Low degree of satisfactory individual pain relief in post-operative pain trials.

BACKGROUND: The majority of clinical trials regarding post-operative pain treatment focuses on the average analgesic efficacy, rather than on efficacy in individual patients. It has been argued, that in acute pain trials, the underlying distributions are often skewed, which makes the average unfit as the only way to measure efficacy. Consequently, dichotomised, individual responder analyses using a predefined 'favourable' response, e.g. Visual Analogue Scale (VAS) pain scores ≤ 30, have recently been suggested as a more clinical relevant outcome. METHODS: We re-analysed data from 16 randomised controlled trials of post-operative pain treatment and from meta-analyses of a systematic review regarding hip arthroplasty. The predefined success criterion was that at least 80% of patients in active treatment groups should obtain VAS < 30 at 6 and 24 h post-operatively. RESULTS: In the analysis of data from the randomised controlled trials, we found that at 6 h post-operatively, 50% (95% CI: 31-69) of patients allocated to active treatment reached the success criterion for pain at rest and 14% (95% CI: 5-34) for pain during mobilisation. At 24 h post-operatively, 60% (95% CI: 38-78) of patients allocated to active treatment reached the success criterion for pain at rest, and 15% (95% CI: 5-36) for pain during mobilisation. Similar results were found for trials from the meta-analyses. CONCLUSION: Our results indicate that for conventional, explanatory trials of post-operative pain, individual patient's achievement of a favourable response to analgesic treatment is rather low. Future pragmatic clinical trials should focus on both average pain levels and individual responder analyses in order to promote effective pain treatment at the individually patient level.

Retracted: Increased risk of stomach and esophageal malignancies in people with AIDS.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief and Authors. The authors recently discovered two programming errors that affected the results in their article on the epidemiology of esophageal and stomach cancers in human immunodeficiency virus infected people. As a result of these errors, the standardized incidence ratios (SIRs) were too high. The corrected SIRs are all lower than the authors reported, and the corrected SIR for stomach cancer is no longer significantly elevated. These errors affect Tables 2-5 in the paper. Because the new findings alter the conclusions, the editors and authors have jointly made the decision to retract the paper. The authors would like to express their sincere regret at the errors in their initial report and any inconvenience or confusion that they created. The corrected results may be obtained by contacting the corresponding author, Dr. Eric A. Engels, by email at engelse@exchange.nih.gov.

Dichlorodiphenyltrichloroethane and risk of hepatocellular carcinoma.

Dichlorodiphenyltrichloroethane (p,p'-DDT), an organochlorine pesticide known to have deleterious health effects in humans, has been linked to hepatocellular carcinoma (HCC) in rodents. A recent study has reported that p,p'-DDT and its most persistent metabolite, dichlorodiphenyldichloroethylene (p,p'-DDE), may also be associated with HCC in humans. To examine whether there is an association between p,p'-DDT and/or p,p'-DDE in a population at high-risk of developing HCC, a nested case-control study was conducted within the 83,794 person Haimen City Cohort in China. Sera and questionnaire data were collected from all participants between 1992 and 1993. This study included 473 persons who developed HCC and 492 who did not, frequency matched on sex, age and area of residence. p,p'-DDT and p,p'-DDE levels were determined by mass spectrometry. Hepatitis B viral infection status (based on hepatitis B virus surface antigen; HBsAg) was also determined. p,p'-DDT and/or p,p'-DDE serum levels were significantly associated with sex, area of residence, occupation, alcohol consumption and cigarette smoking. Adjusting for age, sex, area of residence, HBsAg, family history of HCC, history of acute hepatitis, smoking, alcohol, occupation (farmer vs. other) and levels of p,p'-DDT or p,p'-DDE, odds ratios (OR) and 95% confidence intervals (CI) were calculated via unconditional logistic regression. Overall, the highest quintile of p,p'-DDT was associated with an increased risk of HCC, OR = 2.96 95% CI; 1.19-7.40. There were no statistically significant associations with p,p'-DDE. Overall, these results suggest that recent exposure to p,p'-DDT may increase risk of HCC.

Risk of liver cancer among US male veterans with cirrhosis, 1969-1996.

BACKGROUND: Liver cancer incidence rates in the United States have increased for several decades for reasons that are not entirely clear. Regardless of aetiology, cirrhosis is a strong risk factor for liver cancer. As mortality from cirrhosis has been declining in recent decades, it is possible that the risk of liver cancer among persons with cirrhosis has been affected. METHODS: Data from the US Veterans Affairs medical records database were analysed after adjustment for attained age, race, number of hospital visits, obesity, diabetes, and chronic obstructive pulmonary disease. Hazard ratio (HR) and 95% confidence interval (95% CI) were calculated using Cox proportional hazards modelling. Survival analyses were conducted using age as the time metric and incidence of cirrhosis as a time-dependent covariate. RESULTS: Among 103 257 men with incident cirrhosis, 788 liver cancers developed. The HR of liver cancer was highest among men with viral-related cirrhosis (HR=37.59, 95% CI: 22.57-62.61), lowest among men with alcohol-related cirrhosis (HR=8.20, 95% CI: 7.55-8.91) and intermediate among men with idiopathic cirrhosis (HR=10.45, 95% CI: 8.52-12.81), when compared with those without cirrhosis. Regardless of cirrhosis type, white men had higher HRs than black men. The HR of developing liver cancer increased from 6.40 (95% CI: 4.40-9.33) in 1969-1973 to 34.71 (95% CI: 23.10-52.16) in 1992-1996 for those with cirrhosis compared with those without. CONCLUSION: In conclusion, the significantly increased HR of developing liver cancer among men with cirrhosis compared with men without cirrhosis in the United States may be contributing to the increasing incidence of liver cancer.

Functional characteristics of N8, a new recombinant FVIII.

The aim of this study was to evaluate the in vitro function of the new recombinant factor VIII (FVIII) compound, N8. The specific activity of N8 as measured in a FVIII:C one-stage clot assay was 9300±400 IU mg(-1) based on the analysis of seven individual batches. The ratio between the FVIII:C activity measured in clot and chromogenic assays was 1.00 (95% confidence interval 0.97-1.03). N8 bound to von Willebrand factor with Kd values of 0.2 nm when measured by ELISA and by surface plasmon resonance. FVIIIa cofactor activity was determined from the kinetic parameters of factor IXa-catalysed factor X (FX) activation. The rate of activation of N8 by thrombin as well as Km and kcat for FX activation was in the same range as those observed for Advate®. The rate of activated protein C (APC)-catalysed inactivation was similar for activated N8 and Advate®. N8 improved thrombin generation in a dose-dependent manner and induced similar rates of thrombin generation as Advate® and the plasma-derived FVIII product Haemate®. Using thromboelastography (TEG®), N8 was shown to improve the clot formation and clot stability in whole blood from haemophilia A patients. Comparable potency and efficacy of N8 and Advate® was found based on TEG® parameters. Finally, similar binding profiles to immobilized lipoprotein receptor-related protein (LRP) of N8 and Advate® were observed. The study demonstrated that N8 is fully functional in a variety of assays measuring FVIII activity. No functional differences were found between N8 and comparator compounds.

Stoma-related complications and stoma size - a 2-year follow up.

AIM: The purpose of the study was to prospectively describe stoma configuration and evaluate stoma-related complications and their association with possible risk factors. METHOD: All elective patients (n = 180) operated on with a formation of colostomy, ileostomy or loop-ileostomy between 2003 and 2005 were included in the study. Follow up took place on the ward postoperatively and five times during 2 years after discharge. On these occasions the diameter and height of the stoma were recorded. Complications such as peristomal skin problems, necrosis, leakage caused by a low stoma, stenosis, granuloma formation, prolapse and peristomal hernia formation were evaluated. RESULTS: Most complications occurred 2 weeks after discharge; 53% of patients with colostomies, 79% with loop-ileostomies and 70% of patients with end-ileostomy had one or more complications. The most common complication was skin problems and it was most common in patients with end-ileostomies (60%) and loop-ileostomies (73%). Postoperatively at ward review, the most common complication was necrosis, which occurred in 20% of patients with a colostomy. Granuloma formation was most frequent in colostomies. Almost all patients with an end-ileostomy and loop-ileostomy with a height lower than 20 mm had leakage and skin problems as had half of the patients with a colostomy height lower than 5 mm. CONCLUSION: To prevent stoma-related complications, it is important to produce an adequate height of the stoma, with early and regular follow ups and adjustment of the appliance. To work closely in collaboration with the colorectal surgeons is of utmost important to provide feedback and in turn, to improve stoma outcome.

Interobserver and intraobserver agreement in the evaluation of CT perfusion in ischemic stroke.

Knowledge of interrater reliability in the evaluation of perfusion computed tomography (CTP) studies is very limited even though the method is widely used in the workup of acute stroke. The aims of this study were to estimate the inter- and intraobserver agreement in the evaluation of CTP data and to evaluate the feasibility of the method. The CTP data of 20 consecutive patients (50% were females) aged 68+/-11 years with different categories of acute ischemic stroke were included in this retrospective analysis. Perfusion studies were evaluated independently by six radiologists on two different occasions. The overall inter- and intraobserver agreement was substantial, showing a capital KA, Cyrillic value of 0.65 (95% confidence interval 0.39-0.91). The time required for the post-processing and interpretation ranged from 37 to 460 seconds. Evaluation of manually post-processed CTP data according to the maximum slope model appears to be reliable. Experience and also a short training period increase the reliability of the method and reduce the time needed for delivery of the results to the treating clinician.

Characterisation of invasive group B streptococci based on investigation of surface proteins and genes encoding surface proteins.

The joint distributions of the six genes bca, bac, epsilon/alp1, alp2, alp3 and rib (encoding alpha-C-protein, beta-C-protein, epsilon/Alp1, Alp2, Alp3, and Rib, respectively) and the proteins alpha-C-protein, beta-C-protein and Rib were investigated in invasive isolates of group B streptococcus (GBS). In total, 297 invasive isolates (123 from neonates, 174 from adults) from south-west Sweden were collected during a 13-year period. Genes were detected using multiplex and specific PCRs, and expression of the surface proteins was demonstrated using monoclonal antibodies. The genes studied were found alone or in combinations in 294 (99%) of the invasive isolates. The most common genes were rib (n = 127 isolates, 43%), alp3 (n = 78, 26%) and epsilon/alp1 (n = 42, 14%). The bac gene was never found alone, but was found in combination with one other gene in 36 isolates. The surface proteins studied were detected alone or in combinations in 152 (51%) isolates, with the most common being Rib (n = 80, 27%), alpha-C-protein (n = 68, 23%) and beta-C-protein (n = 24, 8%). Several genes were associated significantly with particular serotypes (e.g., epsilon/alp1 with serotype Ia; bca and bac with serotypes Ib and II; rib with serotype III; alp3 with serotype V). Overall, it was concluded that demonstration of different genes and surface proteins of GBS strains can be useful in epidemiological studies and in formulation of vaccines, but disappointingly, no single gene or surface protein included in the study was sufficiently common for it to be considered as the basis for a successful GBS vaccine.

Osteotropic effects by the neuropeptides calcitonin gene-related peptide, substance P and vasoactive intestinal peptide.

Immunohistochemical phenotypic characterization of skeletal nerve fibers has demonstrated the expression of a restricted number of neuropeptides, including calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP). According to the neuro-osteological hypothesis, such neuropeptides can be released and exert paracrine biological effects on bone cells present close to the nerve endings expressing these signaling molecules. The existence of such interplay is most convincingly shown by the hypothalamic control of bone formation, in the case of leptin stimulation of hypothalamic nuclei mediated by the sympathetic nervous system and inhibitory beta-adrenergic receptors on osteoblasts. In addition to these receptors, osteoblasts and osteoclasts express functional receptors for CGRP, SP and VIP, which can regulate both bone formation and bone resorption. The evidence for these observations is summarized in the present paper.

Improved understanding of the effect of food on drug absorption and bioavailability for lipophilic compounds using an intestinal pig perfusion model.

The purpose of this study was to investigate the relative importance of mechanisms behind the effect of food on the intestinal absorption and bioavailability for low solubility compounds by applying a porcine single-pass perfusion model. Nanoparticle suspensions of the model compounds, danazol and cyclosporine were perfused through the jejunum in isotonic fluid alone (control) and isotonic fluid with a P-glycoprotein (P-gp) inhibitor (verapamil) or dietary and endogenous lipids added. The drugs were also administered as saturated solutions in the isotonic fluid containing lipids. Administration of cyclosporine together with verapamil increased the absorption compared to the control (1.6 times) suggesting an effect on jejunal permeability. However, addition of dietary lipids to the media led to a 50% reduction in the absorption of cyclosporine indicating lack of major effects by P-gp inhibition by lipids in vivo. The absorption of danazol was increased (2.6 times) when administered as a nanosuspension in lipid containing media compared to the control, but decreased (60%) when administered as a solution in the same media. This shows how important dissolution of the drug nanoparticles is in drug absorption. The difference in the effect of lipids in the absorption of cyclosporine and danazol when administered as nanosuspensions may be due to different distribution to the colloidal structures present in the media, thereby rendering the drugs' different diffusion rates in the perfused segment. In conclusion, solubilisation seems to be a more important factor than P-gp inhibition as an explanation for the food-drug interaction observed for several low solubility drugs. In addition, the partition into different colloidal structures seems to play a major role in the dissolution and absorption of poorly soluble drugs.

CD103+CD11b+ mucosal classical dendritic cells initiate long-term switched antibody responses to flagellin.

Antibody responses induced at mucosal and nonmucosal sites demonstrate a significant level of autonomy. Here, we demonstrate a key role for mucosal interferon regulatory factor-4 (IRF4)-dependent CD103+CD11b+ (DP), classical dendritic cells (cDCs) in the induction of T-dependent immunoglobulin G (IgG) and immunoglobulin A (IgA) responses in the mesenteric lymph node (MLN) following systemic immunization with soluble flagellin (sFliC). In contrast, IRF8-dependent CD103+CD11b- (SP) are not required for these responses. The lack of this response correlated with a complete absence of sFliC-specific plasma cells in the MLN, small intestinal lamina propria, and surprisingly also the bone marrow (BM). Many sFliC-specific plasma cells accumulating in the BM of immunized wild-type mice expressed α4ß7+, suggesting a mucosal origin. Collectively, these results suggest that mucosal DP cDC contribute to the generation of the sFliC-specific plasma cell pool in the BM and thus serve as a bridge linking the mucosal and systemic immune system.

Activity and regulation of factor VIIa analogs with increased potency at the endothelial cell surface.

BACKGROUND: Variants of recombinant factor VIIa (rFVIIa) with increased intrinsic activity have been developed to improve efficacy in the treatment of bleeding disorders in the future. The increased potency of FVIIa variants was demonstrated in limited in vitro and in vivo studies. However, further characterization of FVIIa variants is needed to evaluate their potential clinical use. METHODS: In the present study, we investigated the interactions of two FVIIa variants, FVIIa(Q) and FVIIa(DVQ), with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT), and vascular endothelium. TF-FVIIa activity or its inhibition was measured directly in an amidolytic activity assay or for its ability to activate factor X. RESULTS: Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type (WT) FVIIa in the absence of tissue factor (TF). In the presence of TF, TFPI, TFPI-Xa, and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the WT FVIIa failed to generate significant amounts of FXa on unperturbed endothelial cells, FVIIa variants, particularly FVIIa(DVQ), generated a substantial amount of FXa on unperturbed endothelium. Annexin V fully attenuated the FVIIa-mediated activation of FX on unperturbed endothelial cells. On stimulated human umbilical vein endothelial cells, FVIIa and FVIIa variants activated FX at similar rates, and annexin V blocked the activation only partly. AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. Interestingly, despite significant differences observed in FXa generation on unperturbed endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions. CONCLUSION: Overall, the present data suggest that although FVIIa variants generate substantial amounts of FXa, they do not generate excessive thrombin on the surface of endothelium.

Lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis in vitro.

BACKGROUND AND PURPOSE: Two compounds, URB602 and URB754, have been reported in the literature to be selective inhibitors of monoacylglycerol lipase, although a recent study has questioned their ability to prevent 2-arachidonoyl hydrolysis by brain homogenates and cerebellar membranes. In the present study, the ability of these compounds to inhibit monoacylglycerol lipase and fatty acid amide hydrolase has been reinvestigated. EXPERIMENTAL APPROACH: Homogenates and cell lines were incubated with test compounds and, thereafter, with either [(3)H]-2-oleoylglycerol or [(3)H]-anandamide. Labelled reaction products were separated from substrate using chloroform: methanol extraction. KEY RESULTS: In cytosolic fractions from rat brain, URB602 and URB754 inhibited the hydrolysis of 2-oleoylglycerol with IC(50) values of 25 and 48 microM, respectively. Anandamide hydrolysis by brain membranes was not sensitive to URB754, but was inhibited by URB602 (IC(50) value 17 microM). Hydrolysis of 2-oleoylglycerol by human recombinant monoacylglycerol lipase was sensitive to URB602, but not URB754. The lack of selectivity of URB602 for 2-oleoylglycerol compared to anandamide hydrolysis was also observed for intact RBL2H3 basophilic leukaemia cells. C6 glioma expressed mRNA for monoacylglycerol lipase, and hydrolyzed 2-oleoylglycerol in a manner sensitive to inhibition by methyl arachidonoyl fluorophosphonate but not URB754 or URB597. MC3T3-E1 mouse osteoblastic cells, which did not express mRNA for monoacylglycerol lipase, hydrolyzed 2-oleoylglycerol in the presence of URB597, but the hydrolysis was less sensitive to methyl arachidonoyl fluorophosphonate than for C6 cells. CONCLUSIONS AND IMPLICATIONS: The data demonstrate that the compounds URB602 and URB754 do not behave as selective and/or potent inhibitors of monoacylglycerol lipase.

Assessment of dosimetric impact of system specific geometric distortion in an MRI only based radiotherapy workflow for prostate.

Dosimetric errors in a magnetic resonance imaging (MRI) only radiotherapy workflow may be caused by system specific geometric distortion from MRI. The aim of this study was to evaluate the impact on planned dose distribution and delineated structures for prostate patients, originating from this distortion. A method was developed, in which computer tomography (CT) images were distorted using the MRI distortion field. The displacement map for an optimized MRI treatment planning sequence was measured using a dedicated phantom in a 3 T MRI system. To simulate the distortion aspects of a synthetic CT (electron density derived from MR images), the displacement map was applied to CT images, referred to as distorted CT images. A volumetric modulated arc prostate treatment plan was applied to the original CT and the distorted CT, creating a reference and a distorted CT dose distribution. By applying the inverse of the displacement map to the distorted CT dose distribution, a dose distribution in the same geometry as the original CT images was created. For 10 prostate cancer patients, the dose difference between the reference dose distribution and inverse distorted CT dose distribution was analyzed in isodose level bins. The mean magnitude of the geometric distortion was 1.97 mm for the radial distance of 200-250 mm from isocenter. The mean percentage dose differences for all isodose level bins, were ⩽0.02% and the radiotherapy structure mean volume deviations were <0.2%. The method developed can quantify the dosimetric effects of MRI system specific distortion in a prostate MRI only radiotherapy workflow, separated from dosimetric effects originating from synthetic CT generation. No clinically relevant dose difference or structure deformation was found when 3D distortion correction and high acquisition bandwidth was used. The method could be used for any MRI sequence together with any anatomy of interest.

Dose related effects of LPS on endometrial epithelial cell populations from dioestrus cows.

Lipopolysaccharides (LPS) from Gram negative bacteria are involved in the pathogeny of uterine diseases in cows. This study aimed to investigate LPS effects on the growth of bovine endometrial epithelial cells (bEEC) and relationships between LPS response and tissue characteristics. Uteri from 35 females were characterized for parity and stage of oestrous cycle. Densities of glandular tissue (dGT), CD11b+ cells and Ki67+ cells were measured in the endometrial tissue. Cells from 13 dioestrus cows were exposed to 0, 2, 4, 8, 12, 16 or 24µg/mL LPS. Effects of parity and stage of the oestrous cycle on tissue characteristics and effects of LPS dosage, cow and tissue characteristics on changes in cell numbers were analyzed by ANOVA. The dGT was higher in metoestrus and dioestrus samples than in pro-oestrus ones whereas densities of CD11b+ and Ki67+ cells were higher at pro-oestrus (p<0.05-p<0.01). LPS influenced bEEC populations in a dose related manner. An increase in number of live cells was observed for dosages ranging from 2 to 12µg/mL LPS (p<0.0001 vs controls). No effect was found on numbers and frequencies of dead cells. With higher dosages, the numbers of live cells did not increase but the numbers of dead did increase. No relationships were observed between cow or tissue characteristics and growth patterns or frequencies of viable bEEC in controls nor in the response to LPS. To conclude this model is suitable for further studies on dysregulations induced by LPS in endometrial tissue.

Ultrastructure of the chorioallantoic placenta and chorionic vesicles of the lesser bush baby (Galago senegalensis).

The ultrastructure of the chorioallantoic placenta of the lesser bush baby (Galago senegalensis) has been studied. The placenta was shown to be of the diffuse, epitheliochorial and adeciduate type. The trophoblasts of the chorionic villi, other than those lining the chorionic vesicles, were characterized by the presence of many lipid droplets. In the later stage of gestation, the fetal capillaries indented the trophoblastic epithelium reducing the distance between fetal and maternal circulations. In addition chorionic vesicles were observed. The trophoblasts lining the chorionic vesicles have outward bulging apical surfaces. There are clefts between these cells and this region is occupied by microvilli of adjacent cells. Several layers of fusiform cells that did not extend up into the cores of the chorionic vesicle villi formed the outer component of the vesicular wall. Granulated cells were observed within the maternal connective tissue and their possible role is discussed.

Disc filtration for separation of flocs from a moving bed bio-film reactor.

A Discfilter with 10 and 18 microm filter openings, respectively, was placed in parallel to a flotation plant for separation of biological flocs from a post-denitrifying Kaldnes Moving Bed Process, the last treatment step at the municipal wastewater treatment plant at Sjöunda, Malmö, Sweden. The effluent concentrations from the 10 and 18 microm filter were 2-5 and 2-8 mg SS L(-1), respectively, which is comparable to, or better than, the flotation plant. Comparison with experiences from activated sludge plants shows that the Discfilter works especially well after the Kaldnes process. Particle size distribution (PSD) studies show that particles larger than the filter openings of 10 and 18 microm are separated with approximately 90% efficiency, whereas most of the smaller particles pass the filter. This fact indicates that the major particle separation mechanism is physical blocking. These findings point to the possibility of improving the prediction of the separation efficiency by combining measurements of turbidity and suspended solids with particle size analysis.

Overestimation of N-glycoPEGylated factor IX activity in a one-stage factor IX clotting assay owing to silica-mediated premature conversion to activated factor IX.

UNLABELLED: Essentials Nonacog beta pegol (N9-GP) activity is overestimated in clot method using silica-based reagents. Mimicking contact activation phase with silica reveals N9-GP activation before recalcification. Localization of N9-GP to silica facilitates activation by factor XIa and plasma kallikrein. Silica-based reagents to be used with caution when monitoring N9-GP therapy using clot method. SUMMARY: Background Clinical laboratories routinely quantify factor IX (FIX) activity by measurement of the activated partial thromboplastin time (APTT) in a one-stage (OS) clotting assay. This assay can be performed with any of a plethora of differently composed APTT reagents, giving variable recovery when applied to nonacog beta pegol (N9-GP), an N-glycoPEGylated recombinant FIX. Objective To identify the cause of observed overestimations of N9-GP activity in an OS FIX clotting assay when most APTT reagents containing silica are used as the contact activator, and to elucidate the underlying mechanism. Methods Experiments mimicking the contact activation and clotting phases of the OS assay, combined with the use of plasmas with various deficiencies, were employed to shed light on the unique behavior of N9-GP. Confirmatory activations of N9-GP with purified enzymes and physical adsorption to silica particles were studied, and the influence of free polyethylene glycol (PEG) on these processes was investigated. Results N9-GP, but not native FIX, added to FIX-deficient plasma was prematurely converted to activated FIX (FIXa) during the contact activation phase of the clotting assay. Activated FXI (FXIa) and plasma kallikrein (PK) were responsible for the activation of N9-GP, an event that appeared to require the presence of a silica-containing APTT reagent. PEG-dependent adsorption of N9-GP to silica particles could be demonstrated. Conclusions The PEG moiety mediates colocalization of N9-GP with its activators FXIa and PK on silica surfaces, thereby facilitating premature conversion of N9-GP to FIXa during the contact activation phase, and leading to overestimation of the FIX activity in the OS clotting assay.

Stimulation of resorption in cultured mouse calvarial bones by thiazolidinediones.

Dosage-dependent release of 45Ca was observed from prelabeled mouse calvarial bones after treatment with two thiazolidinediones, troglitazone and ciglitazone. Release of 45Ca by ciglitazone was decreased by the osteoclast inhibitors acetazolamide, calcitonin, 3-amino-1-hydroxypropylidene-1,1-bisphosphonate, and IL-4, but not affected by the peroxisome proliferator-activated receptor gamma antagonist, GW 9662, the mitotic inhibitor, hydroxyurea, or indomethacin. Enhanced expression of receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein and decreased osteoprotegerin (OPG) mRNA and protein were noted after ciglitazone treatment of calvariae. Ciglitazone and RANKL each caused increased mRNA expression of osteoclast markers: calcitonin receptor, tartrate-resistant acid phosphatase, cathepsin K, matrix metalloproteinase-9, integrin beta3, and nuclear factor of activated T cells 2. OPG inhibited mRNA expression of RANKL stimulated by ciglitazone, mRNA expression of osteoclast markers stimulated by ciglitazone and RANKL, and 45Ca release stimulated by troglitazone and ciglitazone. Increased expression of IL-1alpha mRNA by ciglitazone was not linked to resorption stimulated by the thiazolidinedione. Ciglitazone did not increase adipogenic gene expression but enhanced osteocalcin mRNA in calvariae. In addition to exhibiting sensitivity to OPG, data indicate that stimulation of osteoclast differentiation and activity by thiazolidinediones may occur by a nonperoxisome proliferator-activated receptor gamma-dependent pathway that does not require cell proliferation, prostaglandins, or IL-1alpha but is characterized by an increased RANKL to OPG ratio.

Tissue factor activates allosteric networks in factor VIIa through structural and dynamic changes.

BACKGROUND: Tissue factor (TF) promotes colocalization of enzyme (factor VIIa) and substrate (FX or FIX), and stabilizes the active conformation of FVIIa. Details on how TF induces structural and dynamic changes in the catalytic domain of FVIIa to enhance its efficiency remain elusive. OBJECTIVE: To elucidate the activation of allosteric networks in the catalytic domain of the FVIIa protease it is when bound to TF. METHODS: Long-timescale molecular dynamics simulations of FVIIa, free and in complex with TF, were executed and analyzed by dynamic network analysis. RESULTS: Allosteric paths of correlated motion from the TF contact point, Met306, in FVIIa to the active site triad can be described and quantified. In particular, the shortest paths from Met306 to Ser344 and His193 are 16% and 8% longer in free FVIIa than in TF-FVIIa, and they encompass previously undiscovered residue-residue interactions that are not likely to be inferred from mutagenesis studies. Furthermore, paths from Met306 to Ile153 (N-terminus) and Trp364, both representing hallmark residues of allostery, are 7% and 37% longer, respectively, in free FVIIa. Thus, there is significantly weaker coupling between the TF contact point and key residues in the catalytic domain of FVIIa, causing the active site triad to disintegrate in the simulation when TF is not present. CONCLUSIONS: These findings complement our current understanding of how the protease FVIIa is stimulated by TF. We demonstrate allosteric networks in the catalytic domain that are activated by TF and help to make FVIIa an efficient catalyst of FIX and FX activation.