ALBERT

Description: JMSE, Vol. 6, Pages 87: Model Uncertainties for Soil-Structure Interaction in Offshore Wind Turbine Monopile Foundations Journal of Marine Science and Engineering doi: 10.3390/jmse6030087 Authors: Lars Vabbersgaard Andersen Thomas Lykke Andersen Lance Manuel Monopiles are the most common type of foundation used for bottom-fixed offshore wind turbines. This investigation concerns the influence of uncertainty related to soil–structure interaction models used to represent monopile–soil systems. The system response is studied for a severe sea state. Three wave-load cases are considered: (i) irregular waves assuming linearity; (ii) highly nonlinear waves that are merged into the irregular wave train; (iii) slamming loads that are included for the nonlinear waves. The extreme response and Fourier amplitude spectra for external moments and mudline bending moments are compared for these load cases where a simpler static pile-cap stiffness and a lumped-parameter model (LPM) are both considered. The fundamental frequency response of the system is well represented by the static pile-cap stiffness model; however, the influence of higher modes (i.e., the second and third modes with frequencies of about 1 Hz and 2 Hz, respectively) is significantly overestimated with the static model compared to the LPM. In the analyzed case, the differences in the higher modes are especially pronounced when slamming loads are not present.

Description: Monopiles are the most common type of foundation used for bottom-fixed offshore wind turbines. This investigation concerns the influence of uncertainty related to soil–structure interaction models used to represent monopile–soil systems. The system response is studied for a severe sea state. Three wave-load cases are considered: (i) irregular waves assuming linearity; (ii) highly nonlinear waves that are merged into the irregular wave train; (iii) slamming loads that are included for the nonlinear waves. The extreme response and Fourier amplitude spectra for external moments and mudline bending moments are compared for these load cases where a simpler static pile-cap stiffness and a lumped-parameter model (LPM) are both considered. The fundamental frequency response of the system is well represented by the static pile-cap stiffness model; however, the influence of higher modes (i.e., the second and third modes with frequencies of about 1 Hz and 2 Hz, respectively) is significantly overestimated with the static model compared to the LPM. In the analyzed case, the differences in the higher modes are especially pronounced when slamming loads are not present.

Description: Introduction: Multiple myeloma is characterized by the accumulation of malignant plasma cells in the bone marrow, and leads most often to bone destruction by osteoclasts and prevention of bone repair by osteoblasts. Bortezomib and glucocorticoids are both powerful anti-myeloma drugs that are used for killing malignant plasma cells in the patients. Furthermore bortezomib has direct anti-osteoclastic and pro-osteoblastic properties that may contribute to bone protection in multiple myeloma, while glucocorticoids have more ambiguous effects on these bone cells and are clearly anti-osteoblastic. Recent clinical trials based on the combination of bortezomib and glucocorticoids drew the attention on the very promising anti-myeloma efficiency of this combination. However, the bone cell response of this combination has not been tested. In order to address this question, we performed an in vitro study, and importantly adapted our in vitro model to mimic the pharmacokinetics of bortezomib and glucocorticoid in the patients. Methods: Myeloma cell lines, primary human osteoclasts and osteoblast-like cells (MC3T3) were pulse-treated or not with clinically relevant doses of bortezomib (12.5, 25 or 50 nM) for 3 hours. Subsequently, the cells were exposed during a 3-day culture to 1.6 μM prednisolone which approximately corresponds to a dose of 50 mg prednisolone in a patient. The impact of the treatment on the cells was determined by survival, activity and gene expression. Results: Bortezomib as a single treatment was very efficient in killing sensitive myeloma cells (OPM2) whereas the more resistant cells (U266) were more efficiently killed in combination with prednisolone. The release of TRAP from primary human osteoclasts, a marker of osteoclastic activation, was strongly inhibited by bortezomib treatment alone, but only in combination with prednisolone did it result in killing of osteoclasts. Survival of osteoblast like cells was uninfluenced by treatment with bortezomib alone. In contrast, as shown previously, prednisolone strongly reduced osteoblast survival. Most importantly however, a 3 hr pre-treatment with bortezomib protected the osteoblasts against the detrimental effects of glucocorticoids. Ongoing investigations by Q-PCR indicate that important markers of osteoblast maturation remain high if the osteoblasts were pre-treated with bortezomib prior to prednisolone exposure. Conclusion: Our study demonstrates in conditions relevant to treatment of myeloma patients, that combining bortezomib and glucocorticoids has a direct synergistic effect against myeloma cells and osteoclasts, and that bortezomib protects directly osteoblasts from the negative impact of glucocorticoids. Thus, the combination of bortezomib and glucocorticoids is not only a powerful treatment of multiple myeloma itself, but also shows promise for treating myeloma bone disease.

Description: Multiple myeloma (MM) leads to high risk for bone pain and fractures. MM-induced bone disease is due to acute degradation of bone matrix by osteoclasts, and absence of repair by bone forming osteoblasts. It is currently treated with bisphosphonates, highly effective bone resorption inhibitors, which do not promote but rather inhibit bone formation and may cause renal damage and osteonecrosis of the jaw. Thus, it is important to reconsider the management of MM bone disease in long-term treatment. Recent preclinical studies reported that the proteasome inhibitor Bortezomib (V) used for the treatment of MM patients can stimulate bone formation, and that in MM patients treated with V, serum levels of bone formation markers are increased. The present study aims at investigating if V may inhibit osteoclast activity. Methods: Osteoclasts were differentiated from pure populations of blood derived CD14-positive monocytes cultured with M-CSF and RANKL for 6–7 days, and treated continuously with V at various concentrations. As prolonged inhibition of proteasome activity has been reported to be toxic for any cell type, and in vivo pharmacodynamic studies have shown V to be eliminated from the vascular compartment as soon as 30min after intravenous injection, displaying maximal inhibitory activity of the proteasome within 24 hours subsiding rapidly thereafter, V was also given intermittently, to mimick the in vivo situation. Osteoclast differentiation and activity were assessed by measuring Tartrate-Resistant Acid Phosphatase (TRACP) activity in the medium. Cell viability was determined with Celltiter Blue measuring metabolic activity. To extend our observations to the clinical situation, serum levels of CTX-I, a bone resorption marker, were measured during the 3 days following therapeutic V administration in a single patient. Results: A continuous treatment of cultures with V at 4 nM and higher concentrations proved to be highly toxic for differentiating osteoclasts but also monocytes. A 3-hour-pulse treatment with V followed by a 3-day culture in the absence of V, was not toxic neither to monocytes nor to osteoclasts, even at a concentration as high as 100 nM. This 3-hour pulse was however highly toxic for myeloma cells. Interestingly, a 3-hour pulse with 25 nM V induced a 50% inhibition of the resorptive activity of osteoclasts, as assessed by culturing them for 3 days on bone slices and measuring the formation of resorption pits. The release of TRACP in the medium was inhibited to a similar extent within the first 24 hours post-pulse, but tended to return to the control level during the next 2 days. This 3-hour pulse with 25 nM V inhibited strongly RANKL-induced translocation of NF-KB in the osteoclast nuclei, an event dependent on proteasome function and critical for osteoclastic activity. Serum CTX-I levels decreased during the first 48 hours after each V injection (n = 3), and tended to increase again after 72 hours suggesting a partial recovery of osteoclast activity between each administration. Conclusions: Our results suggest that Bortezomib temporarily inhibits osteoclast activity in vitro and in vivo. This effect is linked to RANKL-induced translocation of NF-KB in the osteoclast nuclei and proteasome function. Since recent reports suggested that formation of new bone requires at least a transient activity of osteoclasts transient inhibition of osteoclasts could be an advantage compared to the more persistent inhibition of osteoclast activity by bisphosphonate.

Description: Background: Bone degradation in multiple myeloma (MM) is a result of increased bone degradation by osteoclasts that is not compensated for by bone forming osteoblasts. Ideally new drugs used for treatment of MM should target not only the myeloma cells but also the imbalance between bone resorption and bone formation. Statins have been shown to inhibit myeloma cell proliferation and induce apoptosis in vitro. Furthermore statins have been shown to stimulate osteoblasts and inhibit osteoclasts both in vitro and in animal models. Statins are normally used at doses around 20–80 mg/day, but in order to reach serum concentrations that can match the in vitro experiments MM patients were treated with 15 mg/kg/day of Simvastatin (HD-Sim) divided in two daily doses in this study. This high dose has previously been found to be safe for MM patients (Haematologica 2006, 91,542–545) Patients and methods: Six patients with advanced MM have been included in this pilot study, 4 males and 2 females with an average age of 68 years and an average duration of disease of 43 months. The patients were treated with 2 cycles of HD-Sim for seven days followed by a break of 21 days in a 4-weeks cycle. Two of the patients were treated with bisphosphonates during the study, and 4 had previously been treated with bisphosphonates. Endpoints are change in concentrations of markers of osteoclast activity (TRAP) or bone resorption (CTX, NTX, ICTP) or markers of bone formation (Osteocalcin and PINP). Cholesterol, OPG and DDK-1 were also measured. Results: Two patients completed the protocol with two cycles of HD-Sim at full dose, 2 patients were reduced to 7.5 mg/kg/day simvastatin in cycle 2 due to nausea and diarrhea and 2 patients left the protocol after 3 weeks (deaths not related to high dose simvastatin). All patients experienced gastrointestinal toxicity grade 1–2. Myalgia and other muscular symptoms grade 1–2 were reported by 5 patients but were not associated with an increase in creatin kinase. TRAP and NTX activity in serum increased for all 6 patients during the seven days of treatment with HD-Sim indicating that bone resorption may have been stimulated rather than inhibited. The other markers of bone resorption and the bone formation markers showed no change. All patients responded with a significantly reduced level of cholesterol in serum. None of the patients showed any reduction in free monoclonal light chains or monoclonal proteins in serum during treatment with HD-Sim and 2 of the 4 patients completing the protocol showed progression of diseases. Conclusion: This pilot study of HD-Sim in advanced MM has been terminated due to lack of response and evidence from two markers of osteoclast activity (TRAP) and bone resorption (NTX) that HD-Sim may be harmful rather than beneficial in MM.

Description: Acute leukemias are remarkably heterogeneous as evidenced by the increasing number of recurring genetic and epigenetic abnormalities, especially the presence or absence of balanced translocations, which have been shown to be of independent prognostic significance. Thus, In a recent single center study we analyzed 250 AML patients for a series a genetic alteration and found that balanced translocations (identified by a multiplex PCR reaction; Pallisgaard et al, BLOOD, 1998 and Olesen et al, Brit. J. Hem. in press) were present in 17% of the patients, FLT3 internal tandem duplication in 24%, and MLL partial tandem duplication in 4%. In addition, we delineated the presence of promoter hypermethylation for the p15 (71%), MDR1 (4%), E-cadherin (CDH1) (64%), and Estrogen receptor (ER) (40%) genes by bisulfite DGGE (Aggerholm, Cancer Res. 1999) as well as the abnormal expression by RQ-PCR of genes related to increased chemotherapy resistance (MDR1 and MRP1) as well as resistance to undergo apoptosis (FAS, Bcl2, BAX and CASPASE3). Here we have performed gene expression profiling focusing on patients negative for all these molecular lesions. Out of the 250 patients, 8 were determined to be molecularly negative. Global gene expression analysis (Affymetrix human genome chip (U133A)) was performed on the 6 samples, from which high-quality RNA could be harvested. Figure Figure As will be seen from the Figure, all samples could be easily distinguished from TEL/AML pre-B ALL samples, and also in 4/6 cases clustered differently from AML/ETO+ and CBF/MYH11+ AML groups. Interestingly, as seen from the dendrogram, the 6 samples separated in three clusters, one also containing a CBF/MYH11+ patient (4 cases), one consisting of one single patient, while the last patient clustered together with the AML/ETO cases. While these data suggest that the gene expression in the molecularly negative patients can be different from that in CBF leukemias, it gives no clues for the leukemogenetic events in these patients. To that end, we analyzed the gene expression data and found 24 genes to be more than 5-fold overexpressed and 26 genes to be underexpressed compared to CBF AMLs. Of special notice was the increased usage of the prostaglandin/leukotriene metabolism pathway in two patients with strikingly similar global gene expression (arrows 3 and 4 from right on Fig.) including the prostaglandin I2 (prostacyclin) synthase, and the prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) genes, which were more than 10-fold increased. Also noteworthy was the upregulation of LR8 protein IQ motif containing GTPase activating protein 1 in the remaining 4 cases. Applied together with novel global cytogenetic techniques, these data will form a platform for the identification of leukemogenesis in AML patients with no demonstrable genetic aberrations.ßßß

Description: Although myeloma development and enhanced bone resorption are intimately related, the mechanism responsible of this relation is not known. In vitro studies have stressed the critical role of direct cell contacts between myeloma cells and osteoclast. In vivo, however, little is known about the organization of the cells present at osteolytic lesions, because of the complexity of cell-cell interactions in the bone marrow of myeloma patients. Therefore, we conducted an immunohistochemical study with multiple stainings allowing the simultaneous identification of different cell types at resorption sites of bone marrow biopsies of myeloma patients. The biopsies showed that in average 1% of the bone surface was lined by mature multinucleated TRAP+ osteoclasts, but that only 6% of these osteoclasts showed direct contacts with myeloma cells. The biopsies showed also TRAP+ mononucleated pre-osteoclasts in the bone marrow compartment, and 40% of these pre-osteoclasts showed direct contacts with myeloma cells. Bone marrow pre-osteoclasts show thus much more frequent contacts with myeloma cells, compared with mature osteoclasts lining bone surfaces. These respective values remained unchanged, whether the myeloma cells were identified through CD138 or through light chain expression (counts in biopsies from 13 patients). Importantly, we found that 80% of the osteoclasts lining the bone surfaces, were separated from the bone marrow compartment by a specialized cell wall (seen in all biopsies of the 15 patients, that were analyzed). This wall consists of a single layer of (sometimes very) flattened cells lining the bone marrow, and expressing NCAM, propeptide of type III collagen, and osteocalcin, but not CD34. When performing 3D reconstructions by using serial sections, this wall appeared as a continuous roof covering the bone surfaces undergoing remodeling, and connected to the bone lining cells at its periphery. Furthermore, CD34 staining revealed that capillaries are abundant at the bone marrow side of this cell wall, and that some show an opening into the wall. These capillaries may thus allow communication between the bone marrow and the bone surfaces undergoing remodeling, and render the bone remodeling compartments vascular-like. In addition, the TRAP+ preosteoclast detected in the bone marrow space, were positioned along the capillaries leading to the vascular/remodeling compartments. In conclusion, this study shows that in vivo, interactions between myeloma cells and osteoclasts are mediated only rarely through direct cell contacts, and identifies for the first time unique cell arrangements that are likely to play a role in these interactions: a specialized cell wall separates the bone marrow from the vascular/remodeling compartments in most resorption sites (80% of the osteoclasts), and the cells of this wall are thus in a privileged situation to control myeloma-osteoclast interactions; capillaries connect the marrow cavity with the vascular/remodeling compartment, thereby allowing guidance of pre-osteoclasts from the bone marrow to the resorption sites; the generation of these pre-osteoclasts may be stimulated by the high incidence of their direct contacts with myeloma cells in the bone marrow.

Description: Gene expression analysis of 8 different genes in homogenized bone marrow trephine biopsies from 47 newly diagnosed, untreated MM patients with different degrees of osteolytic bone disease (OBD) and 19 MGUS patients were analysed by quantitative PCR technique. The analysed genes represent both genes suspected to be involved in osteoblast inhibition (DKK1, HGF, Frizzled-related protein B, Syndecan-1) and osteoclast activation (RANKL, MIP-1α), and genes involved in the regulation of these genes (OPG, RANK). The expression levels of these 8 genes in the biopsies represent the net gene expression in bone marrow microenvironment including MM cells, hematopoietic cells, stromal cells, osteoblasts, and osteoclasts. OBD was graded into no, limited, or advanced by standard radiography. The expression level of DKK1 increased significantly with more advanced OBD (p=0.005), whereas the expression level between the MGUS group and the MM group with no/limited OBD was not significantly different (p= 0.097). Syndecan-1 was expressed in all BM-samples from MGUS and MM patients. Syndecan-1 expression was significantly increased with more advanced OBD and was significantly different between the three bone morbidity groups. The expression of Syndecan-1 was, furthermore, closely correlated to PC numbers in the BM samples (p〈 0.0001). HGF and FRPB expressions in the MM group with no/low OBD and MM group with advanced OBD was not statistically different but HGF and FRPB expression was significantly increased in the MM groups compared with the MGUS group. OPG expression showed a trend towards being decreased with more advanced OBD. There were no correlation between the degree of OBD and the expression levels of MIP1a, RANKL, and RANK. Thus, in this BM microenvironment expression profile study we found that inhibitors of the Wnt signalling pathways, DKK1 and Frizzled-related protein B, and the inhibition of osteoblastogenesis by HGF, seem to play an important role in the pathogenesis of OBD in MM. In the contrary, we did not find a significant correlation between up-regulation of the osteoclast activating cytokines, RANKL and MIP1α, with more advanced OBD, and we did not find a correlation between the expression level of the RANKL receptor, RANK, and the degree of OBD.