Controlled delivery of 5-chlorouracil using poly(ortho esters) in filtering surgery for glaucoma.

PURPOSE: To evaluate the antimitotic and toxic effects of 5-chlorouracil (5-CU) and 5-fluorouracil (5-FU) and study their potential to delay filtering bleb closure in the rabbit eye when released by poly(ortho esters) (POE). METHODS: Rabbit Tenon fibroblasts and human conjunctival cells were incubated with various 5-CU and 5-FU concentrations. Antiproliferative effects and toxicity were evaluated at 24 and 72 hours by monotetrazolium, neutral red, and Hoechst tests and cell counting. Mechanisms of cell death were evaluated using TUNEL assay, annexin V binding, immunohistochemistry for anti-apoptosis-inducing factor (AIF) and LEI/L-DNase II. Trabeculectomy was performed in pigmented rabbits. Two hundred microliters of POE loaded with 1% wt/wt 5-FU or 5-CU was injected into the subconjunctival space after surgery. Intraocular pressure (IOP) and bleb persistence were monitored for 150 days. RESULTS: In vitro, 5-FU showed a higher antiproliferative effect and a more toxic effect than 5-CU. 5-FU induced cell necrosis, whereas 5-CU induced mostly apoptosis. The apoptosis induced by 5-CU was driven through a non-caspase-dependent pathway involving AIF and LEI/L-DNase II. In vivo, at 34 days after surgery, the mean IOP in the POE/5-CU-treated group was 83% of the baseline level and only 40% in the POE/5-FU-treated group. At 100 days after surgery, IOP was still decreased in the POE/5-CU group when compared with the controls and still inferior to the preoperative value. The mean long-term IOP, with all time points considered, was significantly (P < 0.0001) decreased in the POE/5-CU-treated group (6.0 +/- 2.4 mm Hg) when compared with both control groups, the trabeculectomy alone group (7.6 +/- 2.9 mm Hg), and the POE alone group (7.5 +/- 2.6 mm Hg). Histologic analysis showed evidence of functioning blebs in the POE-5-CU-treated eyes along with a preserved structure of the conjunctiva epithelium. CONCLUSIONS: The slow release of 5-CU from POE has a longstanding effect on the decrease of IOP after glaucoma-filtering surgery in the rabbit eye. Thus, the slow release of POE/5-CU may be beneficial for the prevention of bleb closure in patients who undergo complicated trabeculectomy.

In vitro and in vivo evaluation of sunitinib eluting beads

Background: Chemoembolization is used to treat liver malignancies. However recurrence occurs frequently, possibly because of neoangiogenesis triggered by ischemia caused by the embolic agent. In this context, the combination of an embolic agent with an anti-angiogenic drug seems appealing. This study characterizes the in vitro loading and release profile of sunitinib eluting beads of different sizes and their pharmacokinetic profile in a rabbit model. Methods: 70-150 μm and 100-300 μm drug eluting beads (DC Bead, Biocompatibles UK) were loaded by incubation in a sunitinib hydrochloride solution. Drug was quantified by spectrophotometry at 430 nm. Drug release was measured over one-week periods and normalized using an internal standard in 30% ethanol in NaCl 0.9%. New-Zealand white rabbits were used. Eight animals received 0.2 ml of 100-300 μm DC Bead loaded with 6 mg of sunitinib in the hepatic artery (group 1) and 4 animals received 6 mg of sunitinib p.o. (group 2). Half of the animals were sacrificed after 6 hours and half after24 hours. Liver enzymes were measured at 0, 6 and 24 hours in both groups. Plasmatic sunitinib concentration was determined by tandem mass spectroscopy (LC MS/MS) at 0, 1, 2, 3, 4, 5, 6 and 24 hours. At sacrifice, the livers were harvested and sunitinib concentration in liver tissue was assessed by LC MS/MS. Results: High drug loading was obtained for both microsphere bead sizes. Particle shrinking was observed with adsorption of sunitinib. Almost complete release of sunitinib was detected under physiological conditions, with very similar release for 70-150 μm and 100-300 μm (t50%=1.2 h) DC Bead. Conclusions: Sunitinib eluting beads are well tolerated by rabbits when administered in the hepatic artery. No unexpected toxicity was observed. Very high drug concentration can be obtained at the site of embolization with minimal systemic passage.

Cell viability and noninvasive in vivo MRI tracking of 3D cell encapsulating self-assembled microcontainers.

Several molecular therapies require the implantation of cells that secrete biotherapeutic molecules and imaging the location and microenvironment of the cellular implant to ascertain its function. We demonstrate noninvasive in vivo magnetic resonance imaging (MRI) of self-assembled microcontainers that are capable of cell encapsulation. Negative contrast was obtained to discern the microcontainer with MRI; positive contrast was obtained in the complete absence of background signal. MRI on a clinical scanner highlights the translational nature of this research. The microcontainers were loaded with cells that were dispersed in an extracellular matrix, and implanted both subcutaneously and in human tumor xenografts in SCID mice. MRI was performed on the implants, and microcontainers retrieved postimplantation showed cell viability both within and proximal to the implant. The microcontainers are characterized by their small size, three dimensionality, controlled porosity, ease of parallel fabrication, chemical and mechanical stability, and noninvasive traceability in vivo.

What treatment for periprosthetic shoulder infection? Results from a multicentre retrospective series.

PURPOSE: Controversy still exists as to the best surgical treatment for periprosthetic shoulder infections. The aim of this multi-institutional study was to review a continuous retrospective series of patients treated in four European centres and to assess the respective eradication rate of various treatment approaches. METHODS: Forty-four patients were available for this retrospective follow-up evaluation. Functional and clinical evaluation of treatment for infection was performed using the Constant-Murley score, visual analogue scale and patient satisfaction Neer score. Erythrocyte sedimentation rate, serum leucocyte count and C-reactive protein were measured and shoulder X-ray examination performed prior to surgery and at the latest follow-up. RESULTS: At a mean follow-up of 41 months (range 24-98), 42 of 44 patients (95.5%) showed no signs of infection recurrence/persistence. Comparable eradication rates were observed after resection arthroplasty (100%; 6/6), two-stage revision (17/17) or permanent antibiotic-loaded spacer implant (93.3%; 14/15). No patient was treated by one-stage revision. On average, both functional and pain scores improved significantly; the worst joint function was observed after resection arthroplasty. CONCLUSIONS: This retrospective analysis conducted on the largest published series of patients to date shows comparable infection eradication rates after two-stage revision, resection arthroplasty or permanent spacer implant for the treatment of septic shoulder prosthesis.

Role of lymph node fibroblasts in T cell priming

SummarySecondary lymphoid organs, such as lymph nodes or spleen, are the only places in our body where primary adaptive immune responses are efficiently elicited. These organs have distinct Β and Τ cell rich zones and Τ lymphocytes constantly migrate from the bloodstream into Τ zones to scan dendritic cells (DCs) for antigens they present. Specialized fibroblasts, the Τ zone reticular cells (HR.Cs), span the Τ zone in the form a three-dimensional network. lK.Cs guide incoming Τ cells in their migration, both chemically, by the secretion of the chemokines CCL19 and CCL21, and physically, by construction of a road system to which also DCs adhere. In this way TRCs are thought to facilitate encounters of Τ cells with antigen-bearing DCs and thereby accelerate the selection of rare antigen-specific Τ cells. The resulting Τ cell activation, proliferation and differentiation all take place within the TRC network. However, the influence of TRCs on Τ cell activation has so fer not been elucidated with the possible reasons being that TRCs represent a relative rare cell population and that mice devoid of TRCs have not been described.To circumvent these technical limitations, we established TRC clones and lines to have an abundant source to functionally characterize TRCs. Both the clones and lines show a fibroblastic phenotype, express a surface marker profile comparable to ex vivo TRCs and produce extracellular matrix molecules. However, expression of Ccl19, Ccl21 and ZL-7 is lost and could not be restored by cytokine stimulation. When these TRC clones or lines were cultured in a three-dimensional cell culture system, their morphology changed and resembled that of in vivo TRCs as they formed networks. By adding Τ cells and antigen-loaded DCs to these cultures we successfully reconstructed lymphoid Τ zones that allowed antigen-specific Τ cell activation.To characterize the role of TRCs in Τ cell priming, TRCs were co-cultured with antigen-specific Τ cells in the presence antigen-loaded DCs. Surprisingly, the presence of TRC lines and ex vivo TRCs inhibited rather than enhanced CD8+ Τ cell activation, proliferation and effector cell differentiation. TRCs shared this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. TRCs were identified as a strong source of nitric oxide (NO) thereby directly dampening Τ cell expansion as well as reducing the Τ cell priming capacity of DCs. The expression of inducible NO synthase (iNOS) was up- regulated in a subset of TRCs by both DC-signals as well as interferon-γ produced by primed CD8+ Τ cells. Importantly, iNOS expression was induced during viral infection in vivo in both lymph node TRCs and DCs. Consistent with a role for NO as a negative regulator, the primary Τ cell response was exaggerated in iNOS-/- mice. Our findings highlight that in addition to their established positive roles in Τ cell responses TRCs and DCs cooperate in a negative feedback loop to attenuate Τ cell expansion during acute inflammation.RésuméLes organes lymphoïdes secondaires, comme les ganglions lymphoïdes ou la rate, sont les seuls sites dans notre corps où la réponse primaire des lymphocytes Β et Τ est initiée efficacement. Ces organes ont des zones différentes, riches en cellules Β ou T. Des lymphocytes Τ circulent constamment du sang vers les zones T, où ils échantillonent la surface des cellules dendritiques (DCs) pour identifier les antigènes qu'ils présentent. Des fibroblastes spécialisés - nommés Τ zone reticular cells (TRCs)' forment un réseau tridimensionnel dans la zone T. Les TRCs guident la migration des cellules Τ par deux moyens: chimiquement, par la sécrétion des chimiokines CCL19 et CCL21 et physiquement, par la construction d'un réseau routier en trois dimensions, auquel adhèrent aussi des DCs. Dans ce? cas, on pense que la présence des TRCs facilite les rencontres entre les cellules Τ et les DCs chargées de l'antigène et accélère la sélection des rares cellules Τ spécifiques. Ensuite, l'activation de cellules T, ainsi que la prolifération et la différenciation se produisent toutes à l'intérieur du réseau des TRCs. L'influence des TRCs sur l'activation des cellules T n'est que très peu caractérisée, en partie parce que les TRCs représentent une population rare et que les souris déficientes dans les TRCs n'ont pas encore été découvertes.Pour contourner ces limitations techniques, nous avons établi des clones et des lignées cellulaires de TRC pour obtenir une source indéfinie de ces cellules permettant leur caractérisation fonctionnelle. Les clones et lignées établis ont un phénotype de fibroblaste, ils expriment des molécules de surface similaires aux TRCs ex vivo et produisent de la matrice extracellulaire. Mais l'expression de Ccl19, Ccl21 et 11-7 est perdue et ne peut pas être rétablie par stimulation avec différentes cytokines. Les clones TRC ou les lignées cultivées en un système tridimensionnel de culture cellulaire, montrent une morphologie changée, qui ressemble à celle de TRC ex vivo inclus la construction de réseaux tridimensionnels.Pour caractériser le rôle des TRC dans l'activation des cellules T, nous avons cultivé des TRCs avec des cellules T spécifiques et des DCs chargées avec l'antigène. Etonnamment, la présence des TRC (lignées et ex vivo) inhibait plutôt qu'elle améliorait l'activation, la prolifération et la différenciation des lymphocytes T CDS+. Les TRCs partageaient cette fonction avec des fibr-oblastes des organes non lymphoïdes et des cellules souches du type mésenchymateux. Dans ces conditions, les TRCs sont une source importante d'oxyde nitrique (NO) et par ce fait limitent directement l'expansion des cellules T et réduisent aussi la capacité des DCs à activer les cellules T. L'expression de l'enzyme NO synthase inductible (ïNOS) est régulée à la hausse par des signaux dérivés des DCs et par l'interféron-γ produit par des cellules T de type CD8+ activées. Plus important, l'expression d'iNOS est induite pendant une infection virale in vivo, dans les TRCs et dans les DCs. Par conséquent, la réponse primaire de cellules T est exagérée dans des souris iNOS-/-. Nos résultats mettent en évidence qu'en plus de leur rôle positif bien établi dans la réponse immunitaire, les TRCs et les DCs coopèrent dans une boucle de rétroaction négative pour atténuer l'expansion des cellules T pendant l'inflammation aigiie pour protéger l'intégrité et la fonctionnalité des organes lymphoïdes secondaires.

Intravitreous injection of PLGA microspheres encapsulating GDNF promotes the survival of photoreceptors in the rd1/rd1 mouse.

PURPOSE: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres. METHODS: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections. RESULTS: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres. CONCLUSIONS: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

A new drug delivery system inhibits uveitis in an animal model after cataract surgery.

Cataract surgery is a common ocular surgical procedure consisting in the implantation of an artificial intraocular lens (IOL) to replace the ageing, dystrophic or damaged natural one. The management of postoperative ocular inflammation is a major challenge especially in the context of pre-existing uveitis. The association of the implanted IOL with a drug delivery system (DDS) allows the prolonged intraocular release of anti-inflammatory agents after surgery. Thus IOL-DDS represents an "all in one" strategy that simultaneously addresses both cataract and inflammation issues. Polymeric DDS loaded with two model anti-inflammatory drugs (triamcinolone acetonide (TA) and cyclosporine A (CsA)) were manufactured in a novel way and tested regarding their efficiency for the management of intraocular inflammation during the 3 months following surgery. The study involved an experimentally induced uveitis in rabbits. Experimental results showed that medicated DDS efficiently reduced ocular inflammation (decrease of protein concentration in aqueous humour, inflammatory cells in aqueous humour and clinical score). Additionally, more than 60% of the loading dose remained in the DDS at the end of the experiment, suggesting that the system could potentially cover longer inflammatory episodes. Thus, IOL-DDS were demonstrated to inhibit intraocular inflammation for at least 3 months after cataract surgery, representing a potential novel approach to cataract surgery in eyes with pre-existing uveitis.

FXYD Proteins Reverse Inhibition of the Na+-K+ Pump Mediated by Glutathionylation of Its {beta}1 Subunit.

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

Mineralogical alteration of artificial meteorites during atmospheric entry. The STONE-5 experiment

The generic concept of the artificial meteorite experiment STONE is to fix rock samples bearing microorganisms on the heat shield of a recoverable space capsule and to study their modifications during atmospheric re-entry. The STONE-5 experiment was performed mainly to answer astrobiological questions. The rock samples mounted on the heat shield were used (i) as a carrier for microorganisms and (ii) as internal control to verify whether physical conditions during atmospheric re-entry were comparable to those experienced by "real" meteorites. Samples of dolerite (an igneous rock), sandstone (a sedimentary rock), and gneiss impactite from Haughton Crater carrying endolithic cyanobacteria were fixed to the heat shield of the unmanned recoverable capsule FOTON-M2. Holes drilled on the back side of each rock sample were loaded with bacterial and fungal spores and with dried vegetative cryptoendoliths. The front of the gneissic sample was also soaked with cryptoendoliths. <p>The mineralogical differences between pre- and post-flight samples are detailed. Despite intense ablation resulting in deeply eroded samples, all rocks in part survived atmospheric re-entry. Temperatures attained during re-entry were high enough to melt dolerite, silica, and the gneiss impactite sample. The formation of fusion crusts in STONE-5 was a real novelty and strengthens the link with real meteorites. The exposed part of the dolerite is covered by a fusion crust consisting of silicate glass formed from the rock sample with an admixture of holder material (silica). Compositionally, the fusion crust varies from silica-rich areas (undissolved silica fibres of the holder material) to areas whose composition is "basaltic". Likewise, the fusion crust on the exposed gneiss surface was formed from gneiss with an admixture of holder material. The corresponding composition of the fusion crust varies from silica-rich areas to areas with "gneiss" composition (main component potassium-rich feldspar). The sandstone sample was retrieved intact and did not develop a fusion crust. Thermal decomposition of the calcite matrix followed by disintegration and liberation of the silicate grains prevented the formation of a melt.</p> <p>Furthermore, the non-exposed surface of all samples experienced strong thermal alterations. Hot gases released during ablation pervaded the empty space between sample and sample holder leading to intense local heating. The intense heating below the protective sample holder led to surface melting of the dolerite rock and to the formation of calcium-silicate rims on quartz grains in the sandstone sample. (c) 2008 Elsevier Ltd. All rights reserved.</p>

CD1d-antibody fusion proteins target iNKT cells to the tumor and trigger long-term therapeutic responses.

Despite the well-established antitumor activity of CD1d-restricted invariant natural killer T lymphocytes (iNKT), their use for cancer therapy has remained challenging. This appears to be due to their strong but short-lived activation followed by long-term anergy after a single administration of the CD1d agonist ligand alpha-galactosylceramide (αGC). As a promising alternative, we obtained sustained mouse iNKT cell responses associated with prolonged antitumor effects through repeated administrations of tumor-targeted recombinant sCD1d-antitumor scFv fusion proteins loaded with αGC. Here, we demonstrate that CD1d fusion proteins bound to tumor cells via the antibody fragment specific for a tumor-associated antigen, efficiently activate human iNKT cell lines leading to potent tumor cell lysis. The importance of CD1d tumor targeting was confirmed in tumor-bearing mice in which only the specific tumor-targeted CD1d fusion protein resulted in tumor inhibition of well-established aggressive tumor grafts. The therapeutic efficacy correlated with the repeated activation of iNKT and natural killer cells marked by their release of TH1 cytokines, despite the up-regulation of the co-inhibitory receptor PD-1. Our results demonstrate the superiority of providing the superagonist αGC loaded on recombinant CD1d proteins and support the use of αGC/sCD1d-antitumor fusion proteins to secure a sustained human and mouse iNKT cell activation, while targeting their cytotoxic activity and cytokine release to the tumor site.

Nanoparticles for gene delivery to retinal pigment epithelial cells.

PURPOSE: To evaluate the safety and potential use of poly(lactic) acid (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as vectors for gene transfer to RPE cells. METHODS: Experiments were conducted with primary bovine RPE cells and with the ARPE-19 human RPE cell line. Rhodamine loaded NPs were used to study factors influencing the internalization process by the various RPE cells: concentrations of NPs, duration of contact time, stage of cell culture and ambient temperature. The extent of NPs internalization was evaluated by fluorescence and phase microscopy. Potential NP toxicity was measured by the trypan blue exclusion dye test and the MTT method. Green fluorescent protein (GFP) plasmid or red nuclear fluorescent protein (RNFP) plasmid were sequestered in NPs. The ability ot these "loaded" NPs to generate gene transfection and protein expression in RPE cells was assessed both in vivo and in vitro by fluorescence and confocal microscopy. RESULTS: The extent of NP internalization in cultured cells increases with their concentration reaching a plateau at 1 mg/ml and a contact time of up to 6 h. Temperature and culture stage did not influence the in vitro internalization process. No toxic effects on RPE cells could be detected when these were incubated with up to 4 mg/ml of NPs. In human and bovine RPE cells incubated with GFP loaded NPs, cytoplasmic green fluorescence was observed in 14+/-1.65% of the cultured cells. Incubation with RNFP loaded NPs yielded a nuclear red fluorescence in 18.9+/-1.6% of the cells. These percentage levels of expression initially detected after 48 h of incubation remained unchanged during the following 8 additional days in culture. No significant differences in the extent of cytoplasm or nuclear fluorescence expression were observed between bovine or human RPE cultured cells. In vivo, a preferential RNFP expression within the RPE cell layer was detected after intra vitreous injection of RNFP plasmid loaded NPs. CONCLUSIONS: The ability of PLGA NPs to sequester plasmids, their nontoxic characteristics, and rapid internalization enables gene transfer and expression in RPE cells. These findings may be of potential use when designing future gene therapy strategies for ocular diseases of the posterior segment.

Synthetic calibrators for the analysis of total metanephrines in urine: revisiting the conditions of hydrolysis.

BACKGROUND: The quantification of total (free+sulfated) metanephrines in urine is recommended to diagnose pheochromocytoma. Urinary metanephrines include metanephrine itself, normetanephrine and methoxytyramine, mainly in the form of sulfate conjugates (60-80%). Their determination requires the hydrolysis of the sulfate ester moiety to allow electrochemical oxidation of the phenolic group. Commercially available urine calibrators and controls contain essentially free, unhydrolysable metanephrines which are not representative of native urines. The lack of appropriate calibrators may lead to uncertainty regarding the completion of the hydrolysis of sulfated metanephrines, resulting in incorrect quantification. METHODS: We used chemically synthesized sulfated metanephrines to establish whether the procedure most frequently recommended for commercial kits (pH 1.0 for 30 min over a boiling water bath) ensures their complete hydrolysis. RESULTS: We found that sulfated metanephrines differ in their optimum pH to obtain complete hydrolysis. Highest yields and minimal variance were established for incubation at pH 0.7-0.9 during 20 min. CONCLUSION: Urinary pH should be carefully controlled to ensure an efficient and reproducible hydrolysis of sulfated metanephrines. Synthetic sulfated metanephrines represent the optimal material for calibrators and proficiency testing to improve inter-laboratory accuracy.

Nano-particle vaccination combined with TLR-7 and -9 ligands triggers memory and effector CD8⁺ T-cell responses in melanoma patients.

Optimal vaccine strategies must be identified for improving T-cell vaccination against infectious and malignant diseases. MelQbG10 is a virus-like nano-particle loaded with A-type CpG-oligonucleotides (CpG-ODN) and coupled to peptide(16-35) derived from Melan-A/MART-1. In this phase IIa clinical study, four groups of stage III-IV melanoma patients were vaccinated with MelQbG10, given (i) with IFA (Montanide) s.c.; (ii) with IFA s.c. and topical Imiquimod; (iii) i.d. with topical Imiquimod; or (iv) as intralymph node injection. In total, 16/21 (76%) patients generated ex vivo detectable Melan-A/MART-1-specific T-cell responses. T-cell frequencies were significantly higher when IFA was used as adjuvant, resulting in detectable T-cell responses in all (11/11) patients, with predominant generation of effector-memory-phenotype cells. In turn, Imiquimod induced higher proportions of central-memory-phenotype cells and increased percentages of CD127(+) (IL-7R) T cells. Direct injection of MelQbG10 into lymph nodes resulted in lower T-cell frequencies, associated with lower proportions of memory and effector-phenotype T cells. Swelling of vaccine site draining lymph nodes, and increased glucose uptake at PET/CT was observed in 13/15 (87%) of evaluable patients, reflecting vaccine triggered immune reactions in lymph nodes. We conclude that the simultaneous use of both Imiquimod and CpG-ODN induced combined memory and effector CD8(+) T-cell responses.

Perceptual defense in anxiety disorders

RÉSUMÉ Plusieurs auteurs ont étudié la perception en fonction de la psychopathologie. Dans cette optique, Borgeat, David, Saucier et Dumont en 1994 et Borgeat, Sauvageau, David et Saucier en 1997 ont utilisé la méthode tachistoscopique afin de comparer, dans une étude prospective, la perception de stimuli émotionnels entre des femmes souffrant d'une dépression du post- partum et des femmes non atteintes. Par ailleurs, des études antérieures, notamment les travaux de MacLeod et Rutherford en 1992, avaient montré la possibilité d'un processus de perception différent entre sujets anxieux et non anxieux. L'étude actuelle pose l'hypothèse d'une interaction entre caractéristiques du stimulus et trouble anxieux du sujet. Cette hypothèse suppose donc un processus préconscient de l'information, avec analyse sémantique de cette dernière, à même d'influencer (inhiber ou faciliter) la perception de certains stimuli en fonction de leur charge affective. L'étude que nous présentons a pour but d'évaluer la perception de stimuli anxiogènes chez les patients atteints de troubles anxieux, et plus particulièrement chez des sujets souffrant d'attaque de panique et de troubles obsessionnels-compulsifs. A cette fin, nous avons choisi, contrairement à la plupart des études effectuées jusqu'à présent où la méthode Stroop avait été utilisée, la technique tachistoscopique qui, à notre avis, permet une mesure plus directe de la rapidité du processus perceptuel. Ainsi, trois groupes de sujets ont pris part à l'étude : un groupe contrôle (N = 22), un groupe de patients souffrant d'attaques de panique (N = 21) et un groupe de patients atteints de troubles obsessionnels-compulsifs (N = 20). Ces 63 sujets, âgés entre 18 et 60 ans, ont à la fois répondu au Fear Questionnaire ainsi qu'au Questionnaire Beck 13 pour la dépression et procédé à la reconnaissance de 42 mots (six groupes de sept mots) présentés aléatoirement à l'ordinateur, en cycles successifs de 15 millisecondes. Cinq parmi les six groupes de mots se référaient à un trouble anxieux spécifique, le sixième étant considéré comme un groupe de mots « neutres ». Le temps, en millisecondes, nécessaire à la reconnaissance de chaque mot a été enregistré. Les résultats montrent une lenteur de la part des patients souffrant d'attaques de panique pour la reconnaissance de tous les stimuli par rapport aux sujets contrôle, avec une performance intermédiaire entre les deux groupes pour les patients atteints de troubles obsessionnels-compulsifs. De plus, l'analyse statistique a révélé deux effets d'interaction : les patients atteints d'attaques de panique sont plus rapides à reconnaître le groupe de mots en rapport avec leur angoisse, de même que les patients soufflant de troubles obsessionnels- compulsifs ont un temps moyen de reconnaissance des mots en rapport avec leur trouble plus bas que prévu. Ces résultats amènent à une double conclusion. La première est que les patients anxieux manifestent une défense perceptuelle globale face aux stimuli anxiogènes en général, et que cette défense est plus marquée chez les patients atteints d'attaques de panique que chez les sujets souffrant de troubles obsessionnels-compulsifs. La deuxième est que ces mêmes patients, confrontés à des stimuli en rapport avec leur propre angoisse, montrent une vigilance accrue. Ainsi, ces données évoquent une double stratégie de traitement de l'information chez les patients anxieux : un évitement perceptuel général face à l'information émotionnellement chargée, et un traitement sélectif de l'information ayant un rapport direct avec l'angoisse dont ils soufflent. SUMMARY Prior research by MacLeod and Rutherford (1992) indicates that anxious subjects could have perceptual strategies different from nonanxious subjects. 42 verbal stimuli of six types (disease, social anxiety, panic, agoraphobia, obsessive-compulsive, and neutral) were tachistoscopically presented to three groups of subjects, aged 18 to 60 years: Panic Disorder group (n =21: 13 women and 8 men), and Obsessive-Compulsive Disorder group (n=20: 14 women and 6 men), recruited from an outpatient clinic, and a Control group (n=22: 14 women and 8 men), recruited among students and hospital staff. The times required for correct identification were generally longer for anxious subjects but quicker for stimuli specifically related to their disorder. The data could indicate a two-step perceptual strategy or two distinct ways of perceiving, usually, a generalized perceptual defense for a majority of anxiety-loaded stimuli, but also a selectively facilitated processing for stimuli specific to the disorder.

Downregulation of endotoxin-induced uveitis by intravitreal injection of vasoactive intestinal Peptide encapsulated in liposomes.

PURPOSE: To reestablish the immunosuppressive microenvironment of the eye, disrupted by ocular inflammation during endotoxin-induced uveitis (EIU), by means of intravitreal injection of vasoactive intestinal peptide (VIP) in saline or encapsulated in liposomes, to increase its bioavailability and efficiency. METHODS: EIU was induced in Lewis rats by subcutaneous injection of lipopolysaccharide (LPS). Simultaneously, animals were intravitreally injected with saline, saline/VIP, VIP-loaded liposomes (VIP-Lip), or unloaded liposomes. EIU severity and cellular infiltration were assessed by clinical examination and specific immunostaining. VIP concentration was determined in ocular fluids by ELISA. Ocular expression of inflammatory cytokine and chemokine mRNAs was detected by semiquantitative RT-PCR. Biodistribution of rhodamine-conjugated liposomes (Rh-Lip) was analyzed by immunohistochemistry in eyes and regional cervical lymph nodes (LNs). RESULTS: Twenty-four hours after intravitreal injection of VIP-Lip, VIP concentration in ocular fluids was 15 times higher than after saline/VIP injection. At that time, EIU clinical severity, ocular infiltrating polymorphonuclear leukocytes (PMNs), and, to a lesser extent, ED1(+) macrophages, as well as inflammatory cytokine and chemokine mRNA expression, were significantly reduced in VIP-Lip-injected rats compared with rats injected with saline/VIP, unloaded liposomes, or saline. Rh-Lip was distributed in vitreous, ciliary body, conjunctiva, retina, and sclera. It was internalized by macrophages and PMNs, and VIP colocalized with liposomes at least up to 14 days after injection. In cervical LNs, resident macrophages internalized VIP-Rh-Lip, and some adjacent lymphocytes showed VIP expression. CONCLUSIONS: VIP was efficient at reducing EIU only when formulated in liposomes, which enhanced its immunosuppressive effect and controlled its delivery to all tissues affected by or involved in ocular inflammation.