Efficacy and safety of a T-type calcium channel blocker in patients with neuropathic pain: A proof-of-concept, randomized, double-blind and controlled trial.

BACKGROUND: T-type calcium channels have been shown to play an important role in the initiation and maintenance of neuropathic pain and represent a promising therapeutic target for new analgesic treatments. Ethosuximide (ETX), an anticonvulsant and a T-type channel blocker has shown analgesic effect in several chronic pain models but has not yet been evaluated in patients with neuropathic pain. METHODS: This proof-of-concept, multicentre, double-blind, controlled and randomized trial compared the efficacy and safety of ETX (given as add-on therapy) to an inactive control (IC) in 114 patients with non-diabetic peripheral neuropathic pain. After a 7-day run-in period, eligible patients aged over 18 years were randomly assigned (1:1) to ETX or IC for 6 weeks. The primary outcome was the difference between groups in the pain intensity (% of change from the baseline to end of treatment) assessed in the intention-to-treat population. This study is registered with EudraCT (2013-004801-26) and ClinicalTrials.gov (NCT02100046). RESULTS: The study was stopped during the interim analysis due to the high number of adverse events in the active treatment group. ETX failed to reduce total pain and showed a poor tolerance in comparison to IC. In the per-protocol analysis, ETX significantly reduced pain intensity by 15.6% (95% CI -25.8; -5.4) from baseline compared to IC (-7.8%, 95% CI -14.3; -1.3; p = 0.033), but this result must be interpreted with caution because of a small subgroup of patients. CONCLUSION: Ethosuximide did not reduce the severity of neuropathic pain and induces, at the doses used, many adverse events. SIGNIFICANCE: This article shows that ETX is not effective to treat neuropathic pain. Nevertheless, per-protocol analysis suggests a possible analgesic effect of ETX. Thus, our work adds significant knowledge to preclinical and clinical data on the benefits of T-type calcium channel inhibition for the treatment of neuropathic pain.

Laboratory Studies of Nonlinear Optical Signals for Caries Detection.

Multiphoton confocal microscopy and nonlinear spectroscopy are used to investigate the caries process in dentin. Although dentin is a major calcified tissue of the teeth, its organic phase comprises type I collagen fibers. Caries drive dentin demineralization and collagen denaturation. Multiphoton microscopy is a powerful imaging technique: the biological materials are transparent to infrared frequencies and can be excited to penetration depths inaccessible to 1-photon confocal microscopy. The laser excitation greatly reduces photodamage to the sole focal region, and the signal-to-noise ratio is improved significantly. The method has been used to follow pathologic processes involving collagen fibrosis or collagen destruction based on their 2-photon excited fluorescence (2PEF) emission and second harmonic generation (SHG). Combining multiphoton imaging with nonlinear spectroscopy, we demonstrate that both 2PEF and SHG intensity of human dentin are strongly modified during the tooth caries process, and we show that the ratio between SHG and 2PEF signals is a reliable parameter to follow dental caries. The ratio of the SHG/2PEF signals measured by nonlinear optical spectroscopy provides valuable information on the caries process, specifically on the degradation of the organic matrix of dentin. The goal is to bring these nonlinear optical signals to clinical application for caries diagnosis.

Retrograde Trafficking Inhibitor of Shiga Toxins Reduces Morbidity and Mortality of Mice Infected with Enterohemorrhagic Escherichia coli.

The most deadly outbreak of Escherichia coli O104:H4 occurred in Europe in 2011. Here, we evaluated the effects of the retrograde trafficking inhibitor Retro-2(cycl) in a murine model of E. coli O104:H4 infection. Systemic treatment with Retro-2(cycl) significantly reduced body weight loss and improved clinical scores and survival rates for O104:H4-infected mice. The present data established that Retro-2(cycl) contributes to the protection of mice against O104:H4 infection and may represent a novel approach to limit Shiga toxin-producing Escherichia coli (STEC)-induced toxicity.

High discordance rates between sub-areolar and peri-tumoural breast lymphoscintigraphy.

OBJECTIVE: To test the hypothesis that sub-areolar (SA) lymphoscintigraphy (LSG) identifies the same sentinel node as peri-tumoural (PT) injections. BACKGROUND: It is commonly believed that all LSG techniques will identify the same sentinel lymph nodes (SLN) draining the breast. Hybrid imaging technology (SPECT/CT) allows accurate identification of the exact location of SLNs. Using SPECT/CT SA and PT LSG techniques were compared. METHOD: In a multi-centre trial 39 patients sequentially underwent LSG (SA followed by PT) separated by 2-7 days. Patients were referred by 4 surgeons to 3 LSG centres, with standardization of isotope (99mTc-antimony sulfide colloid), LSG and SPECT/CT evaluation techniques. LSG were evaluated for SLN concordance and degree of discordance in the axilla and internal mammary nodes (IMN). RESULTS: 39 eligible patients, median age 62 years, were recruited. Successful axillary SLN mapping for SA and PT injection techniques was 87% and 95% respectively. Successful internal mammary SLN mapping occurred with SA and PT LSG in 5% and 36% respectively. Discordance was identified in the IMN (39%) and axilla (21%), with an overall rate of discordance between SA and PT LSG of 56%. CONCLUSIONS: There is a high level of discordance in the localization of SLN by these commonly used LSG injection techniques. This discordance has implications for accuracy of axillary and extra-axillary staging and could impact on patient outcome.

Measuring the international spreading of the knowledge produced by French dental master theses.

We estimated the international spreading of the knowledge produced by French dental master theses by searching for corresponding publications, either as articles in Medline-indexed journals or abstracts of IADR meetings published in the Journal of Dental Research. From the 634 theses defended in 2010 in the 16 French odontology faculties, we found only one article, in a journal without impact factor, and six abstracts, over a 3-year period (2009-2011). This corresponds to a spreading rate of 0.6%. The participation rate of French odontology faculties at IADR meetings varies from 37.5% to 81.3% depending on the year. Although there are very few studies available on the matter, it appears that this international spreading rate is much lower than both the one found by Nieminen for odontology in Finland (8.2%) and typical spreading rates of medical theses (from 6% to 41% depending on the country). This great discrepancy could be explained by the lack of specific training provided to the students; the low awareness and little engagement of the students themselves, usually more concerned with their own private practice work; and the inadequate involvement of their supervisors. To tackle the lack of appropriate training, we suggest that a specific course on scientific writing should be offered to the students in the last year of their dental master studies.

Clinical evaluation of the Carisolv chemomechanical caries removal technique according to the site/stage concept, a revised caries classification system.

The objective of the present study was to assess the efficiency and benefit of a chemomechanical system for carious dentin removal, Carisolv, in general practice. A revised caries classification, the site/stage concept, was used to describe the clinical situations of all carious lesions treated. The study was performed by 12 investigators, and 120 carious lesions were treated with Carisolv. Sixty percent of the cases were treated without anaesthesia, and we found a significant correlation between chemomechanical treatment without anaesthesia and absence of pain ( P=0.01). In 78.3% of the cases, carious dentin was totally removed with Carisolv, and in 21.7%, the dentin treatment was completed by drilling. In cases performed with Carisolv alone, the time required to remove carious dentin was 11.1+/-9.51 min (mean+/-SD). Treatment time was equivalent for all sites and increased significantly with each successive stage of lesion progression ( P<0.001). In 82.5% of cases, the clinicians were satisfied with Carisolv, and in 99.2%, so were the patients. We conclude that, using clinical examination methods, Carisolv seems to remove carious dentin at all sites and stages of carious lesions but must be made more efficient for use in general practice.

[Patients, practitioners, faculty and dental esthetics: the same level of perception?]. / Patients, praticiens, enseignants et esthétique dentaire: un même niveau de perception?

The aim of this study was to evaluate the importance of the dental aesthetic for the patients, the dental surgeon and the dental teachers by the study of the consultation reason, the complaints, the post-university congress program, the practical program of the dental students and the programs of the IADR congress. It appears that in odontology, patients ask strongly for aesthetic care, in consultation and litigation. The content of congress and professional literature shows that dental surgeons answer to that request. Only the practical teaching was a bit less but it was recently modify. The research workers are also very interesting for aesthetic care.

Microleakage and penetration depth of three types of materials in fissure sealant: self-etching primer vs etching: an in vitro study.

Clinical preventive procedures must be done after a risk assessment. One of the risk factors is the occlusal morphology of the posterior teeth. These caries-free fissures must be sealed. This first in vitro experimentation of the study evaluated the microleakage and the penetration depth of three types of materials by Vivadent: Helioseal F, Tetric, Tetric Flow. The teeth were etched with phosphoric acid and bonded using a one bottle bonding in order to determine the best material for the sealing of the fissure. The depth of penetration of fuschine dye as well as that of the tested material was measured with a grid. The results, compared to the depth of the fissures, are expressed in percentage of penetration. The results were as follows: penetration of fuschine dye: 0% for the 2 composites, 100% for Helioseal F; penetration of the materials: 96.90% for Helioseal F, 70.82 for Tetric and 86.10 for Tetric Flow (significant difference, Wilcoxon test = 0.0105). In this first in vitro study, Tetric Flow shows no microleakage and is more efficient when compared to Helioseal F and Tetric in obturating deep fissures of non carious bicuspids. The second experiment of the study evaluated the microleakage and the penetration depth of Tetric Flow when it is bonded by two different methods: Group 1: total etch (phosphoric acid) and Scotch-bond 1 (3M), and Group 2: self-etching primer with Prompt (Espe). There was no significant difference (p > 0.03) between classical bonding vs self-etching primer. The self-etching primer Prompt is very efficient vs phosphoric acid in obturating the fissures of non carious bicuspids with Tetric Flow. It is concluded that for prevention by sealing, using a flowable ceromer (Tetric Flow) with the self-etching (Prompt), is a really good technique.

[Water absorption of five dental resins used in bonded restorations]. / Absorption d'eau de cinq matériaux à base de résine utilisés en Dentisterie restauratrice collée.

The actual restorative dentistry need to bond material which are under the constraint of saliva likely, as all liquid, to enter inside the product with time and to modify its characteristics. In this study, we compare the behaviour of five materials opposite water absorption, in vitro, until one year: two composite resins (Tetric et Pertac II), two ceromer (ceromer (Tetric ceram et Tetric flow) and one compomer (Hytac(r)). Each pastille weight is expressed in percentage of initial weight. All materials loose weight in the first hours except Tetric ceram which stay stable. At 48 h, all materials except Pertac II get back their initial weight. At long-term, all the materials are stable with a profit of 1% for Hytac, 0.5% for Tetric, Tetric ceram et Tetric flow and a loss of à 0.3% for Pertac II. As a result of this study, we understand why the clinical used of Hytac must be done following strict conditions.

Prolonged display or rapid internalization of the IgG-binding protein ZZ anchored to the surface of cells using the diphtheria toxin T domain.

We have shown previously that the diphtheria toxin transmembrane domain (T) may function as a membrane anchor for soluble proteins fused at its C-terminus. Binding to membranes is triggered by acidic pH. Here, we further characterized this anchoring device. Soluble proteins may be fused at the N-terminus of the T domain or at both extremities, without modifying its membrane binding properties. This allows one to choose the orientation of the protein to be attached to the membrane. Maximum binding to the cell surface is reached within 1 h. Anchoring occurs on cells previously treated with proteinase K, suggesting that T interacts with the lipid phase of the membrane without the help of cell surface proteins. Binding does not permeabilize cells or affect cell viability, despite the fact that it permeabilizes liposomes and alters their structure. When attached to L929 fibroblasts, the proteins are not internalized and remain displayed at their surface for more than 24 h. When bound to K562 myeloid cells, the molecules are internalized and degraded. Thus, depending on the cell type, soluble proteins may be anchored to the surface of cells by the T domain for an extended time or directed towards an internalization pathway.

Comet assay and early apoptosis.

The comet assay is a single cell gel electrophoresis test currently used as a qualitative and quantitative genotoxicity test. However, some of the results from this comet assay and current knowledge on apoptosis lead us to suspect the presence of some false positive results. The aim of this study was to ascertain if apoptotic cells can yield comet images that might distort the interpretation of the results. Using Jurkat cells, that hardly express Fas antigen, and apoptosis induction with anti-Fas antibody, it was possible to show that apoptosis can generate typical comet pictures as soon as the cells enter the apoptosis process. Therefore, comet images cannot be interpreted as a genotoxicity indicator when an apoptosis risk is present. Yopro-1 staining, that is also nearly immediate after apoptosis induction, can be used to balance comet assay results.

Improving standards in the treatment of acute otitis externa by the use of a treatment protocol and open access to aural toilet.

A prospective audit of the procedure and outcome in the management of acute otitis externa was undertaken in our unit. The first cycle demonstrated a heterogeneous approach and clinical isolation of junior staff. A questionnaire survey of local general practitioners highlighted clinical confusion over the use of topical medication and a need for improved access to facilities for aural toilet. General practitioner liaison and education was an essential component in formulating a change in practice. In particular, open access for aural toilet was introduced and utilization encouraged. Following changes in practice, the second cycle of the audit showed that treatment protocols were effective and adhered to by junior staff.

[Anchoring cytokines to cancer cells using diphtheria toxin: better than immunotherapy by gene transfer?]. / Ancrer des cytokines aux cellules cancéreuses à l'aide de la toxine diphtérique: mieux que l'immunothérapie par transfert de gène?

Many cytokines are able to stimulate the antitumor immune response. However, in order to avoid the toxic effects due to systemic injection, it is necessary to concentrate the cytokines in the tumor microenvironment. Current methods, based on the transfer of cytokine genes into tumor cells, still suffer drawbacks. We describe an alternative approach using recombinant cytokines genetically conjugated to a membrane anchor derived from diphtheria toxin. Interleukin-2 anchored to lymphoma and melanoma cells remained displayed on their surface and were not internalized. Injection of these cell preparations to mice led to an immune response able to prevent or slow tumor growth following tumor challenge.

Anchoring antibodies to membranes using a diphtheria toxin T domain-ZZ fusion protein as a pH sensitive membrane anchor.

We have constructed a fusion protein, T-ZZ, in which the IgG-Fc binding protein ZZ was fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). While soluble at neutral pH, T-ZZ retained the capacity of the T domain to bind to phospholipid membranes at acidic pH. Once anchored to the membrane, the ZZ part of the protein was capable of binding mouse monoclonal or rabbit polyclonal IgG. Our results show that the T-ZZ protein can function as a pH sensitive membrane anchor for the linkage of IgG to the membrane of lipid vesicles, adherent and non-adherent cells.

The diphtheria toxin transmembrane domain as a pH sensitive membrane anchor for human interleukin-2 and murine interleukin-3.

We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T-(Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine-dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.

Clostridium perfringens epsilon-toxin acts on MDCK cells by forming a large membrane complex.

Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in animals, which is characterized by edema in several organs due to an increase in blood vessel permeability. The Madin-Darby canine kidney (MDCK) cell line has been found to be susceptible to epsilon-toxin (D. W. Payne, E. D. Williamson, H. Havard, N. Modi, and J. Brown, FEMS Microbiol. Lett. 116:161-168, 1994). Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K+ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37 labeled C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37 degrees C was more stable than that formed at 4 degrees C, since it was not dissociated by 5% sodium dodecyl sulfate and boiling.

Characterization and receptor specific toxicity of two diphtheria toxin-related interleukin-3 fusion proteins DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3.

We have constructed two fusion proteins, DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3, in which the receptor-binding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB389-(Gly4Ser)2-mIL-3 was up to 5 times more toxic than DAB389-mIL-3, depending on the cell line (0.8 x 10(-10) M < IC50 < 3 x 10(-10) M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptor-positive pluripotent hematopoietic stem cells prior to bone marrow transplantation.