The MyD88 protein 88 pathway is differently involved in immune responses induced by distinct substrains of Leishmania major.

Host resistance to Leishmania major is highly dependent on the development of a Th1 immune response. The TLR adaptator myeloid differentiation protein 88 (MyD88) has been implicated in the Th1 immune response associated with the resistant phenotype observed in C57BL/6 mice after infection with L. major. To investigate whether the MyD88 pathway is differentially used by distinct substrains of parasites, MyD88(-/-) C57BL/6 mice were infected with two substrains of L. major, namely L. major LV39 and L. major IR75. MyD88(-/-) mice were susceptible to both substrains of L. major, although with different kinetics of infection. The mechanisms involved during the immune response associated with susceptibility of MyD88(-/-) mice to L. major is however, parasite substrain-dependent. Susceptibility of MyD88(-/-) mice infected with L. major IR75 is a consequence of Th2 immune-deviation, whereas susceptibility of MyD88(-/-) mice to infection with L. major LV39 resulted from an impaired Th1 response. Depletion of regulatory T cells (Treg) partially restored IFN-gamma secretion and the Th1 immune response in MyD88(-/-) mice infected with L. major LV39, demonstrating a role of Treg activity in the development of an impaired Th1 response in these mice.

Impact of the phosphatidylinositide 3-kinase signaling pathway on the cardioprotection induced by intermittent hypoxia.

BACKGROUND: Exposure to intermittent hypoxia (IH) may enhance cardiac function and protects heart against ischemia-reperfusion (I/R) injury. To elucidate the underlying mechanisms, we developed a cardioprotective IH model that was characterized at hemodynamic, biochemical and molecular levels. METHODS: Mice were exposed to 4 daily IH cycles (each composed of 2-min at 6-8% O2 followed by 3-min reoxygenation for 5 times) for 14 days, with normoxic mice as controls. Mice were then anesthetized and subdivided in various subgroups for analysis of contractility (pressure-volume loop), morphology, biochemistry or resistance to I/R (30-min occlusion of the left anterior descending coronary artery (LAD) followed by reperfusion and measurement of the area at risk and infarct size). In some mice, the phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin was administered (24 µg/kg ip) 15 min before LAD. RESULTS: We found that IH did not induce myocardial hypertrophy; rather both contractility and cardiac function improved with greater number of capillaries per unit volume and greater expression of VEGF-R2, but not of VEGF. Besides increasing the phosphorylation of protein kinase B (Akt) and the endothelial isoform of NO synthase with respect to control, IH reduced the infarct size and post-LAD proteins carbonylation, index of oxidative damage. Administration of wortmannin reduced the level of Akt phosphorylation and worsened the infarct size. CONCLUSION: We conclude that the PI3K/Akt pathway is crucial for IH-induced cardioprotection and may represent a viable target to reduce myocardial I/R injury.

Mechanism of hcnA mRNA recognition in the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens.

In the plant-beneficial bacterium Pseudomonas fluorescens CHA0, the expression of antifungal exoproducts is controlled by the GacS/GacA two-component system. Two RNA binding proteins (RsmA, RsmE) ensure effective translational repression of exoproduct mRNAs. At high cell population densities, GacA induces three small RNAs (RsmX, RsmY, RsmZ) which sequester both RsmA and RsmE, thereby relieving translational repression. Here we systematically analyse the features that allow the RNA binding proteins to interact strongly with the 5' untranslated leader mRNA of the P. fluorescens hcnA gene (encoding hydrogen cyanide synthase subunit A). We obtained evidence for three major RsmA/RsmE recognition elements in the hcnA leader, based on directed mutagenesis, RsmE footprints and toeprints, and in vivo expression data. Two recognition elements were found in two stem-loop structures whose existence in the 5' leader region was confirmed by lead(II) cleavage analysis. The third recognition element, which overlapped the hcnA Shine-Dalgarno sequence, was postulated to adopt either an open conformation, which would favour ribosome binding, or a stem-loop structure, which may form upon interaction with RsmA/RsmE and would inhibit access of ribosomes. Effective control of hcnA expression by the Gac/Rsm system appears to result from the combination of the three appropriately spaced recognition elements.

STAT3α interacts with nuclear GSK3beta and cytoplasmic RISK pathway and stabilizes rhythm in the anoxic-reoxygenated embryonic heart.

Activation of the Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway is known to play a key role in cardiogenesis and to afford cardioprotection against ischemia-reperfusion in adult. However, involvement of JAK2/STAT3 pathway and its interaction with other signaling pathways in developing heart transiently submitted to anoxia remains to be explored. Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30 min) and reoxygenation (80 min) with or without the antioxidant MPG, the JAK2/STAT3 inhibitor AG490 or the PhosphoInositide-3-Kinase (PI3K)/Akt inhibitor LY-294002. Time course of phosphorylation of STAT3α(tyrosine705) and Reperfusion Injury Salvage Kinase (RISK) proteins [PI3K, Akt, Glycogen Synthase Kinase 3beta (GSK3beta), Extracellular signal-Regulated Kinase 2 (ERK2)] was determined in homogenate and in enriched nuclear and cytoplasmic fractions of the ventricle. STAT3 DNA-binding was determined. The chrono-, dromo- and inotropic disturbances were also investigated by electrocardiogram and mechanical recordings. Phosphorylation of STAT3α(tyr705) was increased by reoxygenation, reduced (~50%) by MPG or AG490 but not affected by LY-294002. STAT3 and GSK3beta were detected both in nuclear and cytoplasmic fractions while PI3K, Akt and ERK2 were restricted to cytoplasm. Reoxygenation led to nuclear accumulation of STAT3 but unexpectedly without DNA-binding. AG490 decreased the reoxygenation-induced phosphorylation of Akt and ERK2 and phosphorylation/inhibition of GSK3beta in the nucleus, exclusively. Inhibition of JAK2/STAT3 delayed recovery of atrial rate, worsened variability of cardiac cycle length and prolonged arrhythmias as compared to control hearts. Thus, besides its nuclear translocation without transcriptional activity, oxyradicals-activated STAT3α can rapidly interact with RISK proteins present in nucleus and cytoplasm, without dual interaction, and reduce the anoxia-reoxygenation-induced arrhythmias in the embryonic heart.

Itinéraire clinique en traumatologie, une mode ou un besoin [Clinical pathway in traumatology, a fashion or need?].

A clinical route is defined as a "set of methods and instruments to members of a multidisciplinary and Interprofessional team to agree on the tasks for a specific patient population. This is a program of care to ensure the provision of quality care and efficient realization". The University Hospital is not immune to this phenomenon. In the Department of the musculoskeletal system, a first project of this kind concerns the fracture of the proximal femur in the elderly.

Triggering of Bcl-2-related pathway is associated with apoptosis of photoreceptors in Rpe65-/- mouse model of Leber's congenital amaurosis.

Mutations in RPE65 protein is characterized by the loss of photoreceptors, although the molecular pathways triggering retinal cell death remain largely unresolved. The role of the Bcl-2 family of proteins in retinal degeneration is still controversial. However, alteration in Bcl-2-related proteins has been observed in several models of retinal injury. In particular, Bax has been suggested to play a crucial role in apoptotic pathways in murine glaucoma model as well as in retinal detachment-associated cell death. We demonstrated that Bcl-2-related signaling pathway is involved in Rpe65-dependent apoptosis of photoreceptors during development of the disease. Pro-apoptotic Bax alpha and beta isoforms were upregulated in diseased retina. This was associated with a progressive reduction of anti-apoptotic Bcl-2, reflecting imbalanced Bcl-2/Bax ratio as the disease progresses. Moreover, specific translocation of Bax beta from cytosol to mitochondria was observed in Rpe65-deficient retina. This correlated with the initiation of photoreceptor cell loss at 4 months of age, and further increased during disease development. Altogether, these data suggest that Bcl-2-apoptotic pathway plays a crucial role in Leber's congenital amaurosis disease. They further highlight a new regulatory mechanism of Bax-dependent apoptosis based on regulated expression and activation of specific isoforms of this protein.

Arabidopsis jasmonate signaling pathway.

Jasmonates control defense gene expression and male fertility in the model plant Arabidopsis thaliana. In both cases, the involvement of the jasmonate pathway is complex, involving large-scale transcriptional reprogramming. Additionally, jasmonate signaling is hard-wired into the auxin, ethylene, and salicylate signal networks, all of which are under intense investigation in Arabidopsis. In male fertility, jasmonic acid (JA) is the essential signal intervening both at the level of anther elongation and in pollen dehiscense. A number of genes potentially involved in jasmonate-dependent anther elongation have recently been discovered. In the case of defense, at least two jasmonates, JA and its precursor 12-oxo-phytodienoic acid (OPDA), are necessary for the fine-tuning of defense gene expression in response to various microbial pathogens and arthropod herbivores. However, only OPDA is required for full resistance to some insects and fungi. Other jasmonates probably affect yet more physiological responses. A series of breakthroughs have identified the SKP/CULLIN/F-BOX (SCF), CORONATINE INSENSITIVE (COI1) complex, acting together with the CONSTITUTIVE PHOTOMORPHOGENIC 9 (COP9) signalosome, as central regulatory components of jasmonate signaling in Arabidopsis. The studies, mostly involving mutational approaches, have paved the way for suppressor screens that are expected to further extend our knowledge of jasmonate signaling. When these and other new mutants affecting jasmonate signaling are characterized, new nodes will be added to the Arabidopsis Jasmonate Signaling Pathway Connections Map, and the lists of target genes regulated by jasmonates in Arabidopsis will be expanded.

Biochemical characterization of FANCD2 and FANCM in the Fanconi Anaemia pathway

Abstract : The maintenance of genome stability is a challenge for all living organisms. DNA is regularly subjected to chemical alterations by both endogenous and exogenous DNA damaging agents. If left unrepaired, these lesions will create mutations or lead to chromosomal instability. DNA crosslinking agents probably bring about the most toxic lesions. By linking covalently the two strands of DNA, crosslinking agents will impede essential cellular processes such as replication and transcription. Cells from Fanconi anaemia patients are extremely sensitive to these agents. Fanconi anaemia (FA) is a rare chromosomal instability disorder that leads to developmental defects, pancytopenia and cancer susceptibility. FA is a genetically heterogeneous disease with thirteen complementation groups identified. Proteins encoded by the FA genes work together in the FA pathway. Eight of these proteins form the FA core complex (FANC-A, B, C,E, F, G, L and -M), whose integrity is required to monoubiquitinate FANCD2 and FANCI in response to DNA damage. The hypersensitivity of FA cells to crosslinking agents, which perturb the progression of replication forks, has led to the hypothesis that FA proteins play a crucial role in the response to replication stress. However, at the molecular level, the functions of the FA pathway remain largely unknown. Our efforts were first focused on the characterization of FANCD2, "the key effector of the FA pathway". Using different substrates, we found that in vitro, purified hFANCD2 preferentially binds single strand DNA and double strand DNA extremities. Concomitantly, FANCM was identified as a new component of the FA core complex. Moreover FANCM was shown to have specific branch migration activities and probably a role as a "landing platform" on DNA for the other components of the core complex. By using FANCM mutants carrying deletions within the internal domain, we investigated the role of FANCM as a DNA anchor protein for the core complex. We observed that indeed, a specific part of the internal domain of FANCM interacts with components of the core complex. Finally, in collaboration with Weidong Wang's lab we characterized two new components of the FA pathway: FAAP10 and FAAP16. As a heterodimer these two proteins show affinity for dsDNA, and anneal complementary oligonucleotides in vitro. Moreover these proteins can associate with FANCM via a part of its internal domain. We find that FANCM, FAAP 10 and FAAP 16 can co-exist on the branch point of replication and recombination intermediates, and that FAAP10 and FAAP16 stimulate replication fork reversal by FANCM. These results suggest that FANCM may function as a landing platform for the core complex. After loading on DNA, the core complex can activate FANCD2 through monoubiquitination leading to its recruitment to the site of damage. Since ssDNA and double strand breaks are intermediates that are generated as a consequence of collapsed replication forks, FANCD2 by binding to ds DNA ends and ssDNA could protect such structures from the recombination repair machinery and prevent unscheduled recombination events. Alternatively, FANCD2 could avoid nucleases from gaining access to collapsed forks, preserving the DNA in state that can be used as a starting point for resumption of DNA synthesis. The overall comprehension of the FA pathway is far from been complete. Our results unravel new aspects of Fanconi Anaemia, which hopefully in the near future will address keys questions leading to a better understanding of the fascinating Fanconi Anaemia. Résumé : Le maintien de l'intégrité du génome est fondamentale chez tous les organismes vivants. L'ADN est constamment altéré par des composés aussi bien endogènes qu'exogènes. Si ces altérations ne sont pas réparées, elles peuvent conduire à l'apparition de mutations, ainsi qu'à une instabilité génomique accrue. Les lésions les plus sévères qui peuvent survenir sur l'ADN, sont les pontages inter caténaires. Des agents pontants en liant de façon covalente les deux brins d'ADN, vont empêcher le déroulement normal de processus cellulaires essentiels tels que la réplication ou la transcription. La compréhension des mécanismes permettant à la cellule de tolérer et réparer ces lésions est primordiale, notamment dans le cas des patients atteints de l'anémie de Fanconi qui présentent une très grande sensibilité à ces composés pontants. L'anémie de Fanconi est une maladie génétique rare appartenant à un groupe de pathologies associées à une grande instabilité chromosomique. Les patients atteints de l'anémie de Fanconi présentent des malformations du squelette, une pancytopénie et une forte propension à la survenue de cancer. L'anémie de Fanconi est génétiquement très hétérogène. À ce jour, 13 gènes codant pour 13 protéines FANC différentes ont été identifiés. Huit de ces protéines fonctionnent ensemble au sein d'un complexe (nommé le complexe FANC) ayant pour but de monoubiquitiner FANCD2 et FANCI en réponse à la formation de lésions sur l'ADN. L'extrême sensibilité des cellules de patients atteints de l'anémie de Fanconi à ces agents pontant l'ADN suggère l'implication des protéines FANC dans la réponse cellulaire suite à une stress réplicatif. Cependant, le rôle moléculaire exact de ces protéines demeure encore inconnu. Après purification, nous avons observé que FANCD2 était capable de lier l'ADN simple brin, ainsi que les extrémités d'ADN in vitro. Dans le même temps, FANCM fut identifié comme appartenant au complexe FANC. FANCM est décrit comme une translocase capable de promouvoir le déplacement de point de jonction dans des structures d'ADN spécifiques in vitro. De plus, en se liant à l'ADN, FANCM peut agir comme une plateforme pour les autres protéines FANC, leur permettant ainsi d'être adressées à l'ADN. En créant des protéines FANCM recombinantes ayant des délétions dans le domaine interne, nous avons pu observer que certaines protéines du complexe FANC se fixent à des sites spécifiques sur le domaine interne de FANCM. Enfin, au travers d'une collaboration, nous avons été amenés à caractériser deux nouvelles protéines appartenant au complexe FANC : FAAP 10 et FAAP16. Elles s'associent à FANCM par l'intermédiaire du domaine interne, et forment ainsi un hétérotrimére. La présence de FAAP10 et FAAP16 n'affecte pas la liaison de FANCM à l'ADN, mais semble potentialiser son activité de régression in vitro. FANCM semble donc fonctionner comme une plateforme pour les autres composants du complexe FANC. Ces derniers, une fois liés à l'ADN permettent la monoubiquitination de FANCD2 et son recrutement au site lésé de l'ADN. FANCD2 en se liant de façon préférentielle à l'ADN simple brin et aux extrémités d'ADN qui sont générés lors de l'arrêt et du démantèlement d'une fourche de réplication, pourrait protéger ces même fourches de réplication arrêtées, d'évènements de recombinaison aléatoires. Nos résultats apportent de nouveaux éléments concernant les mécanismes moléculaires de l'anémie de Fanconi. Enfin, l'étude de l'anémie de Fanconi permet aussi de mieux comprendre les mécanismes mis en place par la cellule pour tolérer des lésions survenant lors de la réplication.

Inhibition of Notch Pathway Enhances Photoreceptor Commitment From Cultured Retinal Stem Cells

Purpose: Consequently to the principle that photoreceptors have to be at a very precise development stage to be successfully transplanted (MacLaren 2006), we are trying to mimic this development stage in vitro using retinal stem cells. The latter one isolated from the newborn mouse retina, derived from the radial glia population, which were previously isolated and characterized in our laboratory. We developed a protocol to commit these cells to the photoreceptor fate, but even if the percentage of cells expressing photoreceptor markers is high (30%), the differentiation process is incomplete so far (Merhi-Soussi 2006). Methods: In order to ameliorate photoreceptor differentiation, we hypothesized that the Notch pathway may interfere with this process by either promoting glia commitment, or maintaining an undifferentiated state. We are thus using a gamma-secretase inhibitor (DAPT), which inhibits Notch receptor cleavage and thus Notch activation. DAPT was used either during the whole differentiation stimulation, or only during a restricted period in two various retinal stem cell lines (RSC AA and RSC MP1). Results: RT-PCR performed during cell proliferation, showed the same positive expression in both cell lines for the following genes: Math3, Six3, Hes1, NeuroD, Pax6 and Notch1. Additionally, Mash1, Hes5, Prox1, Crx and Otx2 were detected in both cell lines but with a stronger expression in RSC MP1. Opposite results were obtained for Chx10. Nrl, Peripherin/RDS, GFAP and Math5 were detected neither in RSC AA, nor in RSC MP1. The constant presence of DAPT i) leads to a 233% (RSC AA) or 900% (RSC MP1) increase in peripherin/RDS-positive (photoreceptor marker) cells, compared to controls (no DAPT, n=3, P<0.02) along with a 68% (RSC AA) or 80% (RSC MP1) decrease in GFAP- positive cells (n=3, P<0.04), ii) modifies the ratio between uni-/bi- (23%) and multi- (77%) polar peripherin/RDS-positive cells to 45% and 55%, respectively, for both cell lines and iii) reduces by 50% the total cell number during the whole differentiation process for both cell lines. Conclusions: We are now exploring whether this reduction in total cell number is due to inhibition of cell proliferation or to cell death and whether photoreceptor differentiation is promoted instead of glial induction. We also want to confirm the results obtained with DAPT with RSCs isolated from Notch1-loxP mice. Such protocol may help to better mimic photoreceptor development, but this needs to be confirmed by genomic and proteomic profile analyses.

The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/beta-catenin pathway.

The development of chemoresistance represents a major obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood solid tumour. The mechanisms underlying the chemoresistant phenotype in NB were addressed by gene expression profiling of two doxorubicin (DoxR)-resistant vs sensitive parental cell lines. Not surprisingly, the MDR1 gene was included in the identified upregulated genes, although the highest overexpressed transcript in both cell lines was the frizzled-1 Wnt receptor (FZD1) gene, an essential component of the Wnt/beta-catenin pathway. FZD1 upregulation in resistant variants was shown to mediate sustained activation of the Wnt/beta-catenin pathway as revealed by nuclear beta-catenin translocation and target genes transactivation. Interestingly, specific micro-adapted short hairpin RNA (shRNAmir)-mediated FZD1 silencing induced parallel strong decrease in the expression of MDR1, another beta-catenin target gene, revealing a complex, Wnt/beta-catenin-mediated implication of FZD1 in chemoresistance. The significant restoration of drug sensitivity in FZD1-silenced cells confirmed the FZD1-associated chemoresistance. RNA samples from 21 patient tumours (diagnosis and postchemotherapy), showed a highly significant FZD1 and/or MDR1 overexpression after treatment, underlining a role for FZD1-mediated Wnt/beta-catenin pathway in clinical chemoresistance. Our data represent the first implication of the Wnt/beta-catenin pathway in NB chemoresistance and identify potential new targets to treat aggressive and resistant NB.

Targeting the JNK signaling pathway potentiates the antiproliferative efficacy of Rapamycin in LS174T colon cancer cells

RésuméLes récentes thérapies anticancéreuses développées visent principalement à inhiber les protéines mutées et responsables de la croissance des cellules cancéreuses. Dans ce contexte, l'inhibition d'une protéine appelée mTOR est une stratégie prometteuse. En effet, mTOR régule la prolifération et la survie cellulaire et mTOR est fréquemment activé dans les cellules tumorales.De nombreuses études ont démontré l'efficacité anti-tumorale d'inhibiteurs de mTOR telle que la rapamycine aussi bien dans des modèles expérimentaux que chez les patients souffrant de cancers. Ces études ont cependant également démontré que l'inhibition de mTOR induit l'activation d'autres protéines cellulaires qui vont induire la prolifération cellulaire et ainsi limiter l'effet anti-tumoral des inhibiteurs de mTOR. En particulier, la rapamycine induit l'activation de la voie de signalisation PI3K/Akt qui joue un rôle prépondérant dans la croissance cellulaire.Dans ce travail, nous avons étudié l'effet de la rapamycine sur une protéine appelée JNK ainsi que le rôle de JNK sur les effets anti-tumoraux de la rapamycine. JNK est une protéine impliquée dans la survie et la prolifération cellulaire. Elle est activée notamment par la voie de signalisation PI3K/Akt. De ce fait, nous avons émis l'hypothèse que la rapamycine induirait l'activation de JNK, réduisant ainsi l'efficacité anti¬tumorale de la rapamycine. En utilisant une lignée cellulaire tumorale (LS174T) dérivée du cancer colorectal, nous avons observé que la rapamycine induisait l'activation de JNK. Nous avons également observé que l'inhibition de JNK par le SP600125, un inhibiteur chimique de JNK, ou par la surexpression d'un dominant négatif de JNK dans les cellules LS174T potentialisait l'effet anti-tumoral de la rapamycine in vitro ainsi que dans un modèle murin de xénogreffe tumorale in vivo.En conclusion, nous avons observé que l'activation de JNK induite par la rapamycine entraine une réduction de l'effet anti-tumoral de cette dernière. Nous proposons ainsi que l'inhibition simultanée de JNK et de mTOR représente une nouvelle option thérapeutique en oncologie qu'il conviendra de confirmer dans d'autres modèles expérimentaux avant d'être testée dans des études cliniques.

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer.

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.

Human high-density lipoprotein particles prevent activation of the JNK pathway induced by human oxidised low-density lipoprotein particles in pancreatic beta cells.

AIMS/HYPOTHESIS: We explored the potential adverse effects of pro-atherogenic oxidised LDL-cholesterol particles on beta cell function. MATERIALS AND METHODS: Isolated human and rat islets and different insulin-secreting cell lines were incubated with human oxidised LDL with or without HDL particles. The insulin level was monitored by ELISA, real-time PCR and a rat insulin promoter construct linked to luciferase gene reporter. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Prolonged incubation with human oxidised LDL particles led to a reduction in preproinsulin expression levels, whereas the insulin level was preserved in the presence of native LDL-cholesterol. The loss of insulin production occurred at the transcriptional levels and was associated with an increase in activator protein-1 transcriptional activity. The rise in activator protein-1 activity resulted from activation of c-Jun N-terminal kinases (JNK, now known as mitogen-activated protein kinase 8 [MAPK8]) due to a subsequent decrease in islet-brain 1 (IB1; now known as MAPK8 interacting protein 1) levels. Consistent with the pro-apoptotic role of the JNK pathway, oxidised LDL also induced a twofold increase in the rate of beta cell apoptosis. Treatment of the cells with JNK inhibitor peptides or HDL countered the effects mediated by oxidised LDL. CONCLUSIONS/INTERPRETATION: These data provide strong evidence that oxidised LDL particles exert deleterious effects in the progression of beta cell failure in diabetes and that these effects can be countered by HDL particles.

Combinations of vascular endothelial growth factor pathway inhibitors with metronomic chemotherapy: Rational and current status.

Chemotherapy given in a metronomic manner can be administered with less adverse effects which are common with conventional schedules such as myelotoxicity and gastrointestinal toxicity and thus may be appropriate for older patients and patients with decreased performance status. Efficacy has been observed in several settings. An opportunity to improve the efficacy of metronomic schedules without significantly increasing toxicity presents with the addition of anti-angiogenic targeted treatments. These combinations rational stems from the understanding of the importance of angiogenesis in the mechanism of action of metronomic chemotherapy which may be augmented by specific targeting of the vascular endothelial growth factor (VEGF) pathway by antibodies or small tyrosine kinase inhibitors. Combinations of metronomic chemotherapy schedules with VEGF pathway targeting drugs will be discussed in this paper.