Mandibular canal-related parameters interpreted in panoramic images and CBCT of mandibular third molars as risk factors to predict sensory disturbances of the inferior alveolar nerve.

The aim of this study was to determine whether cone beam computed tomography (CBCT) before mandibular third molar removal can improve the risk assessment for neurosensory disturbances of the inferior alveolar nerve (IAN) compared to panoramic radiography (PAN). One hundred and six mandibular third molars examined by PAN and CBCT were removed. A temporary sensory disturbance of the IAN was present in 20 cases; a permanent disturbance was found in one case. Three blinded observers assessed radiographic risk factors in PAN and CBCT images. Positive (PPV) and negative (NPV) predictive values and positive (LR+) and negative (LR-) likelihood ratios were calculated for all parameters for all observers. Inter-observer reproducibility was expressed as both the percentage accordance and the kappa-statistic. Generally, the PPV and LR + were the same for PAN and CBCT, and there was good inter-observer reproducibility. The highest PPV and LR + for PAN were found when part of the roots were positioned inferior to the lower white border line of the canal, and for CBCT when the canal was positioned between the roots of the tooth. In conclusion, parameters assessed in PAN and CBCT are not reliable risk factors for neurosensory disturbances of the IAN, and CBCT appears not to improve the risk assessment.

Salivary IgA from the sublingual compartment as a novel noninvasive proxy for intestinal immune induction.

Whole-saliva IgA appears like an attractive noninvasive readout for intestinal immune induction after enteric infection or vaccination, but has failed to show consistent correlation with established invasive markers and IgA in feces or intestinal lavage. For reference, we measured antibodies in samples from 30 healthy volunteers who were orally infected with wild-type enterotoxigenic Escherichia coli. The response against these bacteria in serum, lavage, and lymphocyte supernatants (antibody-in-lymphocyte-supernatant, ALS) was compared with that in targeted parotid and sublingual/submandibular secretions. Strong correlation occurred between IgA antibody levels against the challenge bacteria in sublingual/submandibular secretions and in lavage (r=0.69, P<0.0001) and ALS (r=0.70, P<0.0001). In sublingual/submandibular secretions, 93% responded with more than a twofold increase in IgA antibodies against the challenge strain, whereas the corresponding response in parotid secretions was only 67% (P=0.039). With >twofold ALS as a reference, the sensitivity of a >twofold response for IgA in sublingual/submandibular secretion was 96%, whereas it was only 67% in the parotid fluid. To exclude that flow rate variations influenced the results, we used albumin as a marker. Our data suggested that IgA in sublingual/submandibular secretions, rather than whole saliva with its variable content of parotid fluid, is a preferential noninvasive proxy for intestinal immune induction.

Economic and health implications of routine CBCT examination before surgical removal of the mandibular third molar in the Danish population.

OBJECTIVES: This epidemiological study aimed to analyse economical and societal consequences in Denmark if CBCT is used routinely as a diagnostic method before removal of the mandibular third molar. Furthermore, the aim was to calculate the excess cancer incidence from this practice. METHODS: 17 representative dental clinics in the regions of Denmark were visited by two observers, who registered the total number of patients in each clinic, the number of removed mandibular third molars from patients' files together with the age and gender of these patients. The data were collected from 2008 to 2014. The total number of removed mandibular third molars in Denmark each year was derived from the collected data and information on patients' contacts with dentists from Statistics Denmark as a sum of contributions from each region. The contribution of a region was obtained as the number of removed mandibular third molars in the selected clinics in the region times the ratio of the number of patients in the selected clinics in the region to the total number of patients with contact to a general practitioner in the region in 2011. Existing knowledge on the costs for panoramic and CBCT imaging was used to calculate total costs. The cancer incidence was calculated from lifetime attributable risk curves based on linear risk assumptions. RESULTS: The selected clinics included 109,686 patients, and 1369 mandibular third molars had been surgically removed. Using data from Statistics Denmark gave an estimated annual number of removed mandibular third molars of 36,882 at a total cost of €6,633,400. The additional cancer incidence was estimated to be approximately 0.46 per year. CONCLUSIONS: The data should be used in a cost-effectiveness analysis of the clinical efficacy of CBCT imaging before removal of mandibular third molars.

Image and surgery-related costs comparing cone beam CT and panoramic imaging before removal of impacted mandibular third molars.

OBJECTIVES: The aim of this prospective clinical study was to derive the absolute and relative costs of cone beam CT (CBCT) and panoramic imaging before removal of an impacted mandibular third molar. Furthermore, the study aimed to analyse the influence of different cost-setting scenarios on the outcome of the absolute and relative costs and the incremental costs related to surgery. METHODS: A randomized clinical trial compared complications following surgical removal of a mandibular third molar, where the pre-operative diagnostic method had been panoramic imaging or CBCT. The resources implied in the two methods were measured with health economic tools. The primary outcome was total costs defined as the sum of absolute imaging costs and incremental surgery-related costs. The basic variables were capital costs, operational costs, radiological costs, radiographic costs, overheads and patient resource utilization. Differences in resources used for surgical and post-surgical management were calculated for each patient. RESULTS: Converted to monetary units, the total costs for panoramic imaging equalized €49.29 and for CBCT examination €184.44. Modifying effects on this outcome such as differences in surgery time, treatment time for complications, pre- and post-surgical medication, sickness absence, specialist treatment and hospitalization were not statistically significant between the two diagnostic method groups. CONCLUSIONS: Costs for a CBCT examination were approximately four times the costs for panoramic imaging when used prior to removal of a mandibular third molar. The use of CBCT did not change the resources used for surgery, post-surgical treatment and patient complication management.

Antithrombotic efficacy of inactivated active site recombinant factor VIIa is shear dependent in human blood.

Several studies have indicated a profound role for factor VII(a) [FVII(a)] in venous and arterial thrombogenesis. In the present study, we quantified the inhibitory efficacy of dansyl-glutamyl-glycyl-arginyl-recombinant FVIIa (DEGR-rFVIIa) on acute thrombus formation. Thrombus formation was elicited by immobilized tissue factor (TF) in a parallel-plate perfusion chamber device at blood flow conditions characterized by wall shear rates of 100 S-1 (veins) and 650 S-1 (medium-sized healthy arteries). Native human blood was drawn directly from an antecubital vein by a pump into a heparin-coated mixing device in which DEGR-rFVIIa (0.09 to 880 nmol/L final plasma concentration) or buffer was mixed homogeneously with flowing blood. Subsequently, the blood was passed over a plastic coverslip coated with TF and phospholipids in the parallel-plate perfusion chamber. Fibrin deposition, platelet-fibrin adhesion, and platelet thrombus volume triggered by this surface were measured by morphometry. DEGR-rFVIIa inhibited thrombus formation in a dose-dependent manner, but the efficacy was shear rate dependent. At a wall shear rate of 100 S-1, the IC50 (50% inhibition) was 30 nmol/L, whereas at 650 S-1, the IC50 was 0.6 nmol/L. Binding studies to immobilized TF under flow conditions using surface plasmon resonance revealed a significantly higher on-rate for DEGR-rFVIIa and FVIIa than for FVII, 2.8 x 10(5), 2.6 x 10(5), and 1.8 x 10(5) M-1 S-1, respectively. This indicates that a contributing factor to the shear-dependent efficacy may be a differential importance of on-rates at arterial and venous blood flow conditions.

A peptide sequence from the EGF-2 like domain of FVII inhibits TF-dependent FX activation.

We have found that synthetic peptides derived from the two epidermal growth factor-like domains of factor VII are inhibitors of tissue factor dependent factor X activation. Inhibition was most pronounced for a constrained sequence of amino acids corresponding to positions 91-102 of factor VII, Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys-Glu-Gin-Tyr-Cys. The biological activity appeared to be localized to the tripeptide 'motif', Glu-Gln-Tyr, within the larger sequence. The cyclic peptide was also an inhibitor of tissue factor induced coagulation of plasma, using lipidated tissue factor or tissue factor expressed on the surface of living cells. However, it did not interfere with intrinsic coagulation. Inhibition of factor X activation was dose-dependent with an IC50 value of 350 microM. Kinetic analyses revealed non-competitive inhibition with respect to factor X and suggested that the peptide sequence interferes with the factor VII/tissue factor/factor X complex formation and function. A pentapeptide analog of the putative pharmacophore was also a dose-dependent inhibitor of factor X activation with an IC50 value of 560 microM, but the tripeptide, Glu-Gin-Tyr, alone was without effect. Our results suggest a direct role for the second epidermal growth factor-like domain of factor VII, and in particular its loop I, in the formation and function of the factor VII/tissue factor/factor X complex.

Retinyl ester storage is altered in liver stellate cells and in HL60 cells transfected with cellular retinol-binding protein type I.

It is suggested that cellular retinol-binding proteins are important for intracellular metabolism of retinol. Retinol bound to cellular retinol-binding proteins may be esterified with long chain fatty acids by the enzyme lecithin: retinol acyltransferase or may be oxidized to retinoic acid metabolites used in the mechanism of action of vitamin A. The aim of this present report was to determine whether altered levels of cellular retinol-binding protein type I influenced retinol storage and activation. Two different cell types have been examined after transfection with vectors producing sense or antisense mRNA for cellular retinol-binding protein type I. When HL60 cells were transfected with the expression vector for sense cellular retinol-binding protein type I high amounts of cellular retinol-binding protein type I mRNA and protein were produced. We observed that HL60 cells esterified less retinol than control cells without cellular retinol-binding protein type I. Cellular retinol-binding protein type I had, however, no effects on the proliferation or differentiation of HL60 cells by retinoids. Liver stellate cells transfected with the vector for sense cellular retinol-binding protein type I esterified more retinol than cells transfected with the expression vector for antisense cellular retinol-binding protein type I, while retinol esterification in control cells was intermediate. In conclusion, our data show that cellular retinol-binding protein type I influences retinol esterification both in liver stellate cells and in HL60 cells.

Characterisation of cell-surface procoagulant activities using a microcarrier model.

A novel model is described for characterisation of cell-surface procoagulant activities and their inhibitors. Microcarrier beads were used to present living cells to recalcified blood plasma in the stirred measuring wells of an electromagnetic coagulometer. By this means the procoagulant activity on the surface of the cells could be automatically determined as clotting time. Procoagulant activity was investigated on normal and transformed cells, and representing hemopoietic, endothelial, muscle and connective tissue phenotypes. The procoagulant activity on each cell type was characterised by the use of specifically immunodepleted plasmas and specific inhibitors, including monoclonal antibodies. The predominant cell surface trigger of coagulation found in this series was tissue factor, and only blood monocytes provided some evidence for direct activation of factor X independent of FVII. Human ECV304 transformed endothelial cells were more closely studied as representative of a cell type constitutively expressing procoagulant. Coagulation mediated by ECV304 cells was found to be strictly dependent on tissue factor, as shown by an inhibitory monoclonal antibody, and on coagulation factors V, VII and X. ECV304 procoagulant activity was strongly inhibited by active-site-inactivated FVIIa, a synthetic peptide inhibitor of FXa (Tenstop) and the thrombin inhibitor, hirudin. While not appropriate for routine clinical assessment of coagulation factor function, we have found this model to be valuable in characterising the procoagulant activity on different cell types and particularly useful as a drug discovery tool in the search for new anticoagulants.

Dietary supplement of omega-3 fatty acids has no effect on acute collagen-induced thrombus formation in flowing native blood.

We have studied the effect of a daily supplement of 2.4 omega-3 fatty acids (omega 3 FAs) to 16 healthy men on acute collagen-induced thrombus formation in flowing non-anticoagulated blood. The supplement was formulated as Triomar capsules, containing 60% omega-3 FAs with an eicosapentaenoic/docosahexaenoic acid ratio of 3/2. A parallel-plate perfusion chamber device was used to study thrombus formation prior to and after 3 months of omega-3 FAs supplement. The wall shear rates at the thrombogenic surface were 650, 2,600 and 10,500 s-1, which are typical for small arteries, slightly stenosed arteries and severely stenosed arteries, respectively. For the latter situation a parallel-plate perfusion chamber with an eccentric stenosis occluding 80% of the cross-sectional area of the blood flow channel was used. The dietary supplement of omega-3 FAs did not cause significant changes in platelet adhesion to collagen or in thrombus volume. However, fibrin deposition was reduced by 34% (P < 0.03) at the highest shear condition (stenosis). Plasma fibrinogen was reduced by 18% (P < 0.0006). Changes in serum concentration of triglycerides, total-cholesterol, LDL- and HDL-cholesterol were not significant. Our data suggest that a moderate intake of omega-3 FAs provides virtually no protection against acute platelet-dependent thrombus formation, irrespective of the shear conditions. However, the significant reduction in plasma levels of fibrinogen following dietary supplementation of omega-3 FAs may be important, since high levels of fibrinogen is associated with cardiovascular disease and thrombosis.

Reduced thrombus formation in native blood of homozygous factor VII-deficient patients at high arterial wall shear rate.

Inhibition of thrombin formation in flowing native blood reduces thrombus formation on subendothelium, dacron, or collagen fibrils at arterial wall shear rates of 450 to 650 s-1. In the present study, we have investigated the role of low levels of factor VII (FVII) in thrombus formation on collagen fibrils at arterial wall shear rates of 650 s-1 (coronary arteries), 2,600 s-1 (mildly stenosed arteries), and 10,510 s-1 (severely stenosed arteries) in parallel-plate perfusion chambers. In the perfusion chamber with the highest wall shear rate, thrombus formation took place at the apex of an eccentric stenosis, which reduced the cross-sectional area of the blood flow channel by 80%, thus simulating thrombus formation at an atherosclerotic plaque rupture. Native blood from 21 healthy volunteers and 12 homozygous FVII-deficient patients was drawn by a pump directly from an antecubital vein over a surface of fibrillar collagen positioned in the respective perfusion chambers. The patients had FVII coagulant activities ranging from 1.3% to 4.5% and FVII antigen levels of 16% to 23% of normal. Immunoaffinity purification of the patients' FVII followed by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and immunoblotting showed a protein with similar molecular mass as normal FVII. In the perfusion studies, a reduction in thrombus volume of 54% of normal (P < .007) at 10,510 s-1 was observed. The deposition of fibrin on the thrombogenic surface and the plasma level of fibrinopeptide A (FPA) in blood samples collected distal to the perfusion chamber were concomitantly reduced (P < .002 and P < .04, respectively). The plasma FPA level was also reduced at 2,600 s-1 (P < .04), but not at 650 s-1. However, at the lower shear conditions, the thrombus volume and the fibrin deposition were within the ranges observed in normal blood. The platelet-collagen adhesion was not affected at any of the three shear conditions. Thus, low plasma levels of FVII result in significantly less formation of thrombin and fibrin in and around growing platelet masses at high shear condition. This may weaken the thrombus stability and reduce platelet recruitment, thereby lowering thrombus volume. In support of this theory, one patient with afibrinogenemia had an 83% reduction in thrombus volume at this high shear condition.

High-dose retinol in children with acute myelogenous leukemia in remission.

In a single-institution study, 23 consecutive children with acute myeloid leukemia (AML) have been treated with a protocol including doxorubicin, cytarabine and 6-thioguanine as induction therapy, followed by four courses of high-dose cytarabine as consolidation. Total duration of chemotherapy was 6-8 months from diagnosis. 21 out of the 23 children achieved complete remission. During remission, the children received 52 mumol (50,000 I.U.) retinol as retinyl palmitate per square meter daily. 14 of the 21 children are still in their first remission with a mean observation time of 36 months. In our study retinyl ester given in doses up to 30 times the recommended daily allowances has not caused any clinical or biochemical side effect for up to 4 yr of therapy.

Intestinal retinol esterification and serum retinol in children with cystic fibrosis.

Children with cystic fibrosis (CF) often have a poor vitamin A status. We found in the present work a reduced level of serum retinol in older children with this disease. As retinol enters intestinal lymph as retinyl esters, the enzyme acyl-CoA:retinol acyltransferase (ARAT) may be of importance for retinol absorption. We have assayed ARAT activity in duodenal mucosal homogenate from children with CF. There was a large variation within the group. However, mean ARAT activity was not significantly decreased, as compared with controls. Thus, the lack of pancreatic enzymes probably is the main reason for reduced vitamin A absorption in CF. In celiac disease and in lactose intolerance--both mucosal disorders--a significant reduction of mean ARAT activity was found.

Liver retinoids and retinol esterification in fetal and pregnant rats at term.

Retinol esterification in fetal rats and their mothers at term was studied in liver microsomes. The esterification rate was 0.28 +/- 0.05 nmol ester formed per milligram protein per minute, a value somewhat lower than that found in their mothers (0.44 +/- 0.11). The fetuses had significant amounts of liver retinoids. Analysis by high-performance liquid chromatography showed that the retinoid store consisted mainly of retinyl ester both in fetal and adult rat livers, but the fetal livers had higher percentages of free retinol and retinyl oleate than the adult livers. The presence of retinol esterification and a retinyl ester store in fetal rat liver at term is in accordance with the view that retinol brought to liver on retinol-binding protein can be taken up and retained there.

The activity of acyl CoA: retinol acyltransferase in the rat: variation with vitamin A status.

Retinol esterification in the small intestine, liver and kidney of rats given a normal diet or a vitamin-A-free diet and of rats given large doses of vitamin A was studied. The active enzyme is a microsomal acyl CoA:retinol acyl transferase (ARAT). In the small intestine ARAT activity was 0.37 nmol ester/mg microsomal protein per min. Large doses of vitamin A increased the activity significantly, while the enzyme activity in the vitamin-A-deficient rats was in the range of that of the controls. Retinoic acid in physiological doses (0.064 mg three times per week) had no influence on ARAT activity. In the liver, ARAT activity of the controls was 0.58 nmol ester/mg microsomal protein per min. The activity was increased after large doses of vitamin A. It was not significantly reduced in vitamin-A-deficient animals. The kidney had a low, but significant ARAT activity, both in normal and vitamin-A-deficient animals and after large doses of vitamin A (range 0.08-0.14 nmol ester/mg microsomal protein per min). The vitamin-A-esterifying enzyme in the small intestine and liver of the rat seems to be influenced by the amount of retinol in the diet.

Microsomal esterification of retinol in human liver.

Recent work has shown that esterification of retinol in microsomes from rat liver, mammary gland and small intestine and from human small intestine is catalyzed by an acyl CoA: retinol acyl transferase (ARAT). The current study demonstrates ARAT activity in human liver microsomes. At optimal incubation conditions the rate of retinyl ester formation due to ARAT (0.37 +/- 0.31 nmole ester formed X mg microsomal protein-1 X minute-1, mean +/- SD, n = 6) suggests that the enzyme is of physiological importance.

Influence of diets on acyl-CoA:cholesterol acyltransferase and on acyl-CoA:retinol acyltransferase in villous and crypt cells from rat small intestinal mucosa and in the liver.

Cholesterol and retinol are both esterified with long-chain fatty acid within the mucosal cells of the small intestine. The reactions are catalyzed by microsomal acyl-CoA:cholesterol and acyl-CoA:retinol acyltransferases (EC 2.3.1.26, and EC 2.3.1.-, respectively). To gain more insight into the physiological importance of these acyltransferases, they were studied in villous and crypt cells from rats either fasting or on diets which varied in fat and cholesterol content. Both enzymes had a higher activity in villous than in crypt cells. The activities in villous cells varied with feeding and fasting and the composition of diet when the animals were killed postprandially. Acyl-CoA:cholesterol acyltransferase activity went up upon cholesterol feeding whereas retinol acyltransferase in the mucosa was reduced by high-fat diets. The liver cholesterol acyltransferase activity varied with diet, it increased with both cholesterol and fat feeding, whereas retinol acyltransferase activity remained relatively constant. The results obtained suggest that different diets are of importance for cholesterol and retinol acyltransferase activities both in the intestinal mucosa and in the liver. The variation in activities of the two acyltransferases suggests that they may be different enzymes.

Retinol esterification by microsomes from the mucosa of human small intestine. Evidence for acyl-Coenzyme A retinol acyltransferase activity.

The mechanism of the intestinal esterification of retinol has been obscure. Recently, an acyl-Coenzyme A (CoA):retinol acyltransferase (ARAT) was found in rat intestinal microsomes, and experiments were therefore conducted to determine whether a corresponding enzyme exists in human small intestine. When microsomes were incubated with [3H]retinol and palmitoyl-CoA, or retinol and [1-14C]palmitoyl-CoA, radioactive retinyl palmitate was formed as identified by alumina column chromatography and reverse-phase high-pressure liquid chromatography. Heating the microsomes for 30 min at 60 degrees C resulted in loss of activity. The esterification was negligible without exogenous acyl-CoA and markedly stimulated by palmitoyl-, oleoyl-, and stearoyl-CoA in concentrations up to 20 microM. The acyl-CoA was successfully replaced by an acyl-CoA generating system, but not by unactivated palmitate (2.5-200 microM). The assay was dependent on the presence of albumin with optimum activity at 2-10 mg/ml. The optimal retinol concentration was 20-30 microM and pH approximately 7.4. The esterifying activity was completely inhibited by 8 mM of taurocholate and to 90% by 1 mM of 5,5'-dithiobis(2-nitrobenzoic acid). Activity was found throughout the small intestine. In jejunum the rate of retinol esterification was: 3.44 +/- 2.24 nmol [3H]retinyl ester formed . mg microsomal protein-1 . min-1 (mean +/- SD, n = 12). The corresponding activity in whole homogenates of biopsies were 1.17 +/- 0.28 (n = 8). It is concluded that human small intestine contains a microsomal acyl-CoA:retinol acyltransferase. Due to its high activity in vitro this enzyme is likely to be responsible for the intestinal esterification of retinol.

Acyl CoA:retinol acyltransferase in rat small intestine: its activity and some properties of the enzymic reaction.

The present study was conducted to examine whether the intestinal esterification of retinol could be due to a microsomal acyl-CoA transferase. When the 'microsomal fraction' of rat mucosa was incubated with [3H]retinol and palmitoyl-CoA or oleoyl-CoA, [3H]retinyl esters were formed as identified by alumina column chromatography and reverse phase high-pressure liquid chromatography (HPLC). Unlabeled retinol and [1-14C]palmitoyl-CoA yielded retinyl[1-14C]palmitate. The esterifying activity was lost when microsomes were heated at 60 degrees C for 30 min. Only negligible activity was observed without exogenous acyl-CoA while 10-20 muM gave optimum activity provided that 2-5 mg/ml of albumin was present. Replacement of acyl-CoA by palmitate gave no esterification, indicating that the activity was not a reversed hydrolase reaction. Optimum pH was 7.1-7.6 and optimal concentration of retinol was 15 muM. With palmitoyl-CoA, the formation of retinyl ester was 1.00 +/- 0.26 nmol . mg protein-1 . min-1 (x +/- SD, n = 4) in rats killed postprandially versus 2.06 +/- 0.66 (n = 5) after 36 hr of fasting. Oleoyl-CoA gave lower activity: 0.52 +/- 0.14 and 1.41 +/- 0.36, respectively. The variation with feeding and fasting was significant (P less than 0.05) and corresponded to that of the intestinal acyl-CoA:cholesterol acryltransferase (ACAT). Inhibition of retinol esterification was observed with taurocholate and the thiol-blocking agent 5,5'-dithiobis (2-nitrobenzoic acid). The data show that rat intestinal microsomes catalyze the formation of retinyl esters by an acyl-CoA:retinol acyltransferase with several properties in common with ACAT located in the same subcellular fraction.