Biocompatibilité des microcapsules d'alginate : purification d'alginate, réaction immunitaire de l'hôte et protection du receveur

L’immuno-isolation des îlots de Langerhans est proposée comme moyen d’effectuer des transplantations sans prise d’immunosuppresseurs par le patient. Cette immuno-isolation, par l’entremise d’une microcapsule composée d’alginate et de poly-L-lysine (microcapsule APA), protège le greffon d’une éventuelle attaque du système immunitaire du receveur grâce à sa membrane semi-perméable. Cette membrane empêche le système immunitaire du receveur de pénétrer la microcapsule tout en laissant diffuser librement les nutriments, le glucose et l’insuline. Avant l’application de cette technique chez l’humain, quelques défis doivent encore être relevés, dont la biocompatibilité de ce système. La biocompatibilité fait ici référence à la biocompatibilité du biomatériau utilisé pour la fabrication des microcapsules, l’alginate, mais aussi la biocompatibilité des microcapsules reliée à leur stabilité. En effet, il a été remarqué que, lors d’implantation in vivo de microcapsules fabriquées avec de l’alginate non purifiée, ceci induisait un phénomène nommé Réaction de l’Hôte contre la Microcapsule (RHM). De plus, il est connu que la stabilité des microcapsules APA peut influencer leur biocompatibilité puisqu’une microcapsule endommagée ou brisée pourrait laisser s’échapper les cellules du greffon chez le receveur. Nous croyons qu’une compréhension des processus d’initiation de la RHM en fonction de l’efficacité des procédés de purification d’alginate (et donc des quantités de contaminants présents dans l’alginate) ainsi que l’augmentation de la stabilité des microcapsules APA pourront améliorer la biocompatibilité de ce dispositif, ce que tente de démontrer les résultats présentés dans cette thèse. En effet, les résultats obtenus suggèrent que les protéines qui contaminent l’alginate jouent un rôle clé dans l’initiation de la RHM et qu’en diminuant ces quantités de protéines par l’amélioration des procédés de purification d’alginate, on améliore la biocompatibilité de l’alginate. Afin d’augmenter la stabilité des microcapsules APA, nous décrivons une nouvelle technique de fabrication des microcapsules qui implique la présence de liaisons covalentes. Ces nouvelles microcapsules APA réticulées sont très résistantes, n’affectent pas de façon négative la survie des cellules encapsulées et confinent les cellules du greffon à l’intérieur des microcapsules. Cette dernière caractéristique nous permet donc d’augmenter la biocompatibilité des microcapsules APA en protégeant le receveur contre les cellules du greffon.

Isolation of monocytes from human peripheral blood using immuno-affinity expanded-bed adsorption

A novel technique for the separation of monocytes from human peripheral blood preparations has been developed. The technique is based on the use of expanded-bed adsorption and a solid perfluorocarbon derivatized with avidin or streptavidin for the indirect positive or negative capture of cells labeled with biotinylated monoclonal antibodies. The perfluorocarbon support was prepared and characterized and the contactor design and operating conditions, that enable cells to be selectively isolated, were investigated. Experiments consisted of applying an immunolabeled pulse of 1 x 10(8) peripheral blood mononuclear cells (PBMCs), isolated by density gradient centrifugation, directly onto a refrigerated expanded bed. The major cell types remaining were T-lymphocytes, B-lymphocytes, and monocytes. Monocytes could be positively adsorbed, following labeling with anti-CD14 mAb, with a clearance of up to 89% and a depletion factor of 7.6. They could also be

Saponins from Quillaja saponaria Molina: Isolation, Characterization and Ability to Form Immuno Stimulatory Complexes (ISCOMs)

ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. They are colloidal particles typically comprised of phospholipids, cholesterol and Quil A, a crude mixture of saponins extracted from the bark of Quillaja saponaria Molina. We have previously shown that ISCOMs can be prepared by ether injection wherein an ether solution of phospholipids and cholesterol in a mass ratio of 5:2 is injected into a solution of Quil A at a mass ratio of 7 lipids: 3 Quil A. The aim of this study was firstly to isolate and characterise discrete fractions of Quil A and secondly to investigate which of these fractions were able to form ISCOMs by the method of ether injection. Six fractions of Quil A were isolated by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and characterised by analytical HPLC, liquid chromatography tandem mass spectrometry (LC-MS) and the qualitative Liebermann- Burchard and Molisch tests for triterpenoids and carbohydrates respectively. ISCOMs were subsequently prepared from the isolated fractions by the method of ether injection and the resulting preparations characterized by photon correlation spectroscopy (PCS) and negative stain transmission electron microscopy (TEM). The molecular weights of the major compounds in the fractions ranged from ∼1200 to ∼2300 Da; all fractions tested positive for triterpenoids and saccharides and four of the fractions were identified as QS-7, QS-17, QS-18 and QS-21 by analysis (LC-MS and analytical HPLC). Injection of ether solutions of lipids into aqueous solutions of QS-17, QS-18 or QS-21 all resulted in homogeneous ISCOM dispersions. The combination of lipids and QS-7 by ether injection produced lamellae and liposomes as the prominent structures and a minor amount of ISCOMs. The remaining two hydrophilic, low molecular weight fractions of Quil A did not produce ISCOMs, instead liposomes and helical structures predominated in the samples.

The isolation and partial biochemical analysis of Sindbis virus proteins

Recently, the amino acid sequences have been reported for several proteins, including the envelope glycoproteins of Sindbis virus, which all probably span the plasma membrane with a common topology: a large N-terminal, extracellular portion, a short region buried in the bilayer, and a short C-terminal intracellular segment. The regions of these proteins buried in the bilayer correspond to portions of the protein sequences which contain a stretch of hydrophobic amino acids and which have other common characteristics, as discussed. Reasons are also described for uncertainty, in some proteins more than others, as to the precise location of some parts of the sequence relative to the membrane.

The signal hypothesis for the transmembrane translocation of proteins is briefly described and its general applicability is reviewed. There are many proteins whose translocation is accurately described by this hypothesis, but some proteins are translocated in a different manner.

The transmembraneous glycoproteins E1 and E2 of Sindbis virus, as well as the only other virion protein, the capsid protein, were purified in amounts sufficient for biochemical analysis using sensitive techniques. The amino acid composition of each protein was determined, and extensive N-terminal sequences were obtained for E1 and E2. By these techniques E1 and E2 are indistinguishable from most water soluble proteins, as they do not contain an obvious excess of hydrophobic amino acids in their N-terminal regions or in the intact molecule.

The capsid protein was found to be blocked, and so its N-terminus could not be sequenced by the usual methods. However, with the use of a special labeling technique, it was possible to incorporate tritiated acetate into the N-terminus of the protein with good specificity, which was useful in the purification of peptides from which the first amino acids in the N-terminal sequence could be identified.

Nanomole amounts of PE2, the intracellular precursor of E2, were purified by an immuno-affinity technique, and its N-terminus was analyzed. Together with other work, these results showed that PE2 is not synthesized with an N-terminal extension, and the signal sequence for translocation is probably the N-terminal amino acid sequence of the protein. This N-terminus was found to be 80-90% blocked, also by Nacetylation, and this acetylation did not affect its function as a signal sequence. The putative signal sequence was also found to contain a glycosylated asparagine residue, but the inhibition of this glycosylation did not lead to the cleavage of the sequence.

Immuno-reactive proteins from Mycobacterium immunogenum useful for serodiagnosis of metalworking fluid hypersensitivity pneumonitis.

Metalworking fluid-associated hypersensitivity pneumonitis (MWF-HP) is a pulmonary disease caused by inhaling microorganisms present in the metalworking fluids used in the industrial sector. Mycobacterium immunogenum is the main etiological agent. Among the clinical, radiological and biological tools used for diagnosis, serological tests are important. The aim of this study was to identify immunogenic proteins in M. immunogenum and to use recombinant antigens for serological diagnosis of MWF-HP. Immunogenic proteins were detected by two-dimensional Western blot and candidate proteins were identified by mass spectrometry. Recombinant antigens were expressed in Escherichia coli and tested by enzyme-linked immunosorbent assay (ELISA) with the sera of 14 subjects with MWF-HP and 12 asymptomatic controls exposed to M. immunogenum. From the 350 spots visualized by two-dimensional gel electrophoresis with M. immunogenum extract, 6 immunogenic proteins were selected to be expressed as recombinant antigens. Acyl-CoA dehydrogenase antigen allowed for the best discrimination of MWF-HP cases against controls with an area under the receiver operating characteristics (ROC) curve of 0.930 (95% CI=0.820-1), a sensitivity of 100% and a specificity of 83% for the optimum threshold. Other recombinant antigens correspond to acyl-CoA dehydrogenase FadE, cytosol aminopeptidase, dihydrolipoyl dehydrogenase, serine hydroxymethyltransferase and superoxide dismutase. This is the first time that recombinant antigens have been used for the serodiagnosis of hypersensitivity pneumonitis. The availability of recombinant antigens makes it possible to develop standardized serological tests which in turn could simplify diagnosis, thus making it less invasive.

Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans

It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of Chironomus tentans provide a suitable model system for studying gene expression in situ, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in C. tentans named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of C. tentans salivary gland cells. Hrp59 is a C. tentans homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in Drosophila cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences. Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1. Hrp65 binds to HEL/UAP56 in C. tentans cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.

Comparison of methods for processing drinking water samples for the isolation of Myocbacterium avium and intracellulare

Several protocols for isolation of mycobacteria from water exist, but there is no established standard method. This study compared methods of processing potable water samples for the isolation of Mycobacterium avium and Mycobacterium intracellulare using spiked sterilized water and tap water decontaminated using 0.005% cetylpyridinium chloride (CPC). Samples were concentrated by centrifugation or filtration and inoculated onto Middlebrook 7H10 and 7H11 plates and Lowenstein-Jensen slants and into mycobacterial growth indicator tubes with or without polymyxin, azlocillin, nalidixic acid, trimethoprim, and amphotericin B. The solid media were incubated at 32°C, at 35°C, and at 35°C with CO2 and read weekly. The results suggest that filtration of water for the isolation of mycobacteria is a more sensitive method for concentration than centrifugation. The addition of sodium thiosulfate may not be necessary and may reduce the yield. Middlebrook M7H10 and 7H11 were equally sensitive culture media. CPC decontamination, while effective for reducing growth of contaminants, also significantly reduces mycobacterial numbers. There was no difference at 3 weeks between the different incubation temperatures.

50 Years of Isolation

The traditional means for isolating applications from each other is via the use of operating system provided “process” abstraction facilities. However, as applications now consist of multiple fine-grained components, the traditional process abstraction model is proving to be insufficient in ensuring this isolation. Statistics indicate that a high percentage of software failure occurs due to propagation of component failures. These observations are further bolstered by the attempts by modern Internet browser application developers, for example, to adopt multi-process architectures in order to increase robustness. Therefore, a fresh look at the available options for isolating program components is necessary and this paper provides an overview of previous and current research on the area.

Vibration isolation for autonomous helicopter flight

The mechanisms of helicopter flight create a unique, high-vibration environment which can play havoc with the accurate operation of on-board sensors. Vibration isolation of electronic sensors from structural borne oscillations is paramount to their reliable and accurate use. Effective isolation is achieved by realising a trade-off between the properties of the suspended instrument package, and the isolation mechanism. This is made more difficult as the weight and size of the sensors and computing hardware decreases with advances in technology. This paper presents a history of the design, challenges, constraints and construction of an integrated isolated vision and sensor platform and landing gear for the CSIRO autonomous X-Cell helicopter. The results of isolation performance and in-flight tests of the platform in autonomous flight are presented.

Ovine bone and marrow derived progenitor cells : isolation, characterization, and osteogenic potential

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) for their potential clinical use in bone engineering. Prior to clinical application, MPC-based treatment concepts need to be evaluated in preclinical, immunocompetent, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, ovine MPC and their osteogenic potential remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than osteoblasts (OB) derived from tibial compact bone as assessed using standard 2D culture. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14-/CD31-/CD45- /CD29+/CD44+/CD166+) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed >90%. Cells displayed a spindle-like morphology and formed an interconnected network. Implanted subcutaneously into NOD/SCID mice on type I collagen coated polycaprolactone-tricalciumphosphate (mPCL-TCP) scaffolds, MPC presented a higher developmental potential than osteoblasts. In summary, this study provides a detailed in vitro characterisation of ovine MPC from a bone engineering perspective and suggests that MPC provide promising means for future bone disease related treatment applications.