Patient-derived follicular lymphoma spheroids recapitulate lymph node signaling and immune profile uncovering galectin-9 as a novel immunotherapeutic target.

Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, constitutes a paradigm of immune tumor microenvironment (TME) contribution to disease onset, progression, and heterogenous clinical outcome. Here we present the first FL-Patient Derived Lymphoma Spheroid (FL-PDLS), including fundamental immune actors and features of TME in FL lymph nodes (LNs). FL-PDLS is organized in disc-shaped 3D structures composed of proliferating B and T cells, together with macrophages with an intermediate M1/M2 phenotype. FL-PDLS recapitulates the most relevant B-cell transcriptional pathways present in FL-LN (proliferation, epigenetic regulation, mTOR, adaptive immune system, among others). The T cell compartment in the FL-PDLS preserves CD4 subsets (follicular helper, regulatory, and follicular regulatory), also encompassing the spectrum of activation/exhaustion phenotypes in CD4 and CD8 populations. Moreover, this system is suitable for chemo and immunotherapy testing, recapitulating results obtained in the clinic. FL-PDLS allowed uncovering that soluble galectin-9 limits rituximab, rituximab, plus nivolumab/TIM-3 antitumoral activities. Blocking galectin-9 improves rituximab efficacy, highlighting galectin-9 as a novel immunotherapeutic target in FL. In conclusion, FL-PDLS maintains the crosstalk between malignant B cells and the immune LN-TME and constitutes a robust and multiplexed pre-clinical tool to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

Listeria monocytogenes prevalence and genomic diversity along the pig and pork production chain.

The facultative intracellular bacterium Listeria monocytogenes (L. monocytogenes) is the causative agent of listeriosis, a severe invasive illness. This ubiquitous species is widely distributed in the environment, but infection occurs almost exclusively through ingestion of contaminated food. The pork production sector has been heavily affected by a series of L. monocytogenes-related foodborne outbreaks in the past around the world. Ready-to-eat (RTE) pork products represent one of the main food sources for strong-evidence listeriosis outbreaks. This pathogen is known to be present throughout the entire pig and pork production chain. Some studies hypothesized that the main source of contamination in final pork products was either living pigs or the food-processing environment. A detailed genomic picture of L. monocytogenes can provide a renewed understanding of the routes of contamination from pig farms to the final products. This review provides an overview of the prevalence, the genomic diversity and the genetic background linked to virulence of L. monocytogenes along the entire pig and pork production chain, from farm to fork.

Patient-derived lymphoma spheroids integrating immune tumor microenvironment as preclinical follicular lymphoma models for personalized medicine.

BACKGROUND: Follicular lymphoma (FL), the most common indolent non-Hodgkin's Lymphoma, is a heterogeneous disease and a paradigm of the contribution of immune tumor microenvironment to disease onset, progression, and therapy resistance. Patient-derived models are scarce and fail to reproduce immune phenotypes and therapeutic responses. METHODS: To capture disease heterogeneity and microenvironment cues, we developed a patient-derived lymphoma spheroid (FL-PDLS) model culturing FL cells from lymph nodes (LN) with an optimized cytokine cocktail that mimics LN stimuli and maintains tumor cell viability. RESULTS: FL-PDLS, mainly composed of tumor B cells (60% on average) and autologous T cells (13% CD4 and 3% CD8 on average, respectively), rapidly organizes into patient-specific three-dimensional (3D) structures of three different morphotypes according to 3D imaging analysis. RNAseq analysis indicates that FL-PDLS reproduces FL hallmarks with the overexpression of cell cycle, BCR, or mTOR signaling related gene sets. FL-PDLS also recapitulates the exhausted immune phenotype typical of FL-LN, including expression of BTLA, TIGIT, PD-1, TIM-3, CD39 and CD73 on CD3+ T cells. These features render FL-PDLS an amenable system for immunotherapy testing. With this aim, we demonstrate that the combination of obinutuzumab (anti-CD20) and nivolumab (anti-PD1) reduces tumor load in a significant proportion of FL-PDLS. Interestingly, B cell depletion inversely correlates with the percentage of CD8+ cells positive for PD-1 and TIM-3. CONCLUSIONS: In summary, FL-PDLS is a robust patient-derived 3D system that can be used as a tool to mimic FL pathology and to test novel immunotherapeutic approaches in a context of personalized medicine.

Druggable redox pathways against Mycobacterium abscessus in cystic fibrosis patient-derived airway organoids.

Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. To date, our knowledge on the host and bacterial determinants driving Mabs pathology in CF patient lung remains rudimentary. Here, we used human airway organoids (AOs) microinjected with smooth (S) or rough (R-)Mabs to evaluate bacteria fitness, host responses to infection, and new treatment efficacy. We show that S Mabs formed biofilm, and R Mabs formed cord serpentines and displayed a higher virulence. While Mabs infection triggers enhanced oxidative stress, pharmacological activation of antioxidant pathways resulted in better control of Mabs growth and reduced virulence. Genetic and pharmacological inhibition of the CFTR is associated with better growth and higher virulence of S and R Mabs. Finally, pharmacological activation of antioxidant pathways inhibited Mabs growth, at least in part through the quinone oxidoreductase NQO1, and improved efficacy in combination with cefoxitin, a first line antibiotic. In conclusion, we have established AOs as a suitable human system to decipher mechanisms of CF-driven respiratory infection by Mabs and propose boosting of the NRF2-NQO1 axis as a potential host-directed strategy to improve Mabs infection control.

A novel patient-derived 3D model recapitulates mantle cell lymphoma lymph node signaling, immune profile and in vivo ibrutinib responses.

Mantle cell lymphoma (MCL), a rare and aggressive B-cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T-cells self-organize in disc-shaped structures, where B and T-cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproduces in vivo responses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features and in vivo responses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

The distribution of neuromuscular junctions depends on muscle pennation, when botulinum neurotoxin receptors and SNAREs expression are uniform in the rat.

BACKGROUND: Botulinum neurotoxins (BoNTs) are used to treat spastic disorders. Depending on muscle size, one or multiple injections are recommended according to labels to target neuromuscular junctions (NMJ). However, information about NMJ distribution and number in muscles, as well as expression of receptors and molecular targets of toxins is scarce in human and animal models. METHODS: Seven muscles from adult rats were used to identify expression of BoNT receptors and SNAREs using immunohistochemistry (IHC), and fluorescent α-Bungarotoxin combined to light-sheet microscopy used to determine their distribution. RESULTS: The location, number, and density of NMJ were muscle specific and mostly dependent on the type of pennation (myofiber orientation). In the Flexor Digitorum Brevis (a very small muscle) NMJ were as numerous as in the Gastrocnemius lateralis. A strong expression of SV2C, Synaptotagmin 2, SNAP25 and VAMP1 were observed in all muscles, and SV2A, Synaptotagmin 1 and VAMP2 were never detected. CONCLUSION: This work highlights the specific distribution of NMJ in muscles which seems to depend on the type of pennation. Detailed observation of myofibers organization might help clinicians to better evaluate the location of NMJ in humans; the molecular phenotyping of NMJ will contribute to better integrate the rat model into research of BoNT therapeutics.

Screening for anti-adipogenic, pro-lipolytic and thermogenic plant extracts by models associating intestinal epithelial cells with human adipose cells.

PURPOSE: Excessive fat mass accumulation in obesity leads to diverse metabolic disorders, increased risks of cardiovascular diseases and in some cases, mortality. The aim of this study was to screen the actions of botanical extracts intended for oral use on human adipose tissue, using an in vitro screening model combining human intestinal cells with human adipose cells. This was to find the most effective extracts on lipid accumulation, UCP1 expression and ATP production in pre-adipocytes and on adipocyte lipolysis. METHODS: In this study, 25 individual plant extracts were screened for their effects on human adipose cells. Consequently, an original in vitro model was set up using the Caco-2 cell line, to mimic the intestinal passage of the extracts and then exposing human adipose cells to them. The biological actions of extracts were thus characterized, and compared with a coffee extract standard. The most effective extracts, and their combinations, were retained for their actions on lipid accumulation, the expression of the thermogenic effector UCP1 and ATP production in pre-adipocytes as well as on lipolysis activity of mature adipocytes. RESULTS: The biphasic culture system combining human Caco-2 cells with human adipose cells was verified as functional using the green coffee extract standard. Out of the 25 plant extracts studied, only 7 and their combinations were retained due to their potent effects on adipose cells biology. The data showed that compared to the coffee extract standard, Immortelle, Catechu, Carrot and Rose hip extracts were the most effective in reducing lipid accumulation and increased UCP1 expression in human pre-adipocytes. CONCLUSION: This study reveals the potential inhibitory effects on lipid accumulation and thermogenic activity of Immortelle, Catechu, Carrot and Rose hip extracts, and for the first time synergies in their combinations, using an in vitro model mimicking as closely as possible, human intestinal passage linked to adipose cells. These findings need to be confirmed by in vivo trials.

3D Model Characterization by 2D and 3D Imaging in t(14;18)-Positive B-NHL: Perspectives for In Vitro Drug Screens in Follicular Lymphoma.

Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20-30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.

Fluorescent Tagged Vaccinia Virus Genome Allows Rapid and Efficient Measurement of Oncolytic Potential and Discovery of Oncolytic Modulators.

As a live biologic agent, oncolytic vaccinia virus has the ability to target and selectively amplify at tumor sites. We have previously reported that deletion of thymidine kinase and ribonucleotide reductase genes in vaccinia virus can increase the safety and efficacy of the virus. Here, to allow direct visualization of the viral genome in living cells, we incorporated the ANCH target sequence and the OR3-Santaka gene in the double-deleted vaccinia virus. Infection of human tumor cells with ANCHOR3-tagged vaccinia virus enables visualization and quantification of viral genome dynamics in living cells. The results show that the ANCHOR technology permits the measurement of the oncolytic potential of the double deleted vaccinia virus. Quantitative analysis of infection kinetics and of viral DNA replication allow rapid and efficient identification of inhibitors and activators of oncolytic activity. Our results highlight the potential application of the ANCHOR technology to track vaccinia virus and virtually any kind of poxvirus in living cells.

Boosting γδ T cell-mediated antibody-dependent cellular cytotoxicity by PD-1 blockade in follicular lymphoma.

Follicular lymphoma (FL) is a common non Hodgkin's lymphoma subtype in which immune escape mechanisms are implicated in resistance to chemo-immunotherapy. Although molecular studies point to qualitative and quantitative deregulation of immune checkpoints, in depth cellular analysis of FL immune escape is lacking. Here, by functional assays and in silico analyses we show that a subset of FL patients displays a 'high' immune escape phenotype. These FL cases are characterized by abundant infiltration of PD1+ CD16+ TCRVγ9Vδ2 γδ T lymphocytes. In a 3D co-culture assay (MALC), γδ T cells mediate both direct and indirect (ADCC in the presence of anti-CD20 mAbs) cytolytic activity against FL cell aggregates. Importantly, PD-1, which is expressed by most FL-infiltrating γδ T lymphocytes with ADCC capacity, impairs these functions. In conclusion, we identify a PD1-regulated γδ T cell cytolytic immune component in FL. Our data provide a treatment rational by PD-1 blockade aimed at boosting γδ T cell anti-tumor functions in FL.

In Vivo Labelling of Adenovirus DNA Identifies Chromatin Anchoring and Biphasic Genome Replication.

Adenoviruses are DNA viruses with a lytic infection cycle. Following the fate of incoming as well as recently replicated genomes during infections is a challenge. In this study, we used the ANCHOR3 technology based on a bacterial partitioning system to establish a versatile in vivo imaging system for adenoviral genomes. The system allows the visualization of both individual incoming and newly replicated genomes in real time in living cells. We demonstrate that incoming adenoviral genomes are attached to condensed cellular chromatin during mitosis, facilitating the equal distribution of viral genomes in daughter cells after cell division. We show that the formation of replication centers occurs in conjunction with in vivo genome replication and determine replication rates. Visualization of adenoviral DNA revealed that adenoviruses exhibit two kinetically distinct phases of genome replication. Low-level replication occurred during early replication, while high-level replication was associated with late replication phases. The transition between these phases occurred concomitantly with morphological changes of viral replication compartments and with the appearance of virus-induced postreplication (ViPR) bodies, identified by the nucleolar protein Mybbp1A. Taken together, our real-time genome imaging system revealed hitherto uncharacterized features of adenoviral genomes in vivo The system is able to identify novel spatiotemporal aspects of the adenovirus life cycle and is potentially transferable to other viral systems with a double-stranded DNA phase.IMPORTANCE Viruses must deliver their genomes to host cells to ensure replication and propagation. Characterizing the fate of viral genomes is crucial to understand the viral life cycle and the fate of virus-derived vector tools. Here, we integrated the ANCHOR3 system, an in vivo DNA-tagging technology, into the adenoviral genome for real-time genome detection. ANCHOR3 tagging permitted the in vivo visualization of incoming genomes at the onset of infection and of replicated genomes at late phases of infection. Using this system, we show viral genome attachment to condensed host chromosomes during mitosis, identifying this mechanism as a mode of cell-to-cell transfer. We characterize the spatiotemporal organization of adenovirus replication and identify two kinetically distinct phases of viral genome replication. The ANCHOR3 system is the first technique that allows the continuous visualization of adenoviral genomes during the entire virus life cycle, opening the way for further in-depth study.

Evaluation by quantitative image analysis of anticancer drug activity on multicellular spheroids grown in 3D matrices.

Pharmacological evaluation of anticancer drugs using 3D in vitro models provides invaluable information for predicting in vivo activity. Artificial matrices are currently available that scale up and increase the power of such 3D models. The aim of the present study was to propose an efficient and robust imaging and analysis pipeline to assess with quantitative parameters the efficacy of a particular cytotoxic drug. HCT116 colorectal adenocarcinoma tumor cell multispheres were grown in a 3D physiological hyaluronic acid matrix. 3D microscopy was performed with structured illumination, whereas image processing and feature extraction were performed with custom analysis tools. This procedure makes it possible to automatically detect spheres in a large volume of matrix in 96-well plates. It was used to evaluate drug efficacy in HCT116 spheres treated with different concentrations of topotecan, a DNA topoisomerase inhibitor. Following automatic detection and quantification, changes in cluster size distribution with a topotecan concentration-dependent increase of small clusters according to drug cytotoxicity were observed. Quantitative image analysis is thus an effective means to evaluate and quantify the cytotoxic and cytostatic activities of anticancer drugs on 3D multicellular models grown in a physiological matrix.

[From bearing arms to nursing care]. / Du métier des armes aux soins infirmiers.

Having benefited from professional redeployment that has fully met his expectations, a former soldier who has become a nurse shares with us his work in a medical/social organization with residents of areas facing job insecurity.

In vivo quantification of epidermis pigmentation and dermis papilla density with reflectance confocal microscopy: variations with age and skin phototype.

Reflectance confocal microscopy (RCM) may help to quantify variations of skin pigmentation induced by different stimuli such as UV radiation or therapeutic intervention. The objective of our work was to identify RCM parameters able to quantify in vivo dermis papilla density and epidermis pigmentation potentially applicable in clinical studies. The study included 111 healthy female volunteers with phototypes I-VI. Photo-exposed and photo-protected anatomical sites were imaged. The effect of age was also assessed. Four epidermis components were specifically investigated: stratum corneum, stratum spinosum, basal epidermal layer and dermo-epidermal junction. Laser power, diameter of corneocytes and upper spinous keratinocytes, brightness of upper spinous and interpapillary spinous keratinocytes, number of dermal papillae and papillary contrast were systematically assessed. Papillary contrast measured at the dermo-epidermal junction appeared to be a reliable marker of epidermis pigmentation and showed a strong correlation with skin pigmentation assessed clinically using the Fitzpatrick's classification. Brightness of upper spinous and interpapillary spinous keratinocytes was not influenced by the skin phototype. The number of dermal papillae was significantly lower in subjects with phototypes I-II as compared with darker skin subjects. A dramatic reduction in the number of dermal papillae was noticed with age, particularly in subjects with fair skin. The method presented here provides a new in vivo investigation tool for quantification of dermis papilla density and epidermal pigmentation. Papillary contrast measured at the dermo-epidermal junction may be selected as a marker of skin pigmentation for evaluation in clinical studies.

An a priori shading correction technique for contact imaging devices.

Digital imaging devices are increasingly used for color measurements in fields such as forensics, the food industry, and ecological and medical research. Especially concerning the skin, in the follow-up of benign or malignant lesions, it is crucial to ensure that the measured colors are accurate and reproducible. Several color calibration methods having been presented over the last few years. However, the choice of illuminant used remains a major source of color misinterpretation, thus, much effort is being spent in trying to evaluate this a posteriori. The device presented overcomes this problem by integrating its own light source, although corrections in lighting heterogeneity are still required. In this paper, we present a lighting modelling technique used for shading correction which improves color consistency (as assessed by ∆E evaluation versus colorimeter), noise filtering, computation time, and memory consumption for this type of device.

A functional variant of the dopamine D3 receptor is associated with risk and age-at-onset of essential tremor.

Familial essential tremor (ET), the most common inherited movement disorder, is generally transmitted as an autosomal dominant trait. A genome-wide scan for ET revealed one major locus on chromosome 3q13. Here, we report that the Ser9Gly variant in the dopamine D(3) receptor gene (DRD3), localized on 3q13.3, is associated and cosegregates with familial ET in 23 out of 30 French families. Sequencing revealed no other nonsynonymous variants in the DRD3-coding sequence and in the first 871 bp of the 5' flanking region. Moreover, Gly-9 homozygous patients presented with more severe and/or earlier onset forms of the disease than heterozygotes. A replication study comparing 276 patients with ET and 184 normal controls confirmed the association of the Gly-9 variant with risk and age-at-onset of ET. In human embryonic kidney (HEK) 293-transfected cells, the Gly-9 variant did not differ from the Ser-9 variant with respect to glycosylation and to anterograde and retrograde trafficking, but dopamine had an affinity that was four to five times higher. With the Gly-9 variant, the dopamine-mediated cAMP response was increased, and the mitogen-associated protein kinase (MAPK) signal was prolonged, as compared with the Ser-9 variant. The gain-of-function produced by the Gly-9 variant may explain why drugs active against tremor in Parkinson's disease (PD) are usually not effective in the treatment of ET and suggests that DRD3 partial agonists or antagonists should be considered as novel therapeutic options for patients with ET.

Measurement of the mechanical properties of the skin using the suction test.

INTRODUCTION: The suction test is commonly used to study the mechanical properties of human skin in vivo. The unevenness of the stress fields complicates obtaining the intrinsic mechanical parameters of the skin in vivo because the values of the local stresses and deformations cannot be calculated directly from the displacements and forces applied by the test apparatus. In general, users only take into account the negative pressure applied and the elevation of the dome of skin drawn up in order to deduce the properties of the skin. This method has the major disadvantage of being dependent on the experimental conditions used: in particular, the size of the suction cup and the negative pressure applied. Here, we propose a full mechanical study of the test to provide rigorous results. We compare the frequently used geometric method (making the thin plate hypothesis), Timoshenko's method (which can take greater plate thicknesses into account) and finally various results obtained by the finite elements (FE) technique. METHODS: The suction test was modelled by FE with large displacements and large deformations both for orthotropic and isotropic plates. The results obtained in the elastic domain for various values of Young's modulus and of applied negative pressure were used as references and were compared with methods using analytical relationships. RESULTS: The geometric method generally used in the interpretation of suction tests gives results, which in certain configurations, are very different from those obtained by FE. The method of Timoshenko is suited to thick plates 'in contact' or embedded round the edge, the elevation of the dome and the tension and flexion stresss are analytically accessible through relationships involving four constants that are dependent on the limit conditions. Comparison with the FE results enabled the optimisation of the coefficients to adapt the relationships to the particular conditions of the suction trials. CONCLUSION: We showed the limits of the geometrical method and proposed a solution, which while remaining simple to use, gives results that are closer to reality both for the calculation of the modulus and for the determination of the state of the stresses obtained.

Characterization of the mechanical properties of skin by inverse analysis combined with the indentation test.

This study proposes a new method to determine the mechanical properties of human skin by the use of the indentation test [Pailler-Mattei, 2004. Caractérisation mécanique et tribologique de la peau humaine in vivo, Ph.D. Thesis, ECL-no. 2004-31; Pailler-Mattei, Zahouani, 2004. Journal of Adhesion Science and Technology 18, 1739-1758]. The principle of the measurements consists in applying an in vivo compressive stress [Zhang et al., 1994. Proceedings of the Institution of Mechanical Engineers 208, 217-222; Bosboom et al., 2001. Journal of Biomechanics 34, 1365-1368; Oomens et al., 1984. Selected Proceedings of Meetings of European Society of Biomechanics, pp. 227-232; Oomens et al., 1987. Journal of Biomechanics 20(9), 877-885] on the skin tissue of an individual's forearm. These measurements show an increase in the normal contact force as a function of the indentation depth. The interpretation of such results usually requires a long and tedious phenomenological study. We propose a new method to determine the mechanical parameters which control the response of skin tissue. This method is threefold: experimental, numerical, and comparative. It consists combining experimental results with a numerical finite elements model in order to find out the required parameters. This process uses a scheme of extended Kalman filters (EKF) [Gu et al., 2003. Materials Science and Engineering A345, 223-233; Nakamura et al., 2000. Acta Mater 48, 4293-4306; Leustean and Rosu, 2003. Certifying Kalman filters. RIACS Technical Report 03.02, 27pp. http://gureni.cs.uiuc.edu/~grosu/download/luta + leo.pdf; Welch and Bishop, An introduction to Kalman filter, University of North Carolina at Chapel Hill, 16p. http://www.cs.unc.edu/~welch/kalman/]. The first results presented in this study correspond to a simplified numerical modeling of the global system. The skin is assumed to be a semi-infinite layer with an isotropic linear elastic mechanical behavior [Zhang et al., 1994. Proceedings of the Institution of Mechanical Engineers 208, 217-222] This analysis will be extended to more realistic models in further works.

Functional interaction between -629C/A, -971G/A and -1337C/T polymorphisms in the CETP gene is a major determinant of promoter activity and plasma CETP concentration in the REGRESS Study.

Cholesteryl ester transfer protein (CETP) plays a key role in the determination of high-density lipoprotein (HDL) levels via its action on intravascular HDL metabolism. The TaqIB polymorphism of the CETP gene is associated with plasma CETP and high-density lipoprotein cholesterol (HDL-C) levels and with premature coronary artery disease. Such associations appear to result from linkage disequilibrium between TaqIB and other functional polymorphisms. To date, only one functional promoter variant, which may explain the effects of TaqIB, has been identified at position -629 in the CETP gene. Here we describe a C/T polymorphism located at position -1337 in the human CETP gene (C allele frequency: 0.684), which is significantly associated with plasma HDL-C and CETP levels (P=0.0001 and P<0.0001, respectively). Transient transfection of a reporter gene construct containing the CETP promoter from -1707/+28 in liver cells (HepG2) revealed that the -1337T allele was expressed to a significantly lower degree (-34%, P<0.0001) than the -1337C allele. In addition, we clearly demonstrated that the -971G/A polymorphism is functional and that its functionality is intimately linked to the presence of the -1337 site. In vitro evaluation of potential interaction between -1337C/T and other functional variants of the CETP gene (-971G/A and -629C/A) demonstrated that these three functional CETP promoter polymorphisms can interact together to determine the overall activity of the CETP gene and thus contribute significantly to variation in plasma CETP mass concentration.

Automatic measurement of dermal thickness from B-scan ultrasound images using active contours.

BACKGROUND/PURPOSE: Measurement of dermal thickness is useful in the evaluation of dermo-cosmetics for assessing not only morphological changes but also mechanical properties of this layer. Our aim was first to standardise the manual dermal thickness measurement procedure on B-scan ultrasound images, then to develop an automatic operator independent method to detect the boundaries of the dermis. MATERIAL AND METHODS: The Dermcup 20 MHz B-scan ultrasound system was used. The method used for detecting the boundaries was adapted from active contour algorithms. The innovative aspect of the method consists in an automatic initialization of the first step of the algorithm. To validate the method, we correlated measurements obtained by the manual and automatic approaches from a set of images from different anatomical sites. RESULTS AND CONCLUSION: The results showed for the two measurement methods, 72% of the images were perfectly correlated. The remaining images required manual initialization of the boundaries by a non-expert operator before the active contour process could be used. Subsequent to this semi-automatic procedure, the correlation was very high.