Angiostatic kinase inhibitors to sustain photodynamic angio-occlusion.

Targeted angiostatic therapy receives major attention for the treatment of cancer and exudative age-related macular degeneration (AMD). Photodynamic therapy (PDT) has been used as an effective clinical approach for these diseases. As PDT can cause an angiogenic response in the treated tissue, combination of PDT with anti-angiogenic compounds should lead to improved therapy. This study was undertaken to test the clinically used small molecule kinase inhibitors Nexavar® (sorafenib), Tarceva® (erlotinib) and Sutent® (sunitinib) for this purpose, and to compare the results to the combination of Visudyne®-PDT with Avastin® (bevacizumab) treatment. When topically applied to the chicken chorioallantoic membrane at embryo development day (EDD) 7, a clear inhibition of blood vessel development was observed, with sorafenib being most efficient. To investigate the combination with phototherapy, Visudyne®-PDT was first applied on EDD11 to close all <100 μm vessels. Application of angiostatics after PDT resulted in a significant decrease in vessel regrowth in terms of reduced vessel density and number of branching points/mm(2) . As the 50% effective dose (ED50) for all compounds was approximately 10-fold lower, Sorafenib outperformed the other compounds. In vitro, all kinase inhibitors decreased the viability of human umbilical vein endothelial cells. Sunitinib convincingly inhibited the in vitro migration of endothelial cells. These results suggest the therapeutic potential of these compounds for application in combination with PDT in anti-cancer approaches, and possibly also in the treatment of other diseases where angiogenesis plays an important role.

Role of Mitogen-Activated Protein Kinases in Myocardial Ischemia-Reperfusion Injury during Heart Transplantation.

In solid organ transplantation, ischemia/reperfusion (IR) injury during organ procurement, storage and reperfusion is an unavoidable detrimental event for the graft, as it amplifies graft inflammation and rejection. Intracellular mitogen-activated protein kinase (MAPK) signaling pathways regulate inflammation and cell survival during IR injury. The four best-characterized MAPK subfamilies are the c-Jun NH2-terminal kinase (JNK), extracellular signal- regulated kinase-1/2 (ERK1/2), p38 MAPK, and big MAPK-1 (BMK1/ERK5). Here, we review the role of MAPK activation during myocardial IR injury as it occurs during heart transplantation. Most of our current knowledge regarding MAPK activation and cardioprotection comes from studies of preconditioning and postconditioning in nontransplanted hearts. JNK and p38 MAPK activation contributes to myocardial IR injury after prolonged hypothermic storage. p38 MAPK inhibition improves cardiac function after cold storage, rewarming and reperfusion. Small-molecule p38 MAPK inhibitors have been tested clinically in patients with chronic inflammatory diseases, but not in transplanted patients, so far. Organ transplantation offers the opportunity of starting a preconditioning treatment before organ procurement or during cold storage, thus modulating early events in IR injury. Future studies will need to evaluate combined strategies including p38 MAPK and/or JNK inhibition, ERK1/2 activation, pre- or postconditioning protocols, new storage solutions, and gentle reperfusion.

Label-free detection of tobramycin in serum by transmission-localized surface plasmon resonance.

In order to improve the efficacy and safety of treatments, drug dosage needs to be adjusted to the actual needs of each patient in a truly personalized medicine approach. Key for widespread dosage adjustment is the availability of point-of-care devices able to measure plasma drug concentration in a simple, automated, and cost-effective fashion. In the present work, we introduce and test a portable, palm-sized transmission-localized surface plasmon resonance (T-LSPR) setup, comprised of off-the-shelf components and coupled with DNA-based aptamers specific to the antibiotic tobramycin (467 Da). The core of the T-LSPR setup are aptamer-functionalized gold nanoislands (NIs) deposited on a glass slide covered with fluorine-doped tin oxide (FTO), which acts as a biosensor. The gold NIs exhibit localized plasmon resonance in the visible range matching the sensitivity of the complementary metal oxide semiconductor (CMOS) image sensor employed as a light detector. The combination of gold NIs on the FTO substrate, causing NIs size and pattern irregularity, might reduce the overall sensitivity but confers extremely high stability in high-ionic solutions, allowing it to withstand numerous regeneration cycles without sensing losses. With this rather simple T-LSPR setup, we show real-time label-free detection of tobramycin in buffer, measuring concentrations down to 0.5 μM. We determined an affinity constant of the aptamer-tobramycin pair consistent with the value obtained using a commercial propagating-wave based SPR. Moreover, our label-free system can detect tobramycin in filtered undiluted blood serum, measuring concentrations down to 10 μM with a theoretical detection limit of 3.4 μM. While the association signal of tobramycin onto the aptamer is masked by the serum injection, the quantification of the captured tobramycin is possible during the dissociation phase and leads to a linear calibration curve for the concentrations over the tested range (10-80 μM). The plasmon shift following surface binding is calculated in terms of both plasmon peak location and hue, with the latter allowing faster data elaboration and real-time display of the results. The presented T-LSPR system shows for the first time label-free direct detection and quantification of a small molecule in the complex matrix of filtered undiluted blood serum. Its uncomplicated construction and compact size, together with the remarkable performances, represent a leap forward toward effective point-of-care devices for therapeutic drug concentration monitoring.

High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates.

The vulnerability to infection of newborns is associated with a limited ability to mount efficient immune responses. High concentrations of adenosine and prostaglandins in the fetal and neonatal circulation hamper the antimicrobial responses of newborn immune cells. However, the existence of mechanisms counterbalancing neonatal immunosuppression has not been investigated. Remarkably, circulating levels of macrophage migration inhibitory factor (MIF), a proinflammatory immunoregulatory cytokine expressed constitutively, were 10-fold higher in newborns than in children and adults. Newborn monocytes expressed high levels of MIF and released MIF upon stimulation with Escherichia coli and group B Streptococcus, the leading pathogens of early-onset neonatal sepsis. Inhibition of MIF activity or MIF expression reduced microbial product-induced phosphorylation of p38 and ERK1/2 mitogen-activated protein kinases and secretion of cytokines. Recombinant MIF used at newborn, but not adult, concentrations counterregulated adenosine and prostaglandin E2-mediated inhibition of ERK1/2 activation and TNF production in newborn monocytes exposed to E. coli. In agreement with the concept that once infection is established high levels of MIF are detrimental to the host, treatment with a small molecule inhibitor of MIF reduced systemic inflammatory response, bacterial proliferation, and mortality of septic newborn mice. Altogether, these data provide a mechanistic explanation for how newborns may cope with an immunosuppressive environment to maintain a certain threshold of innate defenses. However, the same defense mechanisms may be at the expense of the host in conditions of severe infection, suggesting that MIF could represent a potential attractive target for immune-modulating adjunctive therapies for neonatal sepsis.

A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria.

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant "persister" trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2' Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.

A quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes.

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.

Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin.

Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires. However, the affinity of the selected antibodies is usually low and current methods of affinity maturation are complex and time-consuming. In this paper, we describe an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their site-specific biotinylation by the enzyme BirA. Expression vectors have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on selected scFvs. Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-immune and murine immune phage display libraries. The scFvs were first synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni-NTA affinity chromatography. Purified biotinylated scFvs were tetramerized on the streptavidin molecule to create a streptabody (StAb). The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the scFv monomer counterparts. Furthermore, the percentage of direct binding of 125I-labeled StAb or monomeric scFv on CEA-Sepharose beads and on CEA-expressing cells showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%). Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv.

Rôle de l'EGFR dans le cancer pulmonaire non à petites cellules [The role of EGFR in non-small cell lung carcinoma]

EGFR receptor is expressed on most of the non small cell lung carcinoma (NSCLC) cells. Its relative importance in oncogenesis and tumour progression seems to greatly vary among NSCLC. Two molecules targeting differently EGFR are currently used for the treatment of metastatic NSCLC. cetuximab, a monoclonal antibody directed against the extracellular domain of the receptor, leads to a moderate survival benefit when associated with standard first-line chemotherapy. Erlotinib, a small EGFR tyrosine-kinase inhibitor molecule is used in 2nd or 3rd treatment line. Predictive factors for efficiency of these new treatments are subjects of intense research, in order to allow a better selection of the patients who could benefit from such a strategy.

Immunogold Detection of L-glutamate and D-serine in Small Synaptic-Like Microvesicles in Adult Hippocampal Astrocytes.

Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (∼45 and ∼55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

Volvulus de l'intestin grêle comme cause primaire d'abdomen aigu [Volvulus of the small intestine as a cause of primary acute abdomen].

As a cause of small intestine occlusion, volvulus is often a consequence of a band or adhesions. Except in infants, it is rarely the primary cause of symptomatology. Between January 1976 and December 1992, 13 patients (7 women and 6 men, mean age of 56.8 years) were admitted in our department for an acute abdomen due to a spontaneous primary volvulus of the small bowel. Clinical examination and laboratory tests did not help in preoperative diagnosis. All patients underwent an explorative laparotomy. Six patients had had prior abdominal surgery but none of them presented adhesion or band. In 8 patients (62%), detorsion was sufficient. Resection of a segment of small bowel was necessary in 4 patients. Gangrenous of the entire bowel was observed in one patient who rapidly died. Two patients presented minor complications. One patient with Down syndrome died of bronchoaspiration. One patient has been reoperated on one year later for recurrence of the volvulus, and underwent a Noble procedure. We conclude that volvulus of the small bowel is a rare cause of acute abdomen that must be remembered. Early surgery is mandatory to reduce the risk of gangrene, which is known to double the mortality. Laparoscopy will be helpful in early diagnosis and therapy.

Development of a bifunctional CD1d-anti-HER2 scFv fusion molecule to redirect the iNKT cell-mediated immune response to the tumor site

Abstract : Invariant natural killer T lymphocytes (iNKT) are a unique subpopulation of T lymphocytes recognizing glycolipid antigens in the context of the MHC class I-like molecule CD1d. Upon activation with the high affinity ligand α-galactosylceramide (αGalCer), iNKT cells rapidly produce large amounts of the pro-inflammatory cytokine interferon gamma (IFN-γ) and potently activate cells of the innate and adaptive immune response, such as dendritic cells (DCs), NK and T cells. In this context, iNKT cells have been shown to efficiently mediate antitumor activity, and recent research has focused on the manipulation of these cells for antitumor therapies. However, a major drawback of αGalCer as a free drug is that a single injection of this ligand leads to a short-lived iNKT cell activation followed by a long-term anergy, limiting its therapeutic use. In contrast, we demonstrate here that when αGalCer is loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections lead to a sustained iNKT and NK cell activation associated with IFN-γ secretion as well as with DC maturation. Most importantly, when the αGalCer/sCD1d is fused to an anti-HER2 scFv antibody fragment, potent inhibition of experimental lung metastasis and established subcutaneous tumors is obtained when systemic treatment is started two to seven days after the injection of HER2-expressing B16 melanoma cells, whereas at this time free αGalCer has no effect. The antitumor activity of the sCD1d-anti-HER2 fusion protein is associated with HER2-specific tumor localization and accumulation of iNKT, NK and T cells at the tumor site. Importantly, active T cell immunization combined with the sCD1d-anti-HER2 treatment leads to the accumulation of antigen-specific CD8 T cells exclusively in HER2-expressing tumors, resulting in potent tumor inhibition. In conclusion, sustained activation and tumor targeting of iNKT cells by recombinant αGalCer/sCD1d molecules thus may promote a combined innate and adaptive immune response at the tumor site that may prove to be effective in cancer immunotherapy. RESUME : Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines du complexe majeur d'histocompatibilité de classe I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. En revanche, l'étude présentée ici démontre que, si l'αGalCer est chargé sur des molécules récombinantes soluble CD1d (αGalCer/sCDld), des injections répétées aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si on fusionne la molécule αGalCer/sCD1d avec un fragment single-chain (scFv) de l'anticorps anti-HER2, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. De plus, une immunisation active combinée avec le traitement sCD1d-anti-HER2 aboutit à une accumulation des lymphocytes T CD8 spécifiques de l'antigène d'immunisation, ceci exclusivement dans des tumeurs qui expriment l'antigène HER2. Cette combinaison résulte dans une activité anti-tumeur accrue. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules recombinantes αGalCer/sCDld conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer. RESUME POUR UN LARGE PUBLIC : Le cancer est une cause majeure de décès dans le monde. Sur un total de 58 millions de décès enregistrés au niveau mondial en 2005, 7,6 millions (soit 13%) étaient dus au cancer. Les principaux traitements de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. Néanmoins, ces traitements nuisent aussi aux cellules normales de notre corps et parfois, ils ne suffisent pas pour éliminer définitivement une tumeur. L'immunothérapie est l'une des nouvelles approches pour la lutte contre le cancer et elle vise à exploiter la spécificité du système immunitaire qui peut distinguer des cellules normales et tumorales. Une cellule exprimant un marqueur tumoral (antigène) peut être reconnue par le système immunitaire humoral (anticorps) et/ou cellulaire, induisant une réponse spécifique contre la tumeur. L'immunothérapie peut s'appuyer alors sur la perfusion d'anticorps monoclonaux dirigés contre des antigènes tumoraux, par exemple les anticorps dirigés contre les protéines oncogéniques Her-2/neu dans le cancer du sein. Ces anticorps ont le grand avantage de spécifiquement se localiser à la tumeur et d'induire la lyse ou d'inhiber la prolifération des cellules tumorales exprimant l'antigène. Aujourd'hui, six anticorps monoclonaux non-conjugés sont approuvés en clinique. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements sont souvent combinés avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et exploiter par immunisation active le développement de lymphocytes T cytotoxiques (CTL) capables de détruire spécifiquement les cellules malignes. De telles «vaccinations »sont actuellement testées en clinique, mais jusqu'à présent elles n'ont pas abouti aux résultats satisfaisants. Pour obtenir une réponse lymphocytaire T cytotoxique antitumorale, la cellule T doit reconnaître un antigène associé à la tumeur, présenté sous forme de peptide dans un complexe majeur d'histocompatibilité de classe I (CHM I). Cependant les cellules tumorales sont peu efficace dans la présentation d'antigène, car souvent elles se caractérisent par une diminution ou une absence d'expression des molécules d'histocompatibilité de classe I, et expriment peu ou pas de molécules d'adhésion et de cytokines costimulatrices. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL spécifiquement dirigés contre des antigènes tumoraux, les régressions tumorales obtenus grâce à ces vaccinations sont relativement rares. Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines CMH I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. Notre groupe de recherche a donc eu l'idée de développer une nouvelle approche thérapeutique où la réponse immunitaire des cellules iNKT serait prolongée et redirigée vers la tumeur par des anticorps monoclonaux. Concrètement, nous avons produit des molécules récombinantes soluble CD1d (sCD1d) qui, si elles sont chargés avec l'αGalCer (αGalCer/sCDld), aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si la molécule αGalCer/sCD1d est fusionnée avec un fragment single-chain (scFv) de l'anticorps anti-HER2, la réponse immunitaire est redirigée à la tumeur pour autant que les cellules cancéreuses expriment l'antigène HER2. Les molécules αGalCer/sCDld ainsi présentées activent les lymphocytes iNKT. Avec cette stratégie, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées, même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules récombinantes αGalCer/sCD1d conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer.

Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.