Classification of very high spatial resolution imagery using mathematical morphology and support vector machines

We investigate the relevance of morphological operators for the classification of land use in urban scenes using submetric panchromatic imagery. A support vector machine is used for the classification. Six types of filters have been employed: opening and closing, opening and closing by reconstruction, and opening and closing top hat. The type and scale of the filters are discussed, and a feature selection algorithm called recursive feature elimination is applied to decrease the dimensionality of the input data. The analysis performed on two QuickBird panchromatic images showed that simple opening and closing operators are the most relevant for classification at such a high spatial resolution. Moreover, mixed sets combining simple and reconstruction filters provided the best performance. Tests performed on both images, having areas characterized by different architectural styles, yielded similar results for both feature selection and classification accuracy, suggesting the generalization of the feature sets highlighted.

Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier

RESUME L'utilisation de la thérapie génique dans l'approche d'un traitement des maladies oculaires dégénératives, plus particulièrement de la rétinite pigmentaire, semble être très prometteuse (Acland et al. 2001). Parmi les vecteurs développés, les vecteurs lentiviraux (dérivé du virus humain HIV-1), permettent la transduction des photorécepteurs après injection sous-rétinienne chez la souris durant les premiers jours de vie. Cependant l'efficacité du transfert de gène est nettement plus limitée dans ce type cellulaire après injection chez l'adulte (Kostic et al. 2003). L'objet de notre étude est de déterminer si la présence d'une barrière physique produite au cours du développement, située entre les photorécepteurs et l'épithélium pigmentaire ainsi qu'entre les photorécepteurs eux-mêmes, est responsable de: la diminution de l'entrée en masse du virus dans les photorécepteurs, minimisant ainsi son efficacité chez la souris adulte. De précédentes recherches, chez le lapin, ont décrit la capacité d'enzymes spécifiques comme la Chondroïtinase ABC et la Neuraminidase X de modifier la structure de la matrice entourant les photorécepteurs (Inter Photoreceptor Matrix, IPM) par digestion de certains de ses constituants suite à leur injection dans l'espace sous-rétinien (Yao et al. 1990). Considérant l'IPM comme une barrière physique, capable de réduire l'efficacité de transduction des photorécepteurs chez la souris adulte, nous avons associé différentes enzymes simultanément à l'injection sous-rétinienne de vecteurs lentiviraux afin d'améliorer la transduction virale en fragilisant I'IPM, la rendant ainsi plus perméable à la diffusion du virus. L'injection sous-rétinienne de Neuraminidase X et de Chondroïtinase ABC chez la souris induit des modifications structurales de l'IPM qui se manifestent respectivement par la révélation ou la disparition de sites de liaison de la peanut agglutinin sur les photorécepteurs. L'injection simultanée de Neuraminidase X avec le vecteur viral contenant le transgène thérapeutique augmente significativement le nombre de photorécepteurs transduits (environ cinq fois). Nous avons en fait démontré que le traitement enzymatique augmente principalement la diffusion du lentivirus dans l'espace situé entre l'épithélium pigmentaire et les photorécepteurs. Le traitement à la Chondroïtinase ABC n'entraîne quant à elle qu'une légère amélioration non significative de la transduction. Cette étude montre qu'une meilleure connaissance de l'IPM ainsi que des substances capables de la modifier (enzymes, drogues etc.) pourrait aider à élaborer de nouvelles stratégies afin d'améliorer la distribution de vecteurs viraux dans la rétine adulte.

Identification of the minimal promoter region of the mouse NKX5-3, a transcription factor implicated in eye development.

Early ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signalling molecules. Together, these molecules regulate cell proliferation and apoptosis, and specify retinal fate. NKX5-3 is a homeobox transcription factor implicated in eye development. The analysis of the 5'-flanking region of the mouse Nkx5-3 gene revealed a predicted TATA-less promoter sequence between -416 and -166 of the translation start site. To functionally characterise Nkx5-3 promoter activity, serial deletions of the promoter sequence were introduced in pGL-3 basic vector and promoter activity of these 5'- and 3'-deleted constructions was tested in HeLa and CHO cells. Transactivation assays identified a region between -350 and -296 exhibiting promoter-like activity. Combined analysis by deletions and point mutations showed that this sequence, containing multiple Sp1 binding sites was necessary to promote transcriptional activity. Binding of Sp1 to this region was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation, using an antibody specific for Sp1. Altogether, these results demonstrated that the immediate upstream region of Nkx5-3 gene possessed a strong intrinsic promoter activity in vitro, suggesting a potential role in Nkx5-3 transcription in vivo.

Prolonged fluconazole exposure leads to a shift to Candida species with decreased susceptibility in breakthrough candidemia: prospective survey of the Fungal Infection Network of Switzerland

Objectives: To compare the clinical characteristics, species distribution and antifungal susceptibility of Candida bloodstream isolates (BSI) in breakthrough (BTC) vs. non-breakthrough candidemia (NBTC) and to study the effect of prolonged vs. short fluconazole (F) exposure in BTC.Methods: Candida BSI were prospectively collected during 2004- 2006 from 27 hospitals (seven university, 20 affiliated) of the FUNGINOS network. Susceptibility to F, voriconazole (V) and caspofungin (C) was tested in the FUNGINOS mycology reference laboratory by microtitre broth dilution method with the Sensititre YeastOneTM test panel. Clinical data were collected using standardized CRFs. BTC was defined as occurring during antifungal treatment/prophylaxis of at least three days duration prior to the candidemia. Susceptibility of BSI was defined according to 2010/2011 CLSI clinical breakpoints.Results: Out of 567 candidemia episodes, 550 Candida BSI were available. Of these, 43 (7.6%) were from BTC (37/43, 86% were isolated after F exposure). 38 BTC (88.4%) and 315 NBTC (55.6%) occurred in university hospitals (P < 0.001). The majority of patients developing BTC were immunocompromised: higher proportions of haematological malignancies (62.8% in BTC vs. 47.1% in NBTC, P < 0.001), neutropenia (37.2% vs. 11.8%, P < 0.001), acute GvHD (14% vs. 0.2%, P < 0.001), immunosuppressive drugs (74.4% vs. 7.8%, P < 0.001), and mucositis (32.6% vs. 2.3%, P < 0.001) were observed. Other differences between BTC and NBTC were higher proportions of patients with central venous catheters in the 2 weeks preceding candidemia (95.3% vs. 83.4%, P = 0.047) and receiving total parenteral nutrition (62.8% vs. 35.9%, P < 0.001), but a lower proportion of patients treated with gastric proton pump inhibitors (23.3% vs. 72.1%, P < 0.001). Overall mortality of BTC and NBTC was not different (34.9% vs. 31.7%, P = 0.73), while a trend to higher attributable mortality in BTC was found (13.9% vs. 6.9%, P = 0.12). Species identification showed a majority of C. albicans in both groups (51.2% in BTC vs. 62.9% in NBTC, P = 0.26), followed by C. glabrata (18.6% vs. 18.5%), C. tropicalis (2.3% vs. 6.3%) and C. parapsilosis (7.0% vs. 4.7%). Significantly more C. krusei were detected in BTC versus NBTC (11.6% vs. 1.6%, P = 0.002). The geometric mean MIC for F, V and C between BTC and NBTC isolates was not significantly different. However, in BTC there was a significant association between duration of F exposure and the Candida spp.: >10 days of F was associated with a significant shift from susceptible Candida spp. (C. albicans, C. parapsilosis, C. tropicalis, C. famata) to non-susceptible species (C. glabrata, C. krusei, C. norvegensis). Among 21 BTC episodes occurring after £10 days of F, 19% of the isolates were non-susceptible, in contrast to 68.7% in 16 BTC episodes occurring after >10 days of F (P = 0.003).Conclusions: Breakthrough candidemia occurred more often in immunocompromised hosts. Fluconazole administered for >10 days was associated with a shift to non-susceptible Candida spp.. Length of fluconazole exposure should be taken into consideration for the choice of empirical antifungal treatment.

An international university network for online teaching of pathology in Frenchspeaking countries

Introduction: Building online courses is a highly time consuming task for teachers of a single university. Universities working alone create high-quality courses but often cannot cover all pathological fields. Moreover this often leads to duplication of contents among universities, representing a big waste of teacher time and energy. We initiated in 2011 a French university network for building mutualized online teaching pathology cases, and this network has been extended in 2012 to Quebec and Switzerland. Method: Twenty French universities (see & for details), University Laval in Quebec and University of Lausanne in Switzerland are associated to this project. One e-learning Moodle platform (http://moodle.sorbonne-paris-cite.fr/) contains texts with URL pointing toward virtual slides that are decentralized in several universities. Each university has the responsibility of its own slide scanning, slide storage and online display with virtual slide viewers. The Moodle website is hosted by PRES Sorbonne Paris Cité, and financial supports for hardware have been obtained from UNF3S (http://www.unf3s.org/) and from PRES Sorbonne Paris Cité. Financial support for international fellowships has been obtained from CFQCU (http://www.cfqcu.org/). Results: The Moodle interface has been explained to pathology teachers using web-based conferences with screen sharing. The teachers added then contents such as clinical cases, selfevaluations and other media organized in several sections by student levels and pathological fields. Contents can be used as online learning or online preparation of subsequent courses in classrooms. In autumn 2013, one resident from Quebec spent 6 weeks in France and Switzerland and created original contents in inflammatory skin pathology. These contents are currently being validated by senior teachers and will be opened to pathology residents in spring 2014. All contents of the website can be accessed for free. Most contents just require anonymous connection but some specific fields, especially those containing pictures obtained from patients who agreed for a teaching use only, require personal identification of the students. Also, students have to register to access Moodle tests. All contents are written in French but one case has been translated into English to illustrate this communication (http://moodle.sorbonne-pariscite.fr/mod/page/view.php?id=261) (use "login as a guest"). The Moodle test module allows many types of shared questions, making it easy to create personalized tests. Contents that are opened to students have been validated by an editorial committee composed of colleagues from the participating institutions. Conclusions: Future developments include other international fellowships, the next one being scheduled for one French resident from May to October 2014 in Quebec, with a study program centered on lung and breast pathology. It must be kept in mind that these e-learning programs highly depend on teachers' time, not only at these early steps but also later to update the contents. We believe that funding resident fellowships for developing online pathological teaching contents is a win-win situation, highly beneficial for the resident who will improve his knowledge and way of thinking, highly beneficial for the teachers who will less worry about access rights or image formats, and finally highly beneficial for the students who will get courses fully adapted to their practice.

Robust vaccine-elicited cellular immune responses in breast milk following systemic Simian Immunodeficiency Virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys.

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

Consolidation with Radioimmunotherapy May Prolong Survival in First Remission of Mantle Cell Lymphoma Patients Uneligible for Stem Cell Transplantation, An Analysis of the International RIT-Network

Background: Mantle cell lymphoma (MCL) is a rare subtype (3-9%) of Non Hodgkin Lymphoma (NHL) with a relatively poor prognosis (5-year survival < 40%). Although consolidation of first remission with autologous stem cell transplantation (ASCT) is regarded as "golden standard", less than half of the patients may be subjected to this intensive treatment due to advanced age and co-morbidities. Standard-dose non-myeloablative radioimmunotherapy (RIT) seems to be a very efficient approach for treatment of certain NHL. However, there are almost no data available on the efficacy and safety of RIT in MCL. Methods and Patients: In the RIT-Network, a web-based international registry collecting real observational data from RIT-treated patients, 115 MCL patients treated with ibritumomab tiuxetan were recorded. Most of the patients were elderly males with advanced stage of the disease: median age - 63 (range 31-78); males - 70.4%, stage III/IV - 92%. RIT (i.e. application of ibritumomab tiuxetan) was a part of the first line therapy in 48 pts. (43%). Further 38 pts. (33%) received ibritumomab tiuxetan after two previous chemotherapy regimens, and 33 pts. (24%) after completing 3-8 lines. In 75 cases RIT was applied as a consolidation of chemotherapy induced response; the rest of the patients received ibritumomab tiuxetan because of relapse/refractory disease. At the moment follow up data are available for 74 MCL patients. Results: After RIT the patients achieved high response rate: CR 60.8%, PR 25.7%, and SD 2.7%. Only 10.8% of the patients progressed. For survival analysis many data had to be censored since the documentation had not been completed yet. The projected 3-year overall survival (OAS, fig.1 - image 001.gif) after radioimmunotherapy was 72% for pts. subjected to RIT consolidation versus 29% for those treated in relapse/refractory disease (p=0.03). RIT was feasible for almost all patients; only 3 procedure-related deaths were reported in the whole group. The main adverse event was hematological toxicity (grade III/IV cytopenias) showing a median time of recovery of Hb, WBC and Plt of 45, 40 and 38 days respectively. Conclusion: Standard-dose non-myeloablative RIT is a feasible and safe treatment modality, even for elderly MCL pts. Consolidation radioimmunotherapy with ibritumomab tiuxetan may prolong survival of patients who achieved clinical response after chemotherapy. Therefore, this consolidation approach should be considered as a treatment strategy for those, who are not eligible for ASCT. RIT also has a potential role as a palliation therapy in relapsing/resistant cases.

Rpe65-Gene Transfer Using an Integration-Deficient Lentiviral Vector

Purpose: We previously demonstrated efficient retinal rescue of RPE65 mouse models (Rpe65-/- (Bemelmans et al, 2006) and Rpe65R91W/R91W mice) using a HIV1-derived lentiviral vector encoding for the mouse RPE65 cDNA. In order to optimize a lentiviral vector as an alternative tool for RPE65-derived Leber Congenital Amaurosis clinical trials, we evaluated the efficiency of an integration-deficient lentiviral vector (IDLV) encoding the human RPE65 cDNA to restore retinal function in the Rpe65R91W/R91W mice. Methods: An HIV-1-derived lentiviral vector expressing either the hrGFPII or the human Rpe65 cDNA under the control of a 0.8 kb fragment of the human Rpe65 promoter (R0.8) was produced by transient transfection of 293T cells. A LQ-integrase mutant was used to generate the IDLV vectors. IDLV-R0.8-hRPE65 or hrGFPII were injected subretinally into 1 month-old Rpe65R91W/R91W mice. Functional rescue was assessed by ERG (1 and 3 months post-injection) and cone survival by immunohistology. Results: An increased light sensitivity was detected by scotopic ERG in animals injected with IDLV-R0.8-hRPE65 compared to hrGFPII-treated animals or untreated mice. However the improvement was delayed compared to integration-proficient LV and observed at 3 months but not 1 month post-injection. Immunolabelling of cone markers showed an increased number of cones in the transduced area compared to control groups. Conclusions: The IDLV-R0.8-hRPE65 vectors allow retinal improvement in the Rpe65R91W/R91W mice. Both rod function and cone survival were demonstrated even if there is a delay in the rescue as assessed by scotopic ERG. Integration-deficient vectors minimize insertional mutagenesis and thus are safer candidates for human application. Further experiments using large animals are now needed to validate correct gene transfer and expression of the RPE65 gene as well as tolerance of the vector after subretinal injection before envisaging a clinical trial application.

Primary breast lymphoma: patient profile, outcome and prognostic factors. A multicentre Rare Cancer Network study.

BACKGROUND: To asses the clinical profile, treatment outcome and prognostic factors in primary breast lymphoma (PBL). METHODS: Between 1970 and 2000, 84 consecutive patients with PBL were treated in 20 institutions of the Rare Cancer Network. Forty-six patients had Ann Arbor stage IE, 33 stage IIE, 1 stage IIIE, 2 stage IVE and 2 an unknown stage. Twenty-one underwent a mastectomy, 39 conservative surgery and 23 biopsy; 51 received radiotherapy (RT) with (n = 37) or without (n = 14) chemotherapy. Median RT dose was 40 Gy (range 12-55 Gy). RESULTS: Ten (12%) patients progressed locally and 43 (55%) had a systemic relapse. Central nervous system (CNS) was the site of relapse in 12 (14%) cases. The 5-yr overall survival, lymphoma-specific survival, disease-free survival and local control rates were 53%, 59%, 41% and 87% respectively. In the univariate analyses, favorable prognostic factors were early stage, conservative surgery, RT administration and combined modality treatment. Multivariate analysis showed that early stage and the use of RT were favorable prognostic factors. CONCLUSION: The outcome of PBL is fair. Local control is excellent with RT or combined modality treatment but systemic relapses, including that in the CNS, occurs frequently.

Online teaching of inflammatory skin pathology by a French-speaking International University Network.

INTRODUCTION: Developments in technology, web-based teaching and whole slide imaging have broadened the teaching horizon in anatomic pathology. Creating online learning material including many types of media such as radiologic images, whole slides, videos, clinical and macroscopic photographs, is now accessible to most universities. Unfortunately, a major limiting factor to maintain and update the learning material is the amount of resources needed. In this perspective, a French-national university network was initiated in 2011 to build joint online teaching modules consisting of clinical cases and tests. The network has since expanded internationally to Québec, Switzerland and Ivory Coast. METHOD: One of the first steps of the project was to build a learning module on inflammatory skin pathology for interns and residents in pathology and dermatology. A pathology resident from Québec spent 6 weeks in France and Switzerland to develop the contents and build the module on an e-learning Moodle platform under the supervision of two dermatopathologists. The learning module contains text, interactive clinical cases, tests with feedback, virtual slides, images and clinical photographs. For that module, the virtual slides are decentralized in 2 universities (Bordeaux and Paris 7). Each university is responsible of its own slide scanning, image storage and online display with virtual slide viewers. RESULTS: The module on inflammatory skin pathology includes more than 50 web pages with French original content, tests and clinical cases, links to over 45 virtual images and more than 50 microscopic and clinical photographs. The whole learning module is being revised by four dermatopathologists and two senior pathologists. It will be accessible to interns and residents in the spring of 2014. The experience and knowledge gained from that work will be transferred to the next international resident whose work will be aimed at creating lung and breast pathology learning modules. CONCLUSION: The challenges of sustaining a project of this scope are numerous. The technical aspect of whole-slide imaging and storage needs to be developed by each university or group. The content needs to be regularly updated and its accuracy reviewed by experts in each individual domain. The learning modules also need to be promoted within the academic community to ensure maximal benefit for trainees. A collateral benefit of the project was the establishment of international partnerships between French-speaking universities and pathologists with the common goal of promoting pathology education through the use of multi-media technology including whole slide imaging.