Targeting receptor activator of nuclear factor-kappa B as a new therapy for bone metastasis in non-small cell lung cancer.

PURPOSE OF REVIEW: The review aims at comprehensively discussing our current knowledge on bone metastases incidence in non-small cell lung cancer (NSCLC), their related complications as well as clinical impact in patients suffering from advanced disease. RECENT FINDINGS: After evoking the use of zoledronic acid as the established standard of care until recently, the new class of drugs available to prevent skeletal related events and targeting receptor activator of nuclear factor-kappa B (RANK) will be emphasized, reporting on denosumab clinical trials, a RANK-ligand (RANKL) targeting monoclonal antibody. Biological hypothesis regarding their mechanisms of action as well a potential direct impact on tumor cells are described according to the most recent laboratory as well as hypothesis-generating clinical data. SUMMARY: Targeting the RANK pathway is an efficient way to prevent complications of bone metastases in NSCLC. Interesting additional direct effects on tumor biology and evolution are being analyzed and prospectively assessed in clinical trials.

Rôle de la bradykinine dans l'hypotension induite par le fragment actif dérivé du facteur XII [Role of bradykinin in hypotension induced by the active factor derived from factor XII]

The active fragment derived from factor XII (factor XIIf) was purified from human plasma and administered intravenously to normotensive conscious rats. Factor XIIf-mediated hypotension was dose-dependent and augmented by pretreatment with captopril, an inhibitor of the angiotensin I- and bradykinin-processing enzyme. In contrast, factor XIIf-induced hypotension was not enhanced by blockade of the renin-angiotensin system by saralasin, a competitive antagonist of angiotensin II at the vascular receptor level. These results suggest that factor XIIf-mediated hypotension is due to the formation of bradykinin.

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout

Background: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). Methods: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses. Results: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha. Conclusions In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.

Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis.

The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.

Unexpected Role for Connexin37 in Circulating FVIII and Coagulation

The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. Cx37 is expressed in endothelial cells, platelets and megakaryocytes. We have recently shown that Cx37 limits thrombus propensity by permitting intercellular signaling between aggregating platelets. Here, we have performed high throughput phage display to identify potential binding partners for the regulatory intracellular C-terminus of Cx37 (Cx37CT). We retrieved 2 consensus binding motifs for Cx37CT: WHK...[K,R]XP... and FH-K...[K,R]XXP.... Sequence alignment against the NCBI protein database indicated 66% homology of one the selected peptides with FVIII B-domain. We performed cross-linking reactions using BS3 and confirmed that an 11-mer peptide of the FVIII B-domain sequence linked to recombinant Cx37CT. In vitro binding of this peptide to Cx37CT was also confirmed by surface plasmon resonance. The dissociation constant of FVIII B-domain peptides to Cx37CT was ~20 uM. Other peptide sequences, designed upstream or downstream of the FVIII B-domain sequence, showed very low or no affinity for Cx37CT. Finally, in vivo studies revealed that thrombin generation in platelet-poor plasma from Cx37-/- mice (endogenous thrombin potential: 634±11 nM min, mean±SEM) was increased compared to Cx37+/+ mice (427±12, P<0.001). Moreover, partial activated thromboplastin time (aPTT) was shorter in Cx37-/- (39.7±1.5 s) than in Cx37+/+ mice (45.9±1.8, P=0.03), whereas prothrombin time was comparable. The shorter aPTT in Cx37-/- mice correlated with higher circulating FVIII activity (46.0±0.7 vs. 53.5±2.7 s for Cx37+/+, P=0.03). Overall, our data show for the first time a functional interaction between FVIII and Cx37. This interaction may be relevant for the control of FVIII secretion and, thereby, in the regulation of levels of FVIII circulating in blood. In addition, these results may open new perspectives to improve the efficiency of recombinant FVIII manufacturing.

La diminution de l'activité du répresseur ICER dans les adipocytes est responsable de la résistance à l'insuline dirigée par le facteur CREB dans l'obésité [The decreased activity of the repressor ICER in adipocytes is responsible for insulin resistance led by factor CREB in obesity.]

Introduction: Une élévation de l'activité des facteurs de transcription CREBs dans le tissu adipeux est en partie responsable de l'insulino-résistance systémique dans l'obésité. Le facteur «Inducible cAMP early repressor» (ICER) est un répresseur transcriptionnel passif dont le niveau d'expression antagonise l'activité des CREBs. L'objectif de ce travail adipocytaire des CREBs dans l'obésité chez l'Homme et la souris. Matériels et méthodes: Du tissu adipeux blanc (TAB) a été prélevé chez des souris obèses nourries sous une diète normale et des souris obèses nourries sous un régime riche en graisses pendant 12 semaines. Des biopsies de tissu adipeux viscéral (TAV) ont été prélevées chez les sujets humains minces (BMI = 24 ± 0,5 kg/m2) et obèses (BMI > 35 kg/m2). L'expression des gènes est quantifiée par RT-PCR quantitative. L'activité des CREBs et d'ICER est mesurée par des expériences de retard sur gel. L'activité des histones déacétylases est quantifiée par dosage colorimétrique. Résultats: L'expression et l'activité d'ICER sont diminuées dans le TAB des souris obèses, hyper-glycémiques et insulino-résistantes. De même, l'activité d'ICER est réduite dans le TAV des sujets humains obèses. Cette réduction corrèle avec une augmentation de l'activité des CREBs, une réduction de l'expression de Glut4 et de l'adiponectine, à la fois chez l'Homme et la souris. La diminution de l'expression d'ICER n'est observée que dans la fraction adipocytaire du tissu adipeux. L'expression d'ICER est contrôlée par l'activité des HDACs. L'inhibition des HDACs inhibe l'expression d'ICER dans les adipocytes. L'activité totale des HDACs est réduite dans les tissus adipeux chez les souris et chez les sujets humains obèses. Conclusion: La diminution de l'activité d'ICER dans les adipocytes par une modification de l'activité des HDACs serait responsable de l'augmentation de l'activité des CREBs dans l'obésité.

Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis.

Angiogenesis is an important process in chronic inflammatory diseases. We observed that sera from patients with systemic vasculitis stimulated angiogenesis in an in vitro model using human umbilical vein endothelial cells cultured on a basement membrane (Matrigel) substrate. After 40% ammonium sulfate precipitation, angiogenic activity remained in the low molecular weight fraction and could be inactivated by heat. SDS-page of serum FPLC fractions exhibiting maximal angiogenic activity demonstrated two prominent species of 45 and 16-20 kD in patients' sera. These bands were much less apparent in sera obtained from control subjects. Amino-terminal sequencing of the 45-kD protein demonstrated that it was haptoglobin. Purified haptoglobin stimulated angiogenesis in a dose-dependent manner. The angiogenic activity of vasculitis patients' sera was partially inhibited by an antihaptoglobin antibody. Furthermore, serum haptoglobin levels in vasculitis patients correlated both with disease and angiogenic activity. Haptoglobin angiogenic activity was confirmed in two in vivo models using an implanted disc and a subcutaneous injection of basement membrane. Stimulation of angiogenesis is a newly recognized biological function of haptoglobin. The increased levels of haptoglobin found in chronic inflammatory conditions may play an important role in tissue repair. In systemic vasculitis, haptoglobin might also compensate for ischemia by promoting development of collateral vessels.