Transposition of IS1397 in the family Enterobacteriaceae and first characterization of ISKpn1, a new insertion sequence associated with Klebsiella pneumoniae palindromic units.

IS1397 and ISKpn1 are IS3 family members which are specifically inserted into the loop of palindromic units (PUs). IS1397 is shown to transpose into PUs with sequences close or identical to the Escherichia coli consensus, even in other enterobacteria (Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Klebsiella oxytoca). Moreover, we show that homologous intergenic regions containing PUs constitute IS1397 transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn1, described here for the first time, is specific for PUs from K. pneumoniae, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.

Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein.

The highly conserved amino acid sequence ELDKWA of HIV-1 gp41 has been inserted into Escherichia coli MalE protein which had been shown to be an adequate carrier to present foreign epitopes to the immune system. We first investigated whether eight different permissive sites of MalE are able to tolerate an insertion of 7-50 residues encoding this epitope. Secondly, antigenicity of the epitope inserted in MalE protein was estimated from monoclonal antibody 2F5 binding analysis using the BIAcore(R) technology and its immunogenicity in mice was measured as the ability of hybrid proteins to elicit antibodies against a synthetic peptide containing this epitope. This study revealed a good correlation between the antigenicity of the inserted epitope and its immunogenicity. Increasing the length of the inserted epitope, as well as inserting multicopies of this epitope increased both its antigenicity and immunogenicity. However, none of the MalE hybrid proteins tested induced anti-HIV-1 neutralizing antibodies. This study strongly suggests that the capacity of the 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented.

Virtual university applied to telesurgery: from teleeducation to telemanipulation.

UNLABELLED: PROBLEM/BACKGROUND: In order to improve patient care by minimal invasive surgery (MIS), we perfected a Virtual TeleSurgical University that allows for teleeducation, teleconcertation, surgical planning and telemanipulation, through new Virtual Reality and multimedia systems. TOOLS AND METHODS: The organization of this innovative school was federated around three major research programs. First, the TESUS program focused on the teletransmission of medical information, allowing for videoconferencing around the world and telementoring. Next, the WeBS-Surg program is a multimedia continuous surgical education system on internet, that allows for teleeducation and teleconcertation between world experts in MIS. Then, the MASTER program (Minimal Access Surgery by Telecommunications and Robotics) allowed the development of the third millenium Operating room. It included Virtual Reality systems that delineate automatically anatomical and pathological structures of a patients from him CT-scan, and that allow for an interactive surgical planning and force-feed-back simulation. It also included a telesurgical robot named Zeus controlled by surgeons through telemanipulation system. RESULTS: Tests and validation shows that all these systems improved all steps of the surgical procedure: preoperatively due to a better continuous education and a computer assisted surgical planning, and peroperatively due to teleconcertation, telementoring and telemanipulation systems. CONCLUSION: Revolutionary tools for minimal invasive surgery learning, planning and performing are all ready available. These tools represents the first prototype of the computer assisted tele-robotical surgery that will be the future of surgery.

An automatic virtual patient reconstruction from CT-scans for hepatic surgical planning.

UNLABELLED: PROBLEM/BACKGROUND: In order to help hepatic surgical planning we perfected automatic 3D reconstruction of patients from conventional CT-scan, and interactive visualization and virtual resection tools. TOOLS AND METHODS: From a conventional abdominal CT-scan, we have developed several methods allowing the automatic 3D reconstruction of skin, bones, kidneys, lung, liver, hepatic lesions, and vessels. These methods are based on deformable modeling or thresholding algorithms followed by the application of mathematical morphological operators. From these anatomical and pathological models, we have developed a new framework for translating anatomical knowledge into geometrical and topological constraints. More precisely, our approach allows to automatically delineate the hepatic and portal veins but also to label the portal vein and finally to build an anatomical segmentation of the liver based on Couinaud definition which is currently used by surgeons all over the world. Finally, we have developed a user friendly interface for the 3D visualization of anatomical and pathological structures, the accurate evaluation of volumes and distances and for the virtual hepatic resection along a user-defined cutting plane. RESULTS: A validation study on a 30 patients database gives 2 mm of precision for liver delineation and less than 1 mm for all other anatomical and pathological structures delineation. An in vivo validation performed during surgery also showed that anatomical segmentation is more precise than the delineation performed by a surgeon based on external landmarks. This surgery planning system has been routinely used by our medical partner, and this has resulted in an improvement of the planning and performance of hepatic surgery procedures. CONCLUSION: We have developed new tools for hepatic surgical planning allowing a better surgery through an automatic delineation and visualization of anatomical and pathological structures. These tools represent a first step towards the development of an augmented reality system combined with computer assisted tele-robotical surgery.

Extending the CD4(+) T-cell epitope specificity of the Th1 immune response to an antigen using a Salmonella enterica serovar typhimurium delivery vehicle.

We analyzed the CD4 T-cell immunodominance of the response to a model antigen (Ag), MalE, when delivered by an attenuated strain of Salmonella enterica serovar Typhimurium (SL3261*pMalE). Compared to purified MalE Ag administered with adjuvant, the mapping of the peptide-specific proliferative responses showed qualitative differences when we used the Salmonella vehicle. We observed the disappearance of one out of eight MalE peptides' T-cell reactivity upon SL3261*pMalE immunization, but this phenomenon was probably due to a low level of T-cell priming, since it could be overcome by further immunization. The most striking effect of SL3261*pMalE administration was the activation and stimulation of new MalE peptide-specific T-cell responses that were silent after administration of purified Ag with adjuvant. Ag presentation assays performed with MalE-specific T-cell hybridomas showed that infection of Ag-presenting cells by this intracellular attenuated bacterium did not affect the processing and presentation of the different MalE peptides by major histocompatibility complex (MHC) class II molecules and therefore did not account for immunodominance modulation. Thus, immunodominance of the T-cell response to microorganisms is governed not only by the frequency of the available T-cell repertoire or the processing steps in Ag-presenting cells that lead to MHC presentation but also by other parameters probably related to the infectious process and to the bacterial products. Our results indicate that, upon infection by a microorganism, the specificity of the T-cell response induced against its Ags can be much more effective than with purified Ags and that it cannot completely be mimicked by purified Ags administered with adjuvant.

Immune responses induced by recombinant BCG strains according to level of production of a foreign antigen: malE.

A variety of viral, bacterial and parasitic antigens have been expressed in BCG and the capacity of these recombinant bacteria to induce immune responses has been well documented. However, little is known about the parameters influencing the induction of immune responses by recombinant BCG (rBCG), such as level of production and localization of the recombinant antigen. In the present study, we have constructed several rBCG strains expressing the malE gene from Escherichia coli which is either secreted or targeted to the cytoplasm or plasma membrane. Expression of malE was quantified by ELISA and localization was analyzed by flow cytometry. Even when using the same promoter, levels of cytoplasmic or membrane MalE production were far less than those from secreting strains using either mycobacterial or E. coli secretion signals. Stronger and more rapid immune responses were induced by rBCG strains with the highest levels of secreted MalE compared to cytoplasmic or membrane constructs, including both good humoral and proliferative responses in BALB/c, C57BL6 and even C3H mice, previously shown to be poor MalE responders. These results suggest that the levels of foreign antigen production play an important role in the induction of immune responses by rBCG strains.

IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements.

We demonstrate that IS1397, a putative mobile genetic element discovered in natural isolates of Escherichia coli, is active for transposition into the chromosome of E. coli K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to E. coli and Salmonella. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.

CTP:phosphocholine cytidylyltransferase: insights into regulatory mechanisms and novel functions.

A key regulatory enzyme in phosphatidylcholine biosynthesis, CTP:cholinephosphate cytidylyltransferase (CCT), catalyzes the formation of CDP-choline. This review discusses the essential features of CCT and addresses intriguing new insights into the catalytic and regulatory properties of this complex enzyme. Characterization of a lipid-binding segment in rat CCT is described and the role of lipids in CCT activation is discussed. An analysis of the phosphorylation domain is presented and possible physiological rationales for reversible phosphorylation of CCT are discussed. The nuclear localization of CCT is examined in the context of multiple CCT isoforms, as is recent evidence establishing a potential link between CCT activity and vesicular transport.

Short palindromic repetitive DNA elements in enterobacteria: a survey.

We present a survey of short palindromic repetitive elements in enterobacteria. Seven families are presented. Five were already known (RSA, IRU, 29-bp repeats, BIMEs and boxC), and their properties are updated; in particular, a new composite element is shown to include the formerly identified boxC repeats. Two repetitions, YPAL1 and YPAL2, found primarily in Yersinia, are described here for the first time.

Virtual reality applied to hepatic surgery simulation: the next revolution.

OBJECTIVE: This article describes a preliminary work on virtual reality applied to liver surgery and discusses the repercussions of assisted surgical strategy and surgical simulation on tomorrow's surgery. SUMMARY BACKGROUND DATA: Liver surgery is considered difficult because of the complexity and variability of the organ. Common generic tools for presurgical medical image visualization do not fulfill the requirements for the liver, restricting comprehension of a patient's specific liver anatomy. METHODS: Using data from the National Library of Medicine, a realistic three-dimensional image was created, including the envelope and the four internal arborescences. A computer interface was developed to manipulate the organ and to define surgical resection planes according to internal anatomy. The first step of surgical simulation was implemented, providing the organ with real-time deformation computation. RESULTS: The three-dimensional anatomy of the liver could be clearly visualized. The virtual organ could be manipulated and a resection defined depending on the anatomic relations between the arborescences, the tumor, and the external envelope. The resulting parts could also be visualized and manipulated. The simulation allowed the deformation of a liver model in real time by means of a realistic laparoscopic tool. CONCLUSIONS: Three-dimensional visualization of the organ in relation to the pathology is of great help to appreciate the complex anatomy of the liver. Using virtual reality concepts (navigation, interaction, and immersion), surgical planning, training, and teaching for this complex surgical procedure may be possible. The ability to practice a given gesture repeatedly will revolutionize surgical training, and the combination of surgical planning and simulation will improve the efficiency of intervention, leading to optimal care delivery.

[A new concept in surgery of the digestive tract: surgical procedure assisted by computer, from virtual reality to telemanipulation]. / Un nouveau concept en chirurgie digestive: la procédure chirurgicale assistée par ordinateur, de la réalité virtuelle à la télémanipulation.

Surgical simulation increasingly appears to be an essential aspect of tomorrow's surgery. The development of a hepatic surgery simulator is an advanced concept calling for a new writing system which will transform the medical world: virtual reality. Virtual reality extends the perception of our five senses by representing more than the real state of things by the means of computer sciences and robotics. It consists of three concepts: immersion, navigation and interaction. Three reasons have led us to develop this simulator: the first is to provide the surgeon with a comprehensive visualisation of the organ. The second reasons is to allow for planning and surgical simulation that could be compared with the detailed flight-plan for a commercial jet pilot. The third lies in the fact that virtual reality is an integrated part of the concept of computer assisted surgical procedure. The project consists of a sophisticated simulator which must include five requirements: a) visual fidelity, b) interactivity, c) physical properties, d) physiological properties, e) sensory input and output. In this report we describe how to obtain a realistic 3D model of the liver from bi-dimensional 2D medical images for anatomical and surgical training. The introduction of a tumor and the consequent planning and virtual resection is also described, as are force feedback and real-time interaction.

Salmonella abortusovis, strain Rv6, a new vaccinal vehicle for small ruminants.

Salmonella abortusovis strain Rv6 (Sao Rv6) is a live attenuated vaccine used for a few years to protect ewes against abortive salmonellosis. As Salmonellae, particularly Salmonella aro mutants, have considerable potential as vehicles for the presentation of heterologous vaccine antigens, Sao Rv6 was tested in order to develop a vaccinal vehicle for small ruminants. Five vector plasmids were tested in Sao Rv6; these plasmids, which carry Maltose Binding Protein (MBP) expressed as protein, but differ in their promotors, had been previously tested in S. typhimurium strain SL3261, and were transferred into Sao Rv6. The five plasmids were stable in vitro, and the recombinant Sao Rv6 expressed MBP at various levels. Intraperitoneal infection of OF1 mice with the recombinant bacteria did not modify the characteristics of Sao Rv6; dissemination and infection levels were similar in all groups and all mice developed antibodies to Salmonella antigens as measured by ELISA. In contrast, only animals immunized with Sao Rv6 carrying the pNTE plasmid developed a serum antibody response to MBP. This plasmid was then tested in sheep; following subcutaneous immunization with Sao Rv6-pNTE, dissemination and infection levels were not modified in comparison with sheep immunized with Sao Rv6 lacking plasmid. Antibodies specific to MBP were detected in sera of sheep immunized with Sao Rv6-pNTE, purified MBP, and with S. typhimurium SL3261-pNTE as positive controls. These results demonstrate that Sao Rv6 can be used as a vehicle for heterologous antigens in sheep with pNTE as plasmid vector.

Bacterial interspersed mosaic elements (BIMEs) are a major source of sequence polymorphism in Escherichia coli intergenic regions including specific associations with a new insertion sequence.

A significant fraction of Escherichia coli intergenic DNA sequences is composed of two families of repeated bacterial interspersed mosaic elements (BIME-1 and BIME-2). In this study, we determined the sequence organization of six intergenic regions in 51 E. coli and Shigella natural isolates. Each region contains a BIME in E. coli K-12. We found that multiple sequence variations are located within or near these BIMEs in the different bacteria. Events included excisions of a whole BIME-1, expansion/deletion within a BIME-2 and insertions of non-BIME sequences like the boxC repeat or a new IS element, named IS 1397. Remarkably, 14 out of IS 1397 integration sites correspond to a BIME sequence, strongly suggesting that this IS element is specifically associated with BIMEs, and thus inserts only in extragenic regions. Unlike BIMEs, IS 1397 is not detected in all E. coli isolates. Possible relationships between the presence of this IS element and the evolution of BIMEs are discussed.

Expression and immune response to foreign epitopes in bacteria. Perspectives for live vaccine development.

We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different regions of a carrier protein. By using as carriers, two bacterial envelope proteins, the LamB and MalE proteins of E. coli K12, we were able to express the same epitope in different sites of the two proteins and in different compartments of the bacteria. This allowed us to analyze the influence of the localization in E. coli cells of a foreign B-cell epitope on the induction of specific antibody responses, and the role of the molecular environment on the immunological properties of foreign B- or T-cell epitopes, using either purified hybrid proteins or live recombinant bacteria. Several LamB and MalE hybrid proteins were expressed in the aroA attenuated strain of S. typhimurium, SL3261. Immunizations of mice with live recombinant bacteria by the intravenous route showed that it was possible to induce humoral responses against inserted foreign sequences. In order to improve the in vivo stability of the plasmids carrying the different contructions, and to increase the amounts of recombinant LamB and MalE hybrid proteins expressed in vivo, the LamB and malE genes were placed under the control of the anaerobically inducible pnirBpromoter control. The genetic factors susceptible of influencing the immune response to recombinant Salmonella in mice were also studied.

[A new concept in digestive surgery: the computer assisted surgical procedure, from virtual reality to telemanipulation]. / Un nouveau concept en chirurgie digestive: la procédure chirurgicale assistée par ordinateur, de la réalité virtuelle à la télémanipulation.

Surgical simulation increasingly appears to be an essential aspect of tomorrow's surgery. The development of a hepatic surgery simulator is an advanced concept calling for a new writing system which will transform the medical world: virtual reality. Virtual reality extends the perception of our five senses by representing more than the real state of things by the means of computer sciences and robotics. It consists of three concepts: immersion, navigation and interaction. Three reasons have led us to develop this simulator: the first is to provide the surgeon with a comprehensive visualisation of the organ. The second reason is to allow for planning and surgical simulation that could be compared with the detailed flight-plan for a commercial jet pilot. The third lies in the fact that virtual reality is an integrated part of the concept of computer assisted surgical procedure. The project consists of a sophisticated simulator which has to include five requirements: visual fidelity, interactivity, physical properties, physiological properties, sensory input and output. In this report we will describe how to get a realistic 3D model of the liver from bi-dimensional 2D medical images for anatomical and surgical training. The introduction of a tumor and the consequent planning and virtual resection is also described, as are force feedback and real-time interaction.

Oral candidosis in long-term hospital care: comparison of edentulous and dentate subjects.

OBJECTIVE: To obtain information on the oral health status of the elderly living in a medicalized, geriatric institution. DESIGN: A cross-sectional clinical investigation with complementary microbiological studies. SUBJECTS AND METHODS: A cohort of 233 elderly in one long-term care ward; collection of demographic data; clinical examination to determine dental and prosthetic status and health of the oral mucosae; swabs for detection of mucosal and denture colonization by Candida; paraffin stimulated saliva for detection of colonization by mutans streptococci and lactobacilli. MAIN OUTCOME MEASURES: Oral and denture hygiene; oral mucosal health; degree of colonization by Candida, mutans streptococci and lactobacilli. RESULTS: Mean age of the 233 patients was 85.6 +/- 6.9 years; 61% were totally dependent, 62.7% were wearing one or two complete dentures; 19.7% had natural teeth and no denture and 17.6% neither teeth nor denture. Of those wearing dentures 72% had denture stomatitis. Of those with natural teeth 72% were affected by active caries. Yeast counts were significantly correlated with the intensity of the erythema of the palatal mucosa, plaque score of the natural teeth, denture plaque score, and salivary counts of mutans streptococci and lactobacilli. CONCLUSIONS: High oral yeast counts and frequent prevalence of oral candidosis in elderly subjects living in institutions are associated with poor oral hygiene and neglect of denture care.

Control by H-2 genes of the Th1 response induced against a foreign antigen expressed by attenuated Salmonella typhimurium.

Attenuated salmonellae represent an attractive vehicle for the delivery of heterologous protective antigens to the immune system. Here, we have investigated the influence of the genetic background of the host which regulates the growth and elimination of Salmonella cells on the cellular response induced against a foreign antigen delivered by an aroA Salmonella strain. We have tested CD4+ T-cell responses (cell proliferation and cytokine production) in various mouse strains following immunization with Salmonella typhimurium SL3261 expressing a high level of the recombinant Escherichia coli MalE protein. We were able to detect a CD4+ T-cell response against the recombinant MalE protein only in a restricted number of mouse strains, whereas all mice produced good levels of anti-MalE immunoglobulin G antibodies. The Ity gene did not play a major role in these differences in T-cell responses, since both Ity-resistant and -susceptible strains of mice were found to be unresponsive to MalE delivered by recombinant salmonellae. In contrast, when B10 congenic mice were used, a correlation was established between MalE-specific T-cell unresponsiveness and H-2 genes. The discrepancies described in this paper in the ability of various strains of mice to develop an efficient Th1 response against a recombinant antigen displayed by a live Salmonella vaccine underscore the difficulties that can be encountered in the vaccination of human populations by such a strategy.

Expression and biological activity of genetic fusions between MalE, the maltose binding protein from Escherichia coli and portions of CD4, the T-cell receptor of the AIDS virus.

Hybrid molecules between MalE, the periplasmic maltose binding protein of Escherichia coli, and CD4, the human T-lymphocyte receptor for the AIDS virus HIV, have been constructed and purified. We show that CD4 can be fused as multiple repeats to both ends of a single MalE molecule. Hybrid proteins are exported into the periplasm of bacteria, bind monoclonal antibodies directed against CD4, bind HIV gp160, and inhibit HIV binding to CD4+ cells. MalE has been used as a scaffold to graft portions of CD4. Deletion analysis allowed to define a minimal structural domain which folds in a way which is compatible with its biological activity. This minimal part was used to design compact hybrid molecules in which CD4 was inserted internally into MalE.