used gas, CO₂, is known to be well tolerated, but also causes changes in acid-base balance. Helium is an alternative gas for laparoscopy. Although safe, it is not widely used. In this study a method for monitoring peritoneal pH during laparoscopy was evaluated and peritoneal pH during CO₂ and helium pneumoperitoneum was studied as well as its systemic reflection in arterial pH. Methods For these experiments 20 pigs were used, with ten exposed to pneumoperitoneum with CO₂, and ten to helium. Peritoneal and sub-peritoneal pH were continuously measured before and during gas insufflation, during a 30-minute period with a pneumoperitoneum and during a 30-minute recovery period. Arterial blood-gases were collected immediately before gas insufflation, at its completion, at 30 minutes of pneumoperitoneum and after the recovery period. Results Peritoneal pH before gas insufflation was in all animals 7.4. An immediate local drop in pH (6.6) occurred in the peritoneum with CO₂ insufflation. During pneumoperitoneum pH declined further, stabilising at 6.4, but was restored after the recovery period (7.3). With helium, tissue pH increased slightly (7.5) during insufflation, followed by a continuous decrease during pneumoperitoneum and recovery, reaching 7.2. Systemic pH decreased significantly with CO₂ insufflation, and increased slightly during helium insufflation. Systemic pH showed co-variation with intra-peritoneal pH at the the end of insufflation and after 30 minutes of pneumoperitoneum. Conclusions Insufflation of CO₂ into the peritoneal cavity seemed to result in an immediate decrease in peritoneal pH, a response that might influence biological events. This peritoneal effect also seems to influence systemic acid-base balance, probably due to trans-peritoneal absorption.     

Cannabinoids Stimulate Fibroblastic Colony Formation by Bone Marrow Cells Indirectly via CB<Subscript>2</Subscript> Receptors

Recently, the cannabinoid receptors CB₁ and CB₂ were shown to modulate bone formation and resorption in vivo, although little is known of the mechanisms underlying this. The effects of cannabinoids on mesenchymal stem cell (MSC) recruitment in whole bone marrow were investigated using either the fibroblastic colony-forming unit (CFU-f) assay or high-density cultures of whole bone marrow. Levels of the CB₁ and CB₂ receptors were assessed by flow cytometry. Treatment of CFU-f cultures with the endocannabinoid 2-arachidonylglycerol (2-AG) dose-dependently increased fibroblastic and differentiated colony formation along with colony size. The nonspecific agonists CP 55,940 and WIN 55,212 both increased colony numbers, as did the CB₂ agonists BML190 and JWH015. The CB₁-specific agonist ACEA had no effect, whereas the CB₂ antagonist AM630 blocked the effect of the natural cannabinoid tetrahydrocannabivarin, confirming mediation via the CB₂ receptor. Treatment of primary bone marrow cultures with 2-AG stimulated proliferation and collagen accumulation, whereas treatment of subcultures of MSC had no effect, suggesting that the target cell is not the MSC but an accessory cell present in bone marrow. Subcultures of MSCs were negative for CB₁ and CB₂ receptors as shown by flow cytometry, whereas whole bone marrow contained a small population of cells positive for both receptors. These data suggest that cannabinoids may stimulate the recruitment of MSCs from the bone marrow indirectly via an accessory cell and mediated via the CB₂ receptor. This recruitment may be one mechanism responsible for the increased bone formation seen after cannabinoid treatment in vivo.     

p53 expression and apoptosis in liver and spleen during CO<Subscript>2</Subscript> pneumoperitoneum

Backgrounds and aims  Laparoscopic surgery techniques have been increasingly preferred to classic laparotomy by surgeons since 1987. However, this method has some important adverse effects on intra-abdominal organs. The aim of this study is to evaluate the effects of different pressures of CO₂ on apoptosis and p53 expression in cells in liver and spleen. Methods  In total, 30 male Sprague–Dawley rats were used in the study. CO₂ was insufflated into the intra-abdominal cavity via angiocatheter cannule by an insufflator in two different pressures of 10 and 20 mm Hg for 60 min. However, in the control group, only cannule was inserted into the intra-abdominal cavity, but no gas was insufflated. After 60 min, the rats were killed and laparotomy was applied. The liver and spleen were excised. The samples were histologically processed and immunohistochemistry was applied. Results  All the data revealed that the number of apoptotic cells in liver and spleen increases in proportion to CO₂ pressure level. No p53 expression was detected in both organs. Conclusion  CO₂ pressure level and application time may affect on cells living in liver and spleen. High pressure and/or long application time may cause releasing of cytokines and superoxide radicals from these organs’ cells, and transient or serious organ dysfunctions may occur.     

Relevance of ICP and ptiO<Subscript>2</Subscript> for indication and timing of decompressive craniectomy in patients with malignant brain edema

Summary Background. The exact effects of decompressive craniectomy on intracranial pressure (ICP) and cerebral tissue oxygenation (ptiO₂) are still unclear. Therefore, we have monitored ICP and ptiO₂ intra-operatively and correlated these values to different operative steps during craniectomy.Methods. ICP and ptiO₂ values have been monitored both, simultaneously and continuously, in 15 patients with cerebral edema due to posttraumatic or postischemic brain swelling. Indications for craniectomy were an increase in ICP above 25 mmHg or a decrease in ptiO₂ below 10 mmHg resistant to conservative treatment (e.g. mannitol, hyperventilation, adequate arterial blood oxygenation, etc.). In all cases, we performed a fronto-temporo-parietal craniectomy (15×12 cm) and dura enlargement with galea-periosteum. During craniectomy, monitoring of ICP and ptiO₂ in the affected hemisphere was continued. Values were recorded and correlated with the different operative steps.Findings. We performed craniectomy according to our treatment protocol in 5 patients. Prior to surgery, mean ICP values were 25.6 mmHg (range: 23–29 mmHg), mean ptiO₂ values were 5.9 mmHg (range: 2.4–9.5 mmHg), and mean CPP values were 66 mmHg (range: 60–70 mmHg). After removing the bone flap, ICP values dropped to physiological values (mean: 7.4 mmHg), whereas ptiO₂ values increased only slightly (mean: 11 mmHg). Opening of the dura resulted in a further decrease of ICP (mean 4.8 mmHg) and an increase of ptiO₂ to normal limits (mean: 18.8 mmHg). After skin closure, mean ICP was 6.8 mmHg and mean ptiO₂ was 21.7 mmHg, respectively. We found a significant decrease of ICP after craniectomy (p<0.042) and after dura enlargement (p<0.039) as well as a statistically significant increase in ptiO₂ after craniectomy (p<0.043) and after dura enlargement (p<0.041).Conclusion. As a large bone flap in decompressive craniectomy is essential for adequate ICP reduction, the results of the presented cases suggest that dura enlargement is the crucial step to restore adequate brain tissue oxygenation and that ptiO₂ monitoring could be an important tool for timing craniectomy in the future.     

Effect of Vitamins D<Subscript>2</Subscript> and D<Subscript>3</Subscript> Supplement Use on Serum 25OHD Concentration in Elderly Women in Summer and Winter

Vitamin D₂ and D₃ are generally considered equipotent in humans. A few studies have reported that serum 25OHD levels are higher in vitamin D₃- compared with vitamin D₂-supplemented subjects. As both vitamin D₂ and D₃ supplements are commonly used by elderly in United States, in the present study we determined the effect of self-reported vitamin D₂ and vitamin D₃ supplement use on serum total 25OHD levels according to season in elderly women aged 65–77 years. Serum total 25OHD levels were determined in winter and summer in unsupplemented women (N = 307) and in women who reported taking vitamin D₂ (N = 56) and vitamin D₃ (N = 55) supplements by competitive protein binding assay. In vitamin D₂-supplemented women, the contribution of vitamin D₂ and D₃ to the mean serum total 25OHD level was assessed by HPLC. In summer, there were no significant differences in the mean total serum 25OHD levels (ng/ml) among the vitamin D₂ (32 ± 2.1), vitamin D₃ (36.7 ± 1.95), and unsupplemented (32.2 ± 0.95) groups. In winter, the mean serum total 25OHD levels were higher in women on vitamin D₂ (33.6 ± 2.34, P < 0.05) and vitamin D₃ (29.7 ± 1.76, NS) supplements compared with unsupplemented women (27.3 ± 0.72). In vitamin D₂-supplemented women, about 25% of the mean serum total 25OHD was 25OHD₂, in both summer and winter. Twelve percent of unsupplemented women and 3.6% of vitamin D-supplemented women had a mean serum total 25OHD level below 15 ng/ml in winter. In elderly subjects, both vitamin D₂ and Vitamin D₃ supplements may contribute equally to circulating 25OHD levels, with the role of vitamin D supplement use being more predominant during winter. This work was presented in part as an abstract at Second Joint Meeting of the American Society for Bone and Mineral Research and The International Bone and Mineral Society, San Francisco, CA, 1–6 December 1998.     

The effect of different levels of peritoneal CO<Subscript>2</Subscript> pressure on bleeding time of spleen capsule injury

Aims: The authors, in contrast to similar injuries with open surgery, had observed spontaneous hemostasis of relatively large spleen capsule injuries during laparoscopic surgery. Methods: Standard spleen injuries were carried out in 5 anesthetized mongrel dogs at different CO₂ pressures and in open surgery. Bleeding was checked every minute by wiping around the injury but not removing the clot. Bleeding time was measured until no more oozing was detected. At every pressure level 3-3 measurements were done and analyzed. Parenchymal pressure of the spleen and systemic blood pressure were detected with direct catheter implantation. Results: In open surgery the average bleeding time was 15.2 min; at 5, 15, and 25 mmHg CO₂ pressures bleeding times were 12.3, 10.6, and 9.8 min, respectively. The parenchymal pressure of the spleen (5–6 mmHg) rose synchronously with peritoneal pressure, but no significant changes in systemic blood pressure were seen. Conclusion: Peritoneal CO₂ pressure may counterweight the parenchymal pressure of the spleen, thus helping hemostasis. There seems to be an inverse proportion between peritoneal pressure and bleeding time. In case of spleen capsule injury during laparoscopic surgery, chances for spontaneous hemostasis are much better compared to open surgery. Attention must be paid to maintain adequate peritoneal pressure.     

Vitamin K<Subscript>2</Subscript> (menaquinone 4) reduces serum undercarboxylated osteocalcin level as early as 2 weeks in elderly women with established osteoporosis

 Twenty elderly osteoporotic women with vertebral fracture(s) were randomly allocated to two groups; women in the MK₄ group received calcium with menaquinone 4 (MK₄) at a dose of 45 mg/day for 2 weeks, and women in the control group received calcium alone for the same period. Serum intact osteocalcin (OC) and undercarboxylated osteocalcin (uc-OC) levels were measured by immunoradiometric assay and enzyme immunoassay, respectively, at baseline and on the 7th and 14th days following the start of the treatment. There were no differences in the baseline data including age, weight, phylloquinone, menaquinone 4, menaquinone 7, OC, and uc-OC levels between the MK₄ group and the control group. Administration of MK₄ significantly raised the MK₄ level from 0.20 ± 0.10 (mean ± SE) pg/ml to 15.09 ± 5.62 pg/ml (P < 0.04), and reduced serum uc-OC levels from 2.80 ± 0.93 ng/ml to 1.76 ± 0.56 ng/ml (P < 0.05) at the end of the study, respectively. No significant changes in these levels were observed in the control group. Serum OC levels were stable during the period in both groups. In this randomized prospective study, the MK₄ group shows a reduction in the serum uc-OC level within 2 weeks without any significant change in OC, suggesting that the uc-OC is changed to carboxylated OC. This early effect of MK₄ on bone metabolism may be estimated by the measurement of serum uc-OC in elderly osteoporotic women with vertebral fractures. Received: January 21, 2002 / Accepted: November 6, 2002 Offprint requests to: T. Miki     

Reduced T-Cell and Dendritic Cell Function Is Related to Cyclooxygenase-2 Overexpression and Prostaglandin E<Subscript>2</Subscript> Secretion in Patients With Breast Cancer

Background: In several neoplastic diseases, including breast cancer, immunosuppression correlates with disease stage, progression, and outcome. Thus, thorough analysis of immune parameters in breast cancer patients may be beneficial in designing effective anticancer immune-based therapies.Methods: We investigated dendritic cell and T-cell function in breast cancer patients at various stages of the disease and in age-matched controls. We also evaluated cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE₂) levels within the tumor milieu and in the circulation.Results: T cells from cancer patients showed decreased proliferation in response to CD3 antibody stimulation. Analysis of T-cell helper type 1 and 2 cytokines revealed reduced levels of interferon-, tumor necrosis factor-, interleukin (IL)-12, and IL-2 and increased levels of IL-10 and IL-4. Dendritic cells from these patients showed significantly reduced expression of co-stimulatory molecules (B7 and CD40) and demonstrated reduced phagocytic ability, reduced antigen presentation to T cells, and reduced ability to mature in response to lipopolysaccharide. Data revealed increased synthesis of PGE₂, an immune suppressor, along with increased expression of COX-2, a key regulator of PGE₂ synthesis.Conclusions: COX-2–induced PGE₂ may contribute to immunosuppression and may directly block antitumor immunity while promoting tumor growth, providing us with the rationale for using COX-2 inhibition combined with immunotherapy.     

Different actions of vitamin D2 and D3 on bone metabolism in patients treated with phenobarbitone/phenytoin

Summary In 22 epileptic outpatients treated for at least 1 year with phenobarbitone/phenytoin the local and total bone mass, together with serum and urinary indices of calcium metabolism, were measured before and during treatment with either vitamin D₂ or D₃, 4,000 IU daily for 24 weeks. The results showed a distinct difference in the action of the two vitamins on bone metabolism during anticonvulsant treatment. The bone mass increased during treatment with vitamin D₂, whereas the vitamin D₃-treated patients showed unchanged values of bone mass, but an increased excretion rate of calcium, probably caused by increased intestinal calcium absorption. The data demonstrate that vitamins D₂ and D₃ (or their metabolites) have quantitative different effects in patients treated with phenobarbitone/phenytoin.     

Vitamin K<Subscript>2</Subscript> supplementation improves hip bone geometry and bone strength indices in postmenopausal women

Summary Vitamin K mediates the synthesis of proteins regulating bone metabolism. We have tested whether high vitamin K₂ intake promotes bone mineral density and bone strength. Results showed that K₂ improved BMC and femoral neck width, but not DXA-BMD. Hence high vitamin K₂ intake may contribute to preventing postmenopausal bone loss. Introduction Vitamin K is involved in the synthesis of several proteins in bone. The importance of K vitamins for optimal bone health has been suggested by population-based studies, but intervention studies with DXA-BMD as a clinical endpoint have shown contradicting results. Unlike BMC, DXA-BMD does not take into account the geometry (size, thickness) of bone, which has an independent contribution to bone strength and fracture risk. Here we have tested whether BMC and femoral neck width are affected by high vitamin K intake. Methods A randomized clinical intervention study among 325 postmenopausal women receiving either placebo or 45 mg/day of vitamin K₂ (MK-4, menatetrenone) during three years. BMC and hip geometry were assessed by DXA. Bone strength indices were calculated from DXA-BMD, femoral neck width (FNW) and hip axis length (HAL). Results K₂ did not affect the DXA-BMD, but BMC and the FNW had increased relative to placebo. In the K₂-treated group hip bone strength remained unchanged during the 3-year intervention period, whereas in the placebo group bone strength decreased significantly. Conclusions Vitamin K₂ helps maintaining bone strength at the site of the femoral neck in postmenopausal women by improving BMC and FNW, whereas it has little effect on DXA-BMD.     

Bone Parameters are Improved with Intermittent Dosing of Vitamin D<Subscript>3</Subscript> and Calcitonin

Intermittent combination of an anabolic agent to promote bone formation and an antiresorptive agent that would prevent further bone loss is a theoretically attractive approach for restoring bone mass. We tested the potential of intermittently dosed calcitriol and calcitonin (CT) to restore bone properties in ovariectomized (Ovx) rats. Rats had Ovx or sham surgery at 8 weeks old and 4 weeks later were assigned to experimental groups: (1) sham vehicle, (2) Ovx vehicle, (3) Ovx + parathyroid hormone (PTH, 40 μg/kg), and (4) Ovx + calcitriol (2 μg/kg) + CT (2 μg/kg). Group 3 received PTH every week throughout the study, and group 4 received calcitriol at weeks 1, 3, 5, and 7 and CT at weeks 2, 4, 6, and 8. Dosing was carried out for 8 weeks with serum, and micro-computed tomographic analysis was done at 0, 4, and 8 weeks. Femurs and tibias were used for radiological analyses and for mechanical testing. Dosing with PTH improved bone mass and structure of cancellous bone at metaphyses of tibias and femurs as well as properties of cortical bone including geometry and strength. Intermittent dosing with calcitriol and CT was less potent in correcting loss of cancellous bone relative to treatment with PTH and had no effect on cortical bone parameters. However, intermittent dosing with calcitriol and CT was robust enough to improve cancellous bone mass and structure through bone formation without causing deleterious side effects. Our data provide additional evidence that therapies can be devised to ameliorate the skeletal defects associated with established osteoporosis.     

The effect of vitamin K2 on bone metabolism in aged female rats

Reactive oxygen species (ROS) may contribute to aging and osteoporosis resulting from marked decreases in plasma antioxidants in aged osteoporotic women. On the other hand, high-dose vitamin K₂ (menaquinone-4: menatrenone, MK-4) supplementation has been reported to reduce ovariectomy-induced bone loss in rats and to decrease osteoporotic fracture in postmenopausal women. However, the mechanism by which vitamin K₂ prevents osteoporosis is unclear. Recently, vitamin K₂ has been suggested to preserve antioxidant activity as a novel function. Therefore, we investigated the effect of vitamin K₂ on the osteoporosis of aged rats by evaluating the relationships between serum antioxidant levels and bone metabolism. Aged female rats exhibited significantly lower serum alkaline phosphatase activity and osteocalcin level, together with lower serum levels of antioxidants such as 17-estradiol, macrophage migration inhibitory factor (MIF) and glutathione peroxidase (GPx) activity, as compared with young female rats. On the other hand, vitamin K₂ supplementation (500 mg/kg, food intake) for 98 days led to a significantly increased serum vitamin K₂ level (3,045±915 ng/ml in the vitamin K₂ supplemented group vs. 4.6±3.4 ng/ml in the control diet group; P <0.0001) with increased serum alkaline phosphatase activity and MIF level ( P <0.05). Unexpectedly, however, it failed to increase the serum level of antioxidants such as GPx. Nor did it affect bone metabolism markers such as oteocalcin and osteopontin, which were significantly lower than in the young female rats ( P <0.05). Finally, the histomorphometric properties of the proximal tibia in the femur were not altered by vitamin K₂. These results suggest that high-dose vitamin K₂ supplementation neither improves lowered antioxidant levels nor stimulates bone formation in aged rats.