Complete Right Bundle Branch Block as a Predictor of Cardiovascular Events in Type 2 Diabetes.

Complete right bundle branch block (CRBBB) is generally regarded as a clinically insignificant abnormality on an electrocardiogram, although its predictive value for cardiovascular events in type 2 diabetes mellitus (T2DM) is unknown. We examined the association of CRBBB with cardiovascular events during a 6-year follow-up in a single-center cohort study. The Fine-Gray model was used to analyze the independent association between CRBBB and composite cardiovascular events including cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, and hospitalization for heart failure during follow up. We analyzed the data of 370 T2DM patients including 62 patients with pre-existing heart disease. CRBBB was found in 34 patients (9.2%). The composite cardiovascular outcome was recorded in 32 patients. When analyzed with the Fine-Gray model with inverse probability of treatment weighting, CRBBB was significantly associated with a higher risk of the cardiovascular outcome (hazard ratio, 2.55; 95% confidence interval, 1.04 to 6.26; p = 0.041). This association remained significant even after further adjustment for each of the potential confounders. This study suggested that CRBBB was an independent predictor of cardiovascular events in T2DM. Further studies with a larger sample size are warranted.

A case of life-threatening small intestinal bleeding accompanied by lower coagulation factor XIII activity.

We report a case of life-threatening bleeding from the small intestine accompanied by lower coagulation factor XIII (FXIII) activity. A 43-year-old man was admitted because of continuous small intestinal bleeding. He had no family or past history of bleeding. He received high volume transfusion of red blood cells (total 20 unit) for persistent bleeding. All routine coagulation tests were normal, and plasma FXIII activity was 41%. Cross-mixing studies suggested an absence of autoantibodies to FXIII. Contrast-enhanced computed tomography and repeated balloon-assisted enteroscopy could not detect the focus of bleeding. The administration of purified concentrate of FXIII immediately halted the bleeding from the small intestine. Since then, he experienced no bleeding episode and has been maintaining normal FXIII activity.

Safety and pharmacokinetic study of nab-paclitaxel plus carboplatin in chemotherapy-naïve patients with advanced non-small cell lung cancer.

BACKGROUND: Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) is a Cremophor EL-free formulation of paclitaxel newly designed to avoid solvent-related toxicities. We have evaluated the safety, tolerability, pharmacokinetics, and tumor response profile of weekly nab-paclitaxel (100 mg/m(2)) infusion together with administration of carboplatin at an area under the curve (AUC) of 6 every 3 weeks in Japanese patients with advanced non-small cell lung cancer (NSCLC). METHODS: Nab-paclitaxel (100 mg/m(2)) was administered without steroid or antihistamine premedication as a 30-min intravenous infusion once a week in combination with carboplatin at an AUC of 6 on day 1 of repeated 21-day cycles. The pharmacokinetics of both drugs were analyzed, and both adverse events and treatment response were monitored. RESULTS: Eighteen patients were enrolled in the study. The most frequent treatment-related toxicities of grade 3 or 4 were neutropenia (67%), leukopenia (50%), and anemia (22%). No severe hypersensitivity reactions were observed despite the lack of premedication, and no unexpected or new toxicities were detected. Pharmacokinetics analysis did not reveal any substantial drug-drug interactions. Seven partial responses were observed among the 18 evaluable patients, yielding a treatment response rate of 38.9%. CONCLUSIONS: The combination of nab-paclitaxel (100 mg/m(2)) administered weekly and carboplatin at an AUC of 6 every 3 weeks was well tolerated in Japanese patients with advanced NSCLC. This combination therapy also showed promising antitumor activity and was not associated with relevant pharmacokinetic interactions.

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.

Distribution of macrophages, osteoclasts and the B-lymphocyte lineage in osteolytic metastasis of mouse mammary carcinoma.

The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteoclasts in tumoral lesions of mammary carcinoma metastasized to bone of non-immunocompromised mice. Mouse mammary carcinoma cells (BALB/c-MC) were injected through the left cardiac ventricle into 5-week-old female wild-type Balb/c mice. The femora and tibiae of mice with metastasized cancer were extracted, and thereafter processed for histochemical analyses. The foci of metastasized tumor cells occupied the metaphyseal area, and the cell death zones could be identified within the tumor mass. Abundant tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were found among the alkaline phosphatase (ALP)-reactive osteoblastic cell layer that covered the bone surface neighboring the metastatic lesion. In contrast, F4/80-positive macrophages/monocytes were localized adjacent to, or invading the metastatic tissue. In addition, some F4/80-positive cells were found in the aforementioned cell death zones. Unlike F4/80-positive cells, CD45R-positive B-lymphocytes did not accumulate at the surfaces of the tumor lesions, nor infiltrate into them, but were found scattered over bone marrow. Interestingly, some CD45R-positive cells were observed close to TRAP-positive osteoclasts in the stromal tissue surrounding the tumor lesion. Our findings suggest that, in the bone metastatic lesions of non-immunocompromised mice, F4/80-positive macrophages/monocytes accumulated on and/or infiltrated into the tumor nests, while CD45R-positive B-lymphocytes were associated with osteoclasts, rather than attacking metastatic tumor cells.

Involvement of hepatocyte growth factor in the development of bone metastasis of a mouse mammary cancer cell line, BALB/c-MC.

Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of certain cancer cells. After tumors metastasize to bone, they stimulate osteoclastogenesis and expand in the bone tissue. Hepatocyte growth factor (HGF), which was originally identified as a potent mitogen for hepatocytes, promotes tumor growth, invasion and metastasis. HGF is mainly produced by cells of mesenchymal origin, and osteoblasts/osteocytes and bone marrow stromal cells originate from mesenchymal cells. However, it is not clear what effect HGF has on tumor progression in bone metastasis. In the present study, we investigated the roles of HGF in bone metastasis using the mouse mammary cancer cell line BALB/c-MC. Cancer cells injected into hearts of mice metastasized to bone in their hind limbs. HGF immunoreactivity was detected in the stroma surrounding the tumor nests, and blood vessels expressing CD31 (a marker of endothelial cells) were observed in the HGF-positive area. To identify the cells producing HGF, we measured concentration of HGF in culture media. HGF concentration was elevated in osteoblast cultures (3.13+/-0.25 ng/ml), whereas HGF was undetectable (<0.4 ng/ml) in BALB/c-MC and bone marrow cell cultures. HGF concentration in osteoblast cultures increased 2.5-fold in response to 10(-6) M PGE(2). Addition of HGF to BALB/c-MC cultures caused doubling of the cell number. Moreover, Western blot analysis revealed expression of c-Met/HGF receptor by BALB/c-MC. In the Matrigel invasion chamber assay, addition of HGF to the bottom well increased the rate at which BALB/c-MC invaded the bottom well through the membrane. Furthermore, when osteoblasts were cultured in the bottom well, the number of BALB/c-MC cells that invaded the bottom well through the membrane increased 3.7-fold, compared to assays without osteoblasts. Addition of NK4, an inhibitor of HGF, completely abolished the enhancement of the invasive potential of the BALB/c-MC cells in the presence of osteoblasts. These findings suggest that HGF produced by osteoblasts induces migration of cancer cells from sinusoidal capillaries to bone marrow space and stimulates growth of cancer cells in the bone microenvironment. Thus, osteoblasts appear to promote bone metastasis of some cancers via HGF-c-Met signaling.

[Bisphosphonates: the molecular targets and mechanisms of action].

Bisphosphonates directly act on osteoclasts to inhibit bone resorption and used most widely to treat osteoporosis. The compounds can be classified into two groups with different modes of action. Nitrogen containing bisphosphonates exert their effects by inhibiting a key enzyme in the mevalonate pathway. The specific target is the isoprenoid biosynthetic enzyme, farnesyl pyrophosphate synthase, which is indispensable for protein prenylation and activation of intracellular signalling proteins, including the small GTPases Rho, Rac, Cdc42 and Ras. The disruption of the function of these key enzymes may explain the loss of osteoclast activity and induction of apoptosis. Whereas the first generation of bisphosphonates such as etidronate and clodronate (nitrogen deficient bisphosphonates) can be incorporated into nonhydrolysable analogues of ATP that may inhibit ATP-dependent intracellular events. Bisphosphonates are highly effective to inhibit bone resorption and increase bone mineral density, although their precise mechanisms of molecular action are not completely understood.

Biphasic effect of prostaglandin E2 on osteoclast formation in spleen cell cultures: role of the EP2 receptor.

UNLABELLED: We examined the effect of PGE2 on OC formation from spleen cells treated with M-CSF and RANKL. PGE2 decreased OC number at 5-6 days of culture and increased OC number, size, and resorptive activity at 7-8 days. A selective EP2 receptor agonist mimicked these effects. Deletion of the EP2 receptor or depletion of T-cells abrogated the increase in OC number. INTRODUCTION: Prostaglandin E2 (PGE2) has been reported to increase osteoclast (OC) number in spleen cells cultured with RANKL and macrophage-colony-stimulating factor (M-CSF). In this study, we examined the time course of PGE2 effects on spleen cells cultured with RANKL and M-CSF. We then investigated which PGE receptors and cell types were involved in these effects. MATERIALS AND METHODS: Spleen cells were cultured from wildtype C57BL/6 mice and EP2 or EP4 receptor-deficient (-/-) and wildtype (+/+) mice on a mixed genetic background. Spleen cells were cultured with M-CSF and RANKL for 5-9 days with or without PGE2 or selective agonists for the four PGE2 receptors (EP1A, EP2A, EP3A, or EP4A). Some cultures were performed using T-cell-depleted spleen cells. OC number and size were quantitated. OC apoptosis and pit formation were measured at 7 or 8 days. RESULTS: PGE2 decreased the number of OCs formed in the presence of RANKL and M-CSF at 5-6 days of culture and increased OC number at 8-9 days compared with cultures without PGE2. PGE2 also increased OC size at 7 and 8 days, decreased apoptosis of OC at 7 days, and increased pit formation at 8 days. EP1A or EP4A had no effect on OC. EP3A decreased OC number. EP2A mimicked effect of PGE2. EP2(-/-) spleen cells showed no increase in OC number in response to PGE2, whereas deletion of EP4 had no effect. Depletion of T-cells abrogated the late increase of OC number. CONCLUSIONS: We conclude that PGE2 has an initial inhibitory effect on OC formation in spleen cell cultures, possibly mediated by both EP2 and EP3 receptors, and a later stimulatory effect, mediated by the EP2 receptor, possibly acting on T-cells.

[Regulation of calcification by bisphosphonates].

Etidronate, an analogue of first generation of bisphosphonates, has inhibitory actions on mineralization and hydroxyapatite crystal formation, which is therefore used as a drug to treat ectopic ossification in cases with total hip arthroplasty and spinal cord injury. The effect of bisphosphonates to prevent calcification could be associated with the physical property to attach tightly to hydroxyapatite crystals in bone matrix. A recent investigation suggested that bisphosphonates might prevent arterial calcification through a mechanism associated with the enhanced production of parathyroid hormone-related peptide from vascular smooth muscle cells. A derivative of bisphosphonates to inhibit vascular calcification in atherosclerosis is expected and should be developed in the near future.

The effect of deletion of cyclooxygenase-2, prostaglandin receptor EP2, or EP4 in bone marrow cells on osteoclasts induced by mouse mammary cancer cell lines.

The inducible prostaglandin (PG) synthesis enzyme, cyclooxygenase-2 (COX-2), is involved in osteoclast (OC) formation in cocultures of mouse mammary cancer cell lines (MMT060562 or BALB/c-MC) and bone marrow cells through production of PGE(2). There are four PGE(2) receptors but only the EP2 and EP4 receptors are reported to be important for OC formation. We have investigated the role of COX-2, EP2 receptor, and EP4 receptor in marrow cells for osteoclastogenesis in cocultures of cancer cells and bone marrow cells. We cocultured cancer cell lines with bone marrow cells from COX-2 knockout (-/-), EP2 -/- or EP4 -/- mice compared to wild-type mice. In addition, an EP4 receptor antagonist (EP4 RA) was added in some cocultures. Disruption of COX-2 gene in bone marrow cells had no effect on PGE(2) production and OC formation in cocultures with MMT060562, while it abrogated PGE(2) production and OC formation in cocultures with BALB/c-MC. Disruption of the EP2 gene in bone marrow cells had no effect on OC formation in the cocultures, while disruption of the EP4 gene in bone marrow cells abrogated OC formation in the cocultures. Furthermore, EP4 RA suppressed OC formation and prevented the increase in receptor activator of nuclear factor kappaB ligand (RANKL) mRNA levels in the cocultures. We conclude that COX-2 in cancer cells is responsible for PGE(2) and OC production in cocultures with MMT060562, while COX-2 in bone marrow cells, not cancer cells, is responsible for PGE(2) and OC production in cocultures with BALB/c-MC, and EP4 receptors are essential for OC formation in both cocultures.

Involvement of cyclo-oxygenase-2 in osteoclast formation and bone destruction in bone metastasis of mammary carcinoma cell lines.

We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c-MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear. In the present in vitro and in vivo studies, we tested the involvement of cyclo-oxygenase-2 (COX-2), an inducible rate-limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines. Addition of a selective COX-2 inhibitor, JTE-522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose-dependent manner. Northern blotting showed a very high level of COX-2 messenger RNA (mRNA) expression in MMT060562. The mRNA expression was low in BALB/c-MC, but it increased when BALB/c-MC and bone marrow cells were cocultured. The results of immunocytochemistry for COX-2 protein in respective cultures were compatible with the results of COX-2 mRNA. In vivo, BALB/c-MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs. Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae. Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX-2 protein. These findings suggest that COX-2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.