Update of the BIPM comparison BIPM.RI(II)-K1.Ho-166m activity measurements to include the IRA and the NPL and a re-evaluation of the degrees of equivalence for the APMP.RI(II)-K2.Ho-166m comparison

The IRA and the NPL have submitted ampoules of 166Hom to the International Reference System (SIR) for activity comparison at the Bureau International des Poids et Mesures, thus becoming the third and fourth participants since 1989. The five samples of known activity of 166Hom now recorded in the SIR have activities between about 70 kBq and 500 kBq. The new results have enabled a re-evaluation of the key comparison reference value, and the degrees of equivalence between each equivalent activity measured in the SIR and the key comparison reference value (KCRV) have been calculated. The results are given in the form of a matrix for these four NMIs together with the recalculated degrees of equivalence of an APMP regional comparison held in 2000, comparison identifier APMP.RI(II)-K2.Ho-166m for six other NMIs. A graphical presentation is also given.

Human immunoglobulins and Fc fragments promote microtubule assembly via tau proteins and induce conformational changes of neuronal microtubules in vitro.

The influence of human immunoglobulins (Ig) in neuronal cytoskeleton stability was studied in vitro. Here we show that human Ig and Fc fragments stimulate animal and human microtubule assembly by binding to microtubules via tau isoforms. In presence of Ig, microtubules show increased aggregation, twisting and rigidity. Non-immune Ig and Fc fragments promote microtubule assembly in temperature-dependent manner and stabilize microtubules at a molecular ratio of 1 Ig per 4 tubulin dimers. These in vitro data provide an experimental support for an immuno-mediated modulation of the cytoskeleton. In conjunction with previous neuropathological data, they suggest that Ig could participate in early stages of neurodegeneration by affecting the microtubule stability in vivo.

Unusual association of diseases/symptoms: Encephalitis with herpes simplex-2 in the cerebrospinal fluid and anti-RI (ANNA-2) antibodies: an infectious or a paraneoplastic syndrome?

We report on a 70-year-old woman with partial complex status epilepticus who was initially diagnosed with herpes simplex-2 (HSV-2) encephalitis, based on brain magnetic resonance imaging (MRI) findings, cerebrospinal fluid (CSF) lymphocytic pleocytosis and HSV-2 DNA detection by polymerase chain reaction (PCR) in the CSF, but without improvement on intravenous acyclovir. Anti-Ri antibodies were positive and computed tomography (CT) investigations revealed a small cell carcinoma at biopsy suggesting paraneoplastic encephalitis. The outcome was unfavourable and the autopsy showed typical features of paraneoplastic encephalitis but no evidence of viral inclusions. This case report is interesting because: (1) it is the first report of an autopsy proven paraneoplastic widespread encephalitis with anti-Ri antibodies; (2) despite a positive HSV-2 PCR in the CSF, there was no sign of herpetic infections of the nervous system; and (3) it illustrates the fact that if paraneoplastic antibodies are usually good markers of the underlying tumour, they are not always predictive of neurological deficits.

Antigen binding to secretory immunoglobulin A results in decreased sensitivity to intestinal proteases and increased binding to cellular Fc receptors.

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA x antigen immune complexes are selectively transported across Peyer's patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (Fc alphaRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.

Fc block treatment, dead cells exclusion, and cell aggregates discrimination concur to prevent phenotypical artifacts in the analysis of subpopulations of tumor-infiltrating CD11b(+) myelomonocytic cells.

It is well established that cancer cells can recruit CD11b(+) myeloid cells to promote tumor angiogenesis and tumor growth. Increasing interest has emerged on the identification of subpopulations of tumor-infiltrating CD11b(+) myeloid cells using flow cytometry techniques. In the literature, however, discrepancies exist on the phenotype of these cells (Coffelt et al., Am J Pathol 2010;176:1564-1576). Since flow cytometry analysis requires particular precautions for accurate sample preparation and trustable data acquisition, analysis, and interpretation, some discrepancies might be due to technical reasons rather than biological grounds. We used the syngenic orthotopic 4T1 mammary tumor model in immunocompetent BALB/c mice to analyze and compare the phenotype of CD11b(+) myeloid cells isolated from peripheral blood and from tumors, using six-color flow cytometry. We report here that the nonspecific antibody binding through Fc receptors, the presence of dead cells and cell doublets in tumor-derived samples concur to generate artifacts in the phenotype of tumor-infiltrating CD11b(+) subpopulations. We show that the heterogeneity of tumor-infiltrating CD11b(+) subpopulations analyzed without particular precautions was greatly reduced upon Fc block treatment, dead cells, and cell doublets exclusion. Phenotyping of tumor-infiltrating CD11b(+) cells was particularly sensitive to these parameters compared to circulating CD11b(+) cells. Taken together, our results identify Fc block treatment, dead cells, and cell doublets exclusion as simple but crucial steps for the proper analysis of tumor-infiltrating CD11b(+) cell populations.

Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms.

The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was observed. Finally, all formulations proved to be biocompatible with both ISO compliant L929 fibroblasts and human MG63 osteoblast-like cells.

Absorbance enhancement in microplate wells for improved-sensitivity biosensors

A generic optical biosensing strategy was developed that relies on the absorbance enhancement phenomenon occurring in a multiple scattering matrix. Experimentally, inserts made of glass fiber membrane were placed into microplate wells in order to significantly lengthen the trajectory of the incident light through the sample and therefore increase the corresponding absorbance. Enhancement factor was calculated by comparing the absorbance values measured for a given amount of dye with and without the absorbance-enhancing inserts in the wells. Moreover, the dilution of dye in solutions with different refractive indices (RI) clearly revealed that the enhancement factor increased with the ΔRI between the membrane and the surrounding medium, reaching a maximum value (EF>25) when the membranes were dried. On this basis, two H2O2-biosensing systems were developed based on the biofunctionalization of the glass fiber inserts either with cytochrome c or horseradish peroxidase (HRP) and the analytical performances were systematically compared with the corresponding bioassay in solution. The efficiency of the absorbance-enhancement approach was particularly clear in the case of the cytochrome c-based biosensor with a sensitivity gain of 40 folds and wider dynamic range. Therefore, the developed strategy represents a promising way to convert standard colorimetric bioassays into optical biosensors with improved sensitivity.

Modeling resting-state functional networks when the cortex falls asleep: local and global changes.

The transition from wakefulness to sleep represents the most conspicuous change in behavior and the level of consciousness occurring in the healthy brain. It is accompanied by similarly conspicuous changes in neural dynamics, traditionally exemplified by the change from "desynchronized" electroencephalogram activity in wake to globally synchronized slow wave activity of early sleep. However, unit and local field recordings indicate that the transition is more gradual than it might appear: On one hand, local slow waves already appear during wake; on the other hand, slow sleep waves are only rarely global. Studies with functional magnetic resonance imaging also reveal changes in resting-state functional connectivity (FC) between wake and slow wave sleep. However, it remains unclear how resting-state networks may change during this transition period. Here, we employ large-scale modeling of the human cortico-cortical anatomical connectivity to evaluate changes in resting-state FC when the model "falls asleep" due to the progressive decrease in arousal-promoting neuromodulation. When cholinergic neuromodulation is parametrically decreased, local slow waves appear, while the overall organization of resting-state networks does not change. Furthermore, we show that these local slow waves are structured macroscopically in networks that resemble the resting-state networks. In contrast, when the neuromodulator decrease further to very low levels, slow waves become global and resting-state networks merge into a single undifferentiated, broadly synchronized network.

HARNESSING iNKT CELLS WITH RECOMBINANT CD1d FUSION PROTEINS TO REDIRECT INNATE AND ADAPTIVE IMMUNITY TO THE TUMOR

Les thérapies du cancer, comme la radiothérapie et la chimiothérapie, sont couramment utilisées mais ont de nombreux effets secondaires. Ces thérapies invasives pour le patient nécessitent d'être améliorées et de nombreuses avancées ont été faites afin d'adapter et de personnaliser le traitement du cancer. L'immunothérapie a pour but de renforcer le système immunitaire du patient et de le rediriger de manière spécifique contre la tumeur. Dans notre projet, nous activons les lymphocytes Invariant Natural Killer T (iNKT) afin de mettre en place une immunothérapie innovatrice contre le cancer. Les cellules iNKT sont une unique sous-population de lymphocytes T qui ont la particularité de réunir les propriétés de l'immunité innée ainsi qu'adaptative. En effet, les cellules iNKT expriment à leur surface des molécules présentes aussi sur les cellules tueuses NK, caractéristique de l'immunité innée, ainsi qu'un récepteur de cellules T (TCR) qui représente l'immunité adaptative. Les cellules iNKT reconnaissent avec leur TCR des antigènes présentés par la molécule CD1d. Les antigènes sont des protéines, des polysaccharides ou des lipides reconnus par les cellules du système immunitaire ou les anticorps pour engendrer une réponse immunitaire. Dans le cas des cellules iNKT, l'alpha-galactosylceramide (αGC) est un antigène lipidique fréquemment utilisé dans les études cliniques comme puissant activateur. Après l'activation des cellules iNKT avec l'αGC, celles-ci produisent abondamment et rapidement des cytokines. Ces cytokines sont des molécules agissant comme des signaux activateurs d'autres cellules du système immunitaire telles que les cellules NK et les lymphocytes T. Cependant, les cellules iNKT deviennent anergiques après un seul traitement avec l'αGC c'est à dire qu'elles ne peuvent plus être réactivées, ce qui limite leur utilisation dans l'immunothérapie du cancer. Dans notre groupe, Stirnemann et al ont publié une molécule recombinante innovante, composée de la molécule CD1d soluble et chargée avec le ligand αGC (αGC/sCD1d). Cette protéine est capable d'activer les cellules iNKT tout en évitant l'anergie. Dans le système immunitaire, les anticorps sont indispensables pour combattre une infection bactérienne ou virale. En effet, les anticorps ont la capacité de reconnaître et lier spécifiquement un antigène et permettent l'élimination de la cellule qui exprime cet antigène. Dans le domaine de l'immunothérapie, les anticorps sont utilisés afin de cibler des antigènes présentés seulement par la tumeur. Ce procédé permet de réduire efficacement les effets secondaires lors du traitement du cancer. Nous avons donc fusionné la protéine recombinante αGC/CD1d à un fragment d'anticorps qui reconnaît un antigène spécifique des cellules tumorales. Dans une étude préclinique, nous avons démontré que la protéine αGC/sCD1d avec un fragment d'anticorps dirigé contre la tumeur engendre une meilleure activation des cellules iNKT et entraîne un effet anti-tumeur prolongé. Cet effet anti-tumeur est augmenté comparé à une protéine αGC/CD1d qui ne cible pas la tumeur. Nous avons aussi montré que l'activation des cellules iNKT avec la protéine αGC/sCD1d-anti-tumeur améliore l'effet anti- tumoral d'un vaccin pour le cancer. Lors d'expériences in vitro, la protéine αGC/sCD1d-anti- tumeur permet aussi d'activer les cellules humaines iNKT et ainsi tuer spécifiquement les cellules tumorales humaines. La protéine αGC/sCD1d-anti-tumeur représente une alternative thérapeutique prometteuse dans l'immunothérapie du cancer. - Les cellules Invariant Natural Killer T (iNKT), dont les effets anti-tumoraux ont été démontrés, sont de puissants activateurs des cellules Natural Killer (NK), des cellules dendritiques (DC) et des lymphocytes T. Cependant, une seule injection du ligand de haute affinité alpha-galactosylceramide (αGC) n'induit une forte activation des cellules iNKT que durant une courte période. Celle-ci est alors suivie d'une longue phase d'anergie, limitant ainsi leur utilisation pour la thérapie. Comme alternative prometteuse, nous avons montré que des injections répétées d'αGC chargé sur une protéine recombinante de CD1d soluble (αGC/sCD1d) chez la souris entraînent une activation prolongée des cellules iNKT, associée à une production continue de cytokine. De plus, le maintien de la réactivité des cellules iNKT permet de prolonger l'activité anti-tumorale lorsque la protéine αGC/sCD1d est fusionnée à un fragment d'anticorps (scFv) dirigé contre la tumeur. L'inhibition de la croissance tumorale n'est optimale que lorsque les souris sont traitées avec la protéine αGC/sCD1d-scFv ciblant la tumeur, la protéine αGC/sCD1d-scFv non-appropriée étant moins efficace. Dans le système humain, les protéines recombinantes αGC/sCD1d-anti-HER2 et anti-CEA sont capables d'activer et de faire proliférer des cellules iNKT à partir de PBMCs issues de donneurs sains. De plus, la protéine αGC/sCD1d-scFv a la capacité d'activer directement des clones iNKT humains en l'absence de cellules présentatrices d'antigènes (CPA), contrairement au ligand αGC libre. Mais surtout, la lyse des cellules tumorales par les iNKT humaines n'est obtenue que lorsqu'elles sont incubées avec la protéine αGC/sCD1d-scFv anti- tumeur. En outre, la redirection de la cytotoxicité des cellules iNKT vers la tumeur est supérieure à celle obtenue avec une stimulation par des CPA chargées avec l'αGC. Afin d'augmenter les effets anti-tumoraux, nous avons exploité la capacité des cellules iNKT à activer l'immunité adaptive. Pour ce faire, nous avons combiné l'immunothérapie NKT/CD1d avec un vaccin anti-tumoral composé d'un peptide OVA. Des effets synergiques ont été obtenus lorsque les traitements avec la protéine αGC/sCD1d-anti-HER2 étaient associés avec le CpG ODN comme adjuvant pour la vaccination avec le peptide OVA. Ces effets ont été observés à travers l'activation de nombreux lymphocytes T CD8+ spécifique de la tumeur, ainsi que par la forte expansion des cellules NK. Les réponses, innée et adaptive, élevées après le traitement avec la protéine αGC/sCD1d-anti-HER2 combinée au vaccin OVA/CpG ODN étaient associées à un fort ralentissement de la croissance des tumeurs B16- OVA-HER2. Cet effet anti-tumoral corrèle avec l'enrichissement des lymphocytes T CD8+ spécifiques observé à la tumeur. Afin d'étendre l'application des protéines αGC/sCD1d et d'améliorer leur efficacité, nous avons développé des fusions CD1d alternatives. Premièrement, une protéine αGC/sCD1d dimérique, qui permet d'augmenter l'avidité de la molécule CD1d pour les cellules iNKT. Dans un deuxième temps, nous avons fusionné la protéine αGC/sCD1d avec un scFv dirigé contre le récepteur 3 du facteur de croissance pour l'endothélium vasculaire (VEGFR-3), afin de cibler l'environnement de la tumeur. Dans l'ensemble, ces résultats démontrent que la thérapie médiée par la protéine recombinante αGC/sCD1d-scFv est une approche prometteuse pour rediriger l'immunité innée et adaptive vers le site tumoral. - Invariant Natural Killer T cells (iNKT) are potent activators of Natural Killer (NK), dendritic cells (DC) and T lymphocytes, and their anti-tumor activities have been well demonstrated. However, a single injection of the high affinity CD1d ligand alpha-galactosylceramide (αGC) leads to a strong but short-lived iNKT cell activation followed by a phase of long-term anergy, limiting the therapeutic use of this ligand. As a promising alternative, we have demonstrated that when αGC is loaded on recombinant soluble CD1d molecules (αGC/sCD1d), repeated injections in mice led to the sustained iNKT cell activation associated with continued cytokine secretion. Importantly, the retained reactivity of iNKT cell led to prolonged antitumor activity when the αGC/sCD1d was fused to an anti-tumor scFv fragments. Optimal inhibition of tumor growth was obtained only when mice were treated with the tumor-targeted αGC/CD1d-scFv fusion, whereas the irrelevant αGC/CD1d-scFv fusion was less efficient. When tested in a human system, the recombinant αGC/sCD1d-anti-HER2 and -anti-CEA fusion proteins were able to expand iNKT cells from PBMCs of healthy donors. Furthermore, the αGC/sCD1d-scFv fusion had the capacity to directly activate human iNKT cells clones without the presence of antigen-presenting cells (APCs), in contrast to the free αGC ligand. Most importantly, tumor cell killing by human iNKT cells was obtained only when co- incubated with the tumor targeted sCD1d-antitumor scFv, and their direct tumor cytotoxicity was superior to the bystander killing obtained with αGC-loaded APCs stimulation. To further enhance the anti-tumor effects, we exploited the ability of iNKT cells to transactivate the adaptive immunity, by combining the NKT/CD1d immunotherapy with a peptide cancer vaccine. Interestingly, synergistic effects were obtained when the αGC/sCD1d- anti-HER2 fusion treatment was combined with CpG ODN as adjuvant for the OVA peptide vaccine, as seen by higher numbers of activated antigen-specific CD8 T cells and NK cells, as compared to each regimen alone. The increased innate and adaptive immune responses upon combined tumor targeted sCD1d-scFv treatment and OVA/CpG vaccine were associated with a strong delay in B16-OVA-HER2 melanoma tumor growth, which correlated with an enrichment of antigen-specific CD8 cells at the tumor site. In order to extend the application of the CD1d fusion, we designed alternative CD1d fusion proteins. First, a dimeric αGC/sCD1d-Fc fusion, which permits to augment the avidity of the CD1d for iNKT cells and second, an αGC/sCD1d fused to an anti vascular endothelial growth factor receptor-3 (VEGFR-3) scFv, in order to target tumor stroma environment. Altogether, these results demonstrate that the iNKT-mediated immunotherapy via recombinant αGC/sCD1d-scFv fusion is a promising approach to redirect the innate and adaptive antitumor immune response to the tumor site.