Activation of T cells by carbamazepine and carbamazepine metabolites

BACKGROUND: T-cell-mediated hypersensitivity is a rare but serious manifestation of drug therapy. OBJECTIVES: To explore the mechanisms of drug presentation to T cells and the possibility that generation of metabolite-specific T cells may provoke cross-sensitization between drugs. METHODS: A lymphocyte transformation test was performed on 13 hypersensitive patients with carbamazepine, oxcarbazepine, and carbamazepine metabolites. Serial dilution experiments were performed to generate drug (metabolite)-specific T-cell clones to explore the structural basis of the T-cell response and mechanisms of antigen presentation. 3-Dimensional energy-minimized structures were generated by using computer modeling. The role of drug metabolism was analyzed with 1-aminobenzotriazole. RESULTS: Lymphocytes and T-cell clones proliferated with carbamazepine, oxcarbazepine, and some (carbamazepine 10,11 epoxide, 10-hydroxy carbamazepine) but not all stable carbamazepine metabolites. Structure activity studies using 29 carbamazepine (metabolite)-specific T-cell clones revealed 4 patterns of drug recognition, which could be explained by generation of preferred 3-dimensional structural conformations. T cells were stimulated by carbamazepine (metabolites) bound directly to MHC in the absence of processing. The activation threshold for T-cell proliferation varied between 5 minutes and 4 hours. 1-Aminobenzotriazole, which inhibits cytochrome P450 activity, did not prevent carbamazepine-related T-cell proliferation. Substitution of the terminal amine residue of carbamazepine with a methyl group diminished T-cell proliferation. CONCLUSION: These data show that carbamazepine and certain stable carbamazepine metabolites stimulate T cells rapidly via a direct interaction with MHC and specific T-cell receptors. CLINICAL IMPLICATIONS: Some patients with a history of carbamazepine hypersensitivity possess T cells that cross-react with oxcarbazepine, providing a rationale for cross-sensitivity between the 2 drugs.

Evaluation of magnetic resonance colonography at 3.0 Tesla regarding diagnostic accuracy and image quality

OBJECTIVES: To assess magnetic resonance (MR)-colonography (MRC) for detection of colorectal lesions using two different T1w three-dimensional (3D)-gradient-recalled echo (GRE)-sequences and integrated parallel data acquisition (iPAT) at a 3.0 Tesla MR-unit. MATERIALS AND METHODS: In this prospective study, 34 symptomatic patients underwent dark lumen MRC at a 3.0 Tesla unit before conventional colonoscopy (CC). After colon distension with tap water, 2 high-resolution T1w 3D-GRE [3-dimensional fast low angle shot (3D-FLASH), iPAT factor 2 and 3D-volumetric interpolated breathhold examination (VIBE), iPAT 3] sequences were acquired without and after bolus injection of gadolinium. Prospective evaluation of MRC was performed. Image quality of the different sequences was assessed qualitatively and quantitatively. The findings of the same day CC served as standard of reference. RESULTS: MRC identified all polyps >5 mm (16 of 16) in size and all carcinomas (4 of 4) correctly. Fifty percent of the small polyps 0.6). CONCLUSIONS: MRC using 3D-GRE-sequences and iPAT is feasible at 3.0 T-systems. The high-resolution 3D-FLASH was slightly preferred over the 3D-VIBE because of better image quality, although both used sequences showed no statistical significant difference.

Quantitative comparison of 3 enamel-stripping devices in vitro: how precisely can we strip teeth?

INTRODUCTION In this in-vitro study, we aimed to investigate the predictability of the expected amount of stripping using 3 common stripping devices on premolars. METHODS One hundred eighty extracted premolars were mounted and aligned in silicone. Tooth mobility was tested with Periotest (Medizintechnik Gulden, Modautal, Germany) (8.3 ± 2.8 units). The selected methods for interproximal enamel reduction were hand-pulled strips (Horico, Hapf Ringleb & Company, Berlin, Germany), oscillating segmental disks (O-drive-OD 30; KaVo Dental, Biberach, Germany), and motor-driven abrasive strips (Orthofile; SDC Switzerland, Lugano-Grancia, Switzerland). With each device, the operator intended to strip 0.1, 0.2, 0.3, or 0.4 mm on the mesial side of 15 teeth. The teeth were scanned before and after stripping with a 3-dimensional laser scanner. Superposition and measurement of stripped enamel on the most mesial point of the tooth were conducted with Viewbox software (dHal Software, Kifissia, Greece). The Wilcoxon signed rank test and the Kruskal-Wallis test were applied; statistical significance was set at alpha ≤ 0.05. RESULTS Large variations between the intended and the actual amounts of stripped enamel, and between stripping procedures, were observed. Significant differences were found at 0.1 mm of intended stripping (P ≤ 0.05) for the hand-pulled method and at 0.4 mm of intended stripping (P ≤ 0.001 to P = 0.05) for all methods. For all scenarios of enamel reduction, the actual amount of stripping was less than the predetermined and expected amount of stripping. The Kruskal-Wallis analysis showed no significant differences between the 3 methods. CONCLUSIONS There were variations in the stripped amounts of enamel, and the stripping technique did not appear to be a significant predictor of the actual amount of enamel reduction. In most cases, actual stripping was less than the intended amount of enamel reduction.

University and school students' motivation for effortful thinking: Factor structure, reliability, and validity of the German Need for Cognition Scale

Need for cognition (NFC) reflects a relatively stable trait regarding the degree to which one enjoys and engages in cognitive endeavors. We examined whether the previously demonstrated one-dimensional structure of the German NFC Scale could be replicated in three samples of undergraduates and secondary school students. Moreover, we investigated the test-retest reliability of the German NFC Scale, which has not yet been tested. Further, we investigated whether the scale would be valid in a sample of secondary school students. Multigroup confirmatory factor analyses established the one-dimensional factor structure of the long form as well as the short form of the German NFC Scale for undergraduates (N = 559), students of academic track secondary schools (German Gymnasium; N = 555), and students of vocational track secondary schools (German Realschule; N = 486). The scale proved to have a high test-retest reliability in a university student sample (N = 43). For secondary school students, we again found a high test-retest reliability (N = 157), and also found the scale to be valid (N = 181).

Expanding the Mutation Spectrum Affecting αIIbβ3 Integrin in Glanzmann Thrombasthenia: Screening of the ITGA2B and ITGB3 Genes in a Large International Cohort.

We report the largest international study on Glanzmann thrombasthenia (GT), an inherited bleeding disorder where defects of the ITGA2B and ITGB3 genes cause quantitative or qualitative defects of the αIIbβ3 integrin, a key mediator of platelet aggregation. Sequencing of the coding regions and splice sites of both genes in members of 76 affected families identified 78 genetic variants (55 novel) suspected to cause GT. Four large deletions or duplications were found by quantitative real-time PCR. Families with mutations in either gene were indistinguishable in terms of bleeding severity that varied even among siblings. Families were grouped into type I and the rarer type II or variant forms with residual αIIbβ3 expression. Variant forms helped identify genes encoding proteins mediating integrin activation. Splicing defects and stop codons were common for both ITGA2B and ITGB3 and essentially led to a reduced or absent αIIbβ3 expression; included was a heterozygous c.1440-13_c.1440-1del in intron 14 of ITGA2B causing exon skipping in 7 unrelated families. Molecular modeling revealed how many missense mutations induced subtle changes in αIIb and β3 domain structure across both subunits thereby interfering with integrin maturation and/or function. Our study extends knowledge of Glanzmann thrombasthenia and the pathophysiology of an integrin. This article is protected by copyright. All rights reserved.

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.

Critical roles for Rac GTPases in T-cell migration to and within lymph nodes

Naive T cells continuously recirculate between secondary lymphoid tissue via the blood and lymphatic systems, a process that maximizes the chances of an encounter between a T cell and its cognate antigen. This recirculation depends on signals from chemokine receptors, integrins, and the sphingosine-1-phosphate receptor. The authors of previous studies in other cell types have shown that Rac GTPases transduce signals leading to cell migration and adhesion; however, their roles in T cells are unknown. By using both 3-dimensional intravital and in vitro approaches, we show that Rac1- and Rac2-deficient T cells have multiple defects in this recirculation process. Rac-deficient T cells home very inefficiently to lymph nodes and the white pulp of the spleen, show reduced interstitial migration within lymph node parenchyma, and are defective in egress from lymph nodes. These mutant T cells show defective chemokine-induced chemotaxis, chemokinesis, and adhesion to integrin ligands. They have reduced lateral motility on endothelial cells and transmigrate in-efficiently. These multiple defects stem from critical roles for Rac1 and Rac2 in transducing chemokine and sphingosine-1-phosphate receptor 1 signals leading to motility and adhesion.

Neuroelectromagnetic correlates of perceptual closure processes

Perceptual closure refers to the coherent perception of an object under circumstances when the visual information is incomplete. Although the perceptual closure index observed in electroencephalography reflects that an object has been recognized, the full spatiotemporal dynamics of cortical source activity underlying perceptual closure processing remain unknown so far. To address this question, we recorded magnetoencephalographic activity in 15 subjects (11 females) during a visual closure task and performed beamforming over a sequence of successive short time windows to localize high-frequency gamma-band activity (60–100 Hz). Two-tone images of human faces (Mooney faces) were used to examine perceptual closure. Event-related fields exhibited a magnetic closure index between 250 and 325 ms. Time-frequency analyses revealed sustained high-frequency gamma-band activity associated with the processing of Mooney stimuli; closure-related gamma-band activity was observed between 200 and 300 ms over occipitotemporal channels. Time-resolved source reconstruction revealed an early (0–200 ms) coactivation of caudal inferior temporal gyrus (cITG) and regions in posterior parietal cortex (PPC). At the time of perceptual closure (200–400 ms), the activation in cITG extended to the fusiform gyrus, if a face was perceived. Our data provide the first electrophysiological evidence that perceptual closure for Mooney faces starts with an interaction between areas related to processing of three-dimensional structure from shading cues (cITG) and areas associated with the activation of long-term memory templates (PPC). Later, at the moment of perceptual closure, inferior temporal cortex areas specialized for the perceived object are activated, i.e., the fusiform gyrus related to face processing for Mooney stimuli.

Spatial and functional relationships between air conduits and blood capillaries in the pulmonary gas exchange tissue of adult and developing chickens

The documented data regarding the three-dimensional structure of the air capillaries (ACs), the ultimate sites of gas exchange in the avian lung is contradictory. Further, the mode of gas exchange, described as cross-current has not been clearly elucidated. We studied the temporal and spatial arrangement of the terminal air conduits of the chicken lung and their relationship with the blood capillaries (BCs) in embryos as well as the definitive architecture in adults. Several visualization techniques that included corrosion casting, light microscopy as well as scanning and transmission electron microscopy were used. Two to six infundibulae extend from each atrium and give rise to numerous ACs that spread centrifugally. Majority of the ACs are tubular structures that give off branches, which anastomose with their neighboring cognates. Some ACs have globular shapes and a few are blind-ending tapering tubes. During inauguration, the luminal aspects of the ACs are characterized by numerous microvillus-like microplicae, which are formed during the complex processes of cell attenuation and canalization of the ACs. The parabronchial exchange BCs, initially inaugurated as disorganized meshworks, are reoriented via pillar formation to lie predominantly orthogonal to the long axes of the ACs. The remodeling of the retiform meshworks by intussusceptive angiogenesis essentially accomplishes a cross-current system at the gas exchange interface in the adults, where BCs form ring-like patterns around the ACs, thus establishing a cross-current system. Our findings clarify the mode of gas exchange in the parabronchial mantle and illuminate the basis for the functional efficiency of the avian lung.

Feasibility of image-guided radiotherapy based on tomotherapy for the treatment of locally advanced anal carcinoma

The standard of care for locally advanced anal cancer has been concurrent chemoradiation. However, conventional treatment with 3-dimensional radiotherapy is associated with significant toxicity. The feasibility of new radiotherapy techniques such as image-guided radiotherapy (IGRT) in combination with chemotherapy for the treatment of this malignancy was assessed.

Computer-assisted femoral head-neck osteochondroplasty using a surgical milling device an in vitro accuracy study

Surgical navigation might increase the safety of osteochondroplasty procedures in patients with femoroacetabular impingement. Feasibility and accuracy of navigation of a surgical reaming device were assessed. Three-dimensional models of 18 identical sawbone femora and 5 cadaver hips were created. Custom software was used to plan and perform repeated computer-assisted osteochondroplasty procedures using a navigated burr. Postoperative 3-dimensional models were created and compared with the preoperative models. A Bland-Altmann analysis assessing α angle and offset ratio accuracy showed even distribution along the zero line with narrow confidence intervals. No differences in α angle and offset ratio accuracy (P = 0.486 and P = 0.2) were detected between both observers. Planning and conduction of navigated osteochondroplasty using a surgical reaming device is feasible and accurate.

Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

Statistical modeling of the eye for multimodal treatment planning for external beam radiation therapy of intraocular tumors

Ocular anatomy and radiation-associated toxicities provide unique challenges for external beam radiation therapy. For treatment planning, precise modeling of organs at risk and tumor volume are crucial. Development of a precise eye model and automatic adaptation of this model to patients' anatomy remain problematic because of organ shape variability. This work introduces the application of a 3-dimensional (3D) statistical shape model as a novel method for precise eye modeling for external beam radiation therapy of intraocular tumors.

Evaluation of radial distribution of cartilage degeneration and necessity of pre-contrast measurements using radial dGEMRIC in adults with acetabular dysplasia

Background The purpose of the present study was to investigate the radial distribution patterns of cartilage degeneration in dysplastic hips at different stages of secondary osteoarthritis (OA) by using radial delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC), and to assess whether pre-contrast measurements are necessary. Methods Thirty-five hips in 21 subjects (mean age ± SD, 27.6 ± 10.8 years) with acetabular dysplasia (lateral CE angle < 25°) were studied. Severity of OA was assessed on radiographs using Tönnis grading. Pre- (T1pre) and post-contrast T1 (T1Gd) values were measured at 7 sub-regions on radial reformatted slices acquired from a 3-dimensional (3D) T1 mapping sequence using a 1.5 T MR scanner. Values of radial T1pre, T1Gd and ΔR1 (1/T1Gd - 1/T1pre) of subgroups with different severity of OA were compared to those of the subgroup without OA using nonparametric tests, and bivariate linear Pearson correlations between radial T1Gd and ΔR1 were analyzed for each subgroup. Results Compared to the subgroup without OA, the subgroup with mild OA was observed with a significant decrease in T1Gd in the anterosuperior to superior sub-regions (mean, 476 ~ 507 ms, p = 0.026 ~ 0.042) and a significant increase in ΔR1 in the anterosuperior to superoposterior and posterior sub-regions (mean, 0.93 ~ 1.37 s-1, p = 0.012 ~ 0.042). The subgroup with moderate to severe OA was observed with a significant overall decrease in T1Gd (mean, 404 ~ 452 ms, p = 0.001 ~ 0.020) and an increase in ΔR1 (mean, 1.17 ~1.69 s-1, p = 0.001 ~ 0.020). High correlations were observed between radial T1Gd and ΔR1 for all subgroups (r = −0.869 ~ −0.944, p < 0.001). Conclusions Radial dGEMRIC without pre-contrast measurements is useful for evaluating different patterns of cartilage degeneration in the entire hip joint of patients with hip dysplasia, particularly for those in early stages of secondary OA.