Percoll centrifugation eliminates mold contaminants from cell cultures

This work was supported by a grant and research associateship to N.A. from the Medical Research Council of Canada and the National Cancer Institute of Canada.     

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, ^p1YMESRADR⁸, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (^pE315-^pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.     

Fatal Histoplasma capsulatum Mitral Endocarditis in a French Patient Treated for Rheumatoid Arthritis

Histoplasmosis is an infectious disease caused by the inhalation of Histoplasma capsulatum spores, a fungus encountered in many diverse areas around the world. Although this infection is often asymptomatic, it may become dramatic in immunocompromised patients. In November 2005, an endocarditis due to Histoplasma capsulatum was diagnosed in a French woman treated for rheumatoid arthritis and who had traveled to South America 2 years earlier. We confirmed the biological diagnosis by mycological, serological, and histological methods. In spite of receiving the appropriate treatment, the patient died 3 months later of cardiac insufficiency. We report here this additional case of Histoplasma endocarditis, by hoping to help rapid and accurate diagnosis of such infections in their early stages of development, in non-endemic areas.     

Pattern of MHC class I and immune proteasome expression in Walker 256 tumor during growth and regression in Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis

Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.     

Single cell co-amplification of polymorphic markers for the indirect preimplantation genetic diagnosis of hemophilia A,X-linked adrenoleukodystrophy,X-linked hydrocephalus and incontinentia pigmenti loci on Xq28

Preimplantation genetic diagnosis (PGD) first consisted of the selection of female embryos for patients at risk of transmitting X-linked recessive diseases. Advances in molecular biology now allow the specific diagnosis of almost any Mendelian disease. For families with an identified X-linked recessive disease-causing mutation, non-specific diagnosis by sex identification can be considered as a sub-standard method, since it involves the unnecessary disposal of healthy male embryos and reduces success rate by diminishing the pool of embryos eligible for transfer. The most telomeric part of the X-chromosome long arm is a highly gene-rich region encompassing disease genes such as haemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti. We developed five single-cell triplex amplification protocols with microsatellite markers DXS1073, DXS9901 (BGN), G6PD, DXS1108, DXS8087 and F8C-IVS13 located in this Xq terminal region. These tests allow the diagnosis of all diseases previously mentioned providing that the genetic material allowing the identification of the morbid allele can be obtained. The choice of the microsatellite set to use depends on the localisation of the gene responsible for the diagnosed pathology and on the informativity of the markers in particular families. Single-cell amplification efficiency was assessed on single lymphocytes. Amplification rate of the different markers ranged from 89–97% with an allele drop out rate of 2–19 %. So far PGD has been carried out for three carrier females at risk of transmitting X-linked adrenoleukodystrophy, X-linked hydrocephalus and hemophilia A. The latter one is now pregnant.     

Extracellular Bacterial Pathogen Induces Host Cell Surface Reorganization to Resist Shear Stress

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow–generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.