Low doses of ionizing radiation promote tumor growth and metastasis by enhancing angiogenesis.

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

Cytotoxins of the human pathogen Aeromonas hydrophila trigger, via the NLRP3 inflammasome, caspase-1 activation in macrophages.

Aeromonas hydrophila is a Gram-negative pathogen that causes serious infectious disease in humans. A. hydrophila induces apoptosis in infected macrophages, but the host proinflammatory responses triggered by macrophage death are largely unknown. Here, we demonstrate that the infection of mouse macrophages with A. hydrophila triggers the activation of caspase-1 and release of IL-1β. Caspase-1 activation was abrogated in macrophages deficient in Nod-like receptor family, pyrin domain containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), but not NLR family, CARD domain containing 4 (NLRC4). The activation of the NLRP3 inflammasome was mediated by three cytotoxins (aerolysin, hemolysin and multifunctional repeat-in-toxin) produced by A. hydrophila. Our results indicated that the NLRP3 inflammasome senses A. hydrophila infection through the action of bacterial cytotoxins.

Gut-derived signaling molecules and vagal afferents in the control of glucose and energy homeostasis.

PURPOSE OF REVIEW: The control of glucose and energy homeostasis, including feeding behaviour, is tightly regulated by gut-derived peptidic and nonpeptidic endocrine mediators, autonomic nervous signals, as well as nutrients such as glucose. We will review recent findings on the role of the gastrointestinal tract innervation and of portal vein glucose sensors; we will review selected data on the action of gastrointestinally released hormones. RECENT FINDINGS: The involvement of mechanosensory vagal afferents in postprandial meal termination has been clarified using mouse models with selective impairments of genes required for development of mechanosensory fibres. These activate central glucogen-like peptide-1/glucogen-like peptide-2 containing ascending pathways linking the visceroceptive brainstem neurons to hypothalamic nuclei. Mucosal terminals comprise the chemosensory vagal afferents responsive to postprandially released gastrointestinal hormones. The mechanism by which the hepatoportal glucose sensor stimulates glucose utilization by muscles was demonstrated, using genetically modified mice, to be insulin-independent but to require GLUT4 and AMP-kinase. This sensor is a key site of glucogen-like peptide-1 action and plays a critical role in triggering first phase insulin secretion. PeptideYY and ghrelin target intracerebral receptors as they are bidirectionally transported across the blood brain barrier. The anorectic functions of peripherally released peptideYY may however be mediated both via vagal afferents and intracerebral Y2 receptors in the brainstem and arcuate nucleus. SUMMARY: These recent findings demonstrate that the use of improved anatomical and physiological techniques and animal models with targeted gene modifications lead to an improved understanding of the complex role of gastrointestinal signals in the control of energy homeostasis.

Novel relationship between Vegf expression and retinal pigmented epithelium permeability following light damage in the mouse retina

Purpose:In the retina, the balance between pro- and anti-angiogenic factors is critical for angiogenesis control but is also involved in cell survival and maintenance. For instance, the anti-angiogenic factor PEDF is neuroprotective for photoreceptors (PRs) in models of retinal degeneration. We previously reported upregulation of VEGF (24h to 48h post lesion) in the light-damage (LD) model. Furthermore, systemic delivery of PEDF, as well as lentiviral gene transfer of an anti-VEGF antibody rescue PRs from cell death. Studies in vitro show that VEGF induces retinal endothelial cells apoptosis via the alteration of the Akt1/p38 MAPK signalling pathway under hypoxic conditions. Thus, in this study, we investigate the effect of high levels of VEGF on retinal pigmented epithelium (RPE) permeability and molecular targets expression after light-induced PR degeneration. Methods:To characterize the action of VEGF in the retina during the course of LD, we exposed adult Balb/c mice to 5'000 lux for 1h, and we collected neural retinas and eye-cups (containing RPE) at different time points after the LD. We analysed protein expression by Elisa and Western blotting. In order to study RPE cell permeability after the LD we stained β-catenin on flat mounted RPE. Results:In the neural retina, preliminary results indicate that high levels of VEGF induce a significant upregulation of VEGF receptor 2, whereas VEGF receptor 1 expression is decreased. Concomitantly with VEGF upregulation, LD increases the Src phosphorylation between 24h to 48h. Furthermore, we observe that β-catenin translocates to the cytoplasm of RPE cells between 24h to 36h after the lesion, indicating an increase on the RPE permeability, which could contribute indirectly to the deleterious effect of VEGF observed during light-induced PR apoptosis. Conclusions:This study further involves VEGF in LD and highlights the prime importance of angiogenic factor balance for PR survival. Our results suggest that PR apoptosis is augmented by RPE cell permeability, which may induce high level of VEGF and could be deleterious. The specific action of RPE permeability on PR survival and the role of Src in the retina are under investigation.

Intracellular pattern-recognition receptors

The last ten years of research in the field of innate immunity have been incredibly fertile: the transmembrane Toll-like receptors (TLRs) were discovered as guardians protecting the host against microbial attacks and the emerging pathways characterized in detail. More recently, cytoplasmic sensors were identified, which are capable of detecting not only microbial, but also self molecules. Importantly, while such receptors trigger crucial host responses to microbial insult, over-activity of some of them has been linked to autoinflammatory disorders, hence demonstrating the importance of tightly regulating their actions over time and space. Here, we provide an overview of recent findings covering this area of innate and inflammatory responses that originate from the cytoplasm

NKT cells: what's in a name?

Recent years have seen so-called natural killer T (NKT) cells emerge as important regulators of the immune response. The existence of NKT-cell subsets, and other types of T cell that resemble NKT cells, is an ongoing source of confusion in the literature. This perspective article seeks to clarify which cells fall under the NKT-cell umbrella, and which might be best considered as separate.

Failure of neutrophil migration toward infectious focus in severe sepsis: a critical event for the outcome of this syndrome

Sepsis is a systemic inflammatory response commonly caused by bacterial infection. We demonstrated that the outcome of sepsis induced by cecal ligation and puncture (CLP) correlates with the severity of the neutrophil migration failure towards infectious focus. Failure appears to be due to a decrease in the rolling and adhesion of neutrophil to endothelium cells. It seems that neutrophil migration impairment is mediated by the circulating inflammatory cytokines, such as TNF-alpha and IL-8, which induce the nitric oxide (NO) production systemically. It is supported by the fact that intravenous administration of these cytokines reduces the neutrophil migration induced by different inflammatory stimuli, and in severe sepsis the circulating concentrations of the cytokines and chemokines are significantly increased. Moreover, the neutrophil migration failure and the reduction in the rolling/adhesion were not observed in iNOS-/- mice and, aminoguanidine prevented this event. We also demonstrated that the failure of neutrophil migration is a Toll-4 receptor (TLR4) dependent mechanism, since it was not observed in TLR4 deficient mice. Furthermore, it was also observed that circulating neutrophils obtained from septic patients present failure of neutrophil chemotaxis toward fMLP, IL-8, and LTB4 and an increased in sera concentrations of NO3 and cytokines. In conclusion, we demonstrated that, in sepsis, failure of neutrophil migration is critical for the outcome and that NO is involved in the process.

Redirecting NK cells mediated tumor cell lysis by a new recombinant bifunctional protein.

Natural killer (NK) cells are at the crossroad between innate and adaptive immunity and play a major role in cancer immunosurveillance. NK cell stimulation depends on a balance between inhibitory and activating receptors, such as the stimulatory lectin-like receptor NKG2D. To redirect NK cells against tumor cells, we designed bifunctional proteins able to specifically bind tumor cells and to induce their lysis by NK cells, after NKG2D engagement. To this aim, we used the 'knob into hole' heterodimerization strategy, in which 'knob' and 'hole' variants were generated by directed mutagenesis within the CH3 domain of human IgG1 Fc fragments fused to an anti-CEA or anti-HER2 scFv or to the H60 murine ligand of NKG2D, respectively. We demonstrated the capacity of the bifunctional proteins produced to specifically coat tumor cells surface with H60 ligand. Most importantly, we demonstrated that these bifunctional proteins were able to induce an NKG2D-dependent and antibody-specific tumor cell lysis by murine NK cells. Overall, the results show the possibility to redirect NK cytotoxicity to tumor cells by a new format of recombinant bispecific antibody, opening the way of potential NK cell-based cancer immunotherapies by specific activation of the NKG2D receptor at the tumor site.

HLA-Bw4 identifies a population of HIV-infected patients with an increased capacity to control viral replication after structured treatment interruption.

OBJECTIVES: After structured treatment interruption (STI) of treatment for HIV-1, a fraction of patients maintain suppressed viral loads. Prospective identification of such patients might improve HIV-1 treatment, if selected patients are offered STI. METHODS: We analysed the effect of previously identified genetic modulators of HIV-1 disease progression on patients' ability to suppress viral replication after STI. Polymorphisms in the genes killer cell immunoglobulin-like receptor 3DLI (KIR3DL1)/KIR3DS1, human leucocyte antigen B (HLA-B) and HLA Complex P5 (HCP5), and a polymorphism affecting HLA-C surface expression were analysed in 130 Swiss HIV Cohort Study patients undergoing STI. Genotypes were correlated with viral load levels after STI. RESULTS: We observed a statistically significant reduction in viral load after STI in carriers of HLA-B alleles containing either the Bw480Thr or the Bw480Ile epitope (mean adjusted effect on post-STI viral load: -0.82 log HIV-1 RNA copies/ml, P < 0.001; and -1.12 log copies/ml, P < 0.001, respectively). No significant effects were detected for the other polymorphisms analysed. The likelihood of being able to control HIV-1 replication using a prespecified cut-off (viral load increase < 1000 copies/ml) increased from 39% in Bw4-negative patients to 53% in patients carrying Bw4-80Thr, and to 65% in patients carrying Bw4-80Ile (P = 0.02). CONCLUSIONS: These data establish a significant impact of HLA-Bw4 on the control of viral replication after STI.

Probing the interactions of NK cell receptors with ligand expressed in trans and cis.

Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2D(d) ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor-ligand pairs and simplify the analysis of NK cell receptor variants.

New insights into dendritic cell biology and histiocytosis through a transgenic tumor model

SUMMARY The effective development of an immune response depends on the careful interplay and the regulation between innate and adaptive immunity. As the dendritic cells (DCs) are equipped with many receptors, such as Toll-like receptors, which can detect the presence of infection by recognizing different component of bacteria, fungi and even viruses, they are the among the first cells to respond to the infection. Upon pathogen challenge, the DCs interpret the innate system activation as a maturation signal, resulting in the migration of the DCS to a draining lymph node site. There, activated DCs present efficiently antigens to naïve T cells, which are in turn activated and initiate adaptive immunity. Therefore, DCs are the main connectors between innate and adaptive immune systems. In addition to be the most efficient antigen- presenting cells, DCs play a central role in the regulation of immune responses and immune tolerance. Despite extensive research, many aspects related to DC biology are still unsolved and/or controversial. The low frequency of DCs in vivo often hamper study of DC biology and in vitro-derived DCs are not suited to address certain questions, such as the development of DC. We sought of transforming in vivo the DCs through the specific expression of an oncogene, in order to obtain unlimited numbers of these cells. To achieve this goal, transgenic mouse lines expressing the SV40 Large T oncogene under the control of the CD1 1 c promoter were generated. These transgenic mice are healthy until the age of three to four months without alterations in the DC biology. Thereafter transgenic mice develop a fatal disease that shows features of a human pathology, named histiocytosis, involving DCs. We demonstrate that the disease development in the transgenic mice correlates with a massive accumulation of transformed DCs in the affected organs. Importantly, transformed DCs are immature and fully conserve their capacity to mature in antigen presenting cells. We observe hyperproliferation of transformed DCs only in the sick transgenic mice. Surprisingly, transformed DCs do not proliferate in vitro, but transfer of the transformed DCs into immunodeficient or tolerant host leads to tumor formation. Altoghether, the transgenic mouse lines we have generated represent a valuable tumor model for human histiocytosis, and provide excellent tools to study DC biology. RESUME Le développement d'une réponse immunitaire efficace dépend d'une minutieuse interaction et régulation entre l'immunité innée et adaptative. Comme les cellules dendritiques (DCs) sont équipées de nombreux récepteurs, tels que les récepteurs Toll-like, qui peuvent détecter la présence d'une infection en reconnaissant différents composants bactériens, issus de champignons ou même viraux, elles sont parmi les premières cellules à répondre à l'infection. Suite à la stimulation induite par le pathogène, les DCs interprètent l'activation du système immunitaire inné comme un signal de maturation, résultant dans la migration des DCs vers le ganglion drainant le site d'infection. Là, les DCs actives présentent efficacement des antigènes aux cellules T, qui sont à leur tour activées et initient les systèmes d'immunité adaptative. Ainsi, les DCs forment le lien principal entre les réponses immunitaires innées et adaptatives. En plus d'être les cellules présentatrices d'antigènes les plus efficaces, les DCs jouent un rôle central dans la régulation du système immunitaire et dans le phénomène de tolérance. Malgré des recherches intensives, de nombreux aspects liés à la biologie des DCs sont encore irrésolus et/ou controversés. La faible fréquence des DCs in vivo gêne souvent l'étude de la biologie de ces cellules et les DCs dérivées in vitro ne sont pas adéquates pour adresser certaines questions, telles que le développement des DCs. Afin d'obtenir des quantités illimitées de DCs, nous avons songé à transformer in vivo les DC grâce à l'expression spécifique d'un oncogène. Afin d'atteindre ce but, nous avons généré des lignées de souris transgéniques qui expriment l'oncogène SV40 Large T sous le contrôle du promoter CD1 le. Ces souris transgéniques sont saines jusqu'à l'âge de trois à quatre mois et ne présentent pas d'altération dans la biologie des DCs. Ensuite, les souris transgéniques développent une maladie présentant les traits caractéristiques d'une pathologie humaine nommée histiocytose, qui implique les DCs. Nous montrons que le développement de cette maladie corrèle avec une accumulation massive des DCs transformées dans les organes touchés. De plus, les DCs transformées sont immatures et conservent leur capacité à différencier en cellules présentatrices d'antigène. Nous observons une hyper-prolifération des DCs transformées seulement dans les souris transgéniques malades. Etonnament, les DC transformées ne prolifèrent pas in vitro, par contre, le transfert des DCs transformées dans des hôtes immuno-déficients ou tolérant conduit à la formation de tumeurs. Globalement, les lignées de souris transgéniques que nous avons générées représentent un modèle valide pour l'histiocytose humaine, et de plus, offrent d'excellents outils pour étudier la biologie des DCs.

Obesity and survival in operable breast cancer patients treated with adjuvant anthracyclines and taxanes according to pathological subtypes: a pooled analysis.

INTRODUCTION Obesity is an unfavorable prognostic factor in breast cancer (BC) patients regardless of menopausal status and treatment received. However, the association between obesity and survival outcome by pathological subtype requires further clarification. METHODS We performed a retrospective analysis including 5,683 operable BC patients enrolled in four randomized clinical trials (GEICAM/9906, GEICAM/9805, GEICAM/2003-02, and BCIRG 001) evaluating anthracyclines and taxanes as adjuvant treatments. Our primary aim was to assess the prognostic effect of body mass index (BMI) on disease recurrence, breast cancer mortality (BCM), and overall mortality (OM). A secondary aim was to detect differences of such prognostic effects by subtype. RESULTS Multivariate survival analyses adjusting for age, tumor size, nodal status, menopausal status, surgery type, histological grade, hormone receptor status, human epidermal growth factor receptor 2 (HER2) status, chemotherapy regimen, and under-treatment showed that obese patients (BMI 30.0 to 34.9) had similar prognoses to that of patients with a BMI < 25 (reference group) in terms of recurrence (Hazard Ratio [HR] = 1.08, 95% Confidence Interval [CI] = 0.90 to 1.30), BCM (HR = 1.02, 0.81 to 1.29), and OM (HR = 0.97, 0.78 to 1.19). Patients with severe obesity (BMI ≥ 35) had a significantly increased risk of recurrence (HR = 1.26, 1.00 to 1.59, P = 0.048), BCM (HR = 1.32, 1.00 to 1.74, P = 0.050), and OM (HR = 1.35, 1.06 to 1.71, P = 0.016) compared to our reference group. The prognostic effect of severe obesity did not vary by subtype. CONCLUSIONS Severely obese patients treated with anthracyclines and taxanes present a worse prognosis regarding recurrence, BCM, and OM than patients with BMI < 25. The magnitude of the harmful effect of BMI on survival-related outcomes was similar across subtypes.

Role of innate immunity in cardiac inflammation after myocardial infarction.

Over the past two decades, inflammation has emerged as a key pathophysiological process during myocardial infarction. It develops consecutively to the activation of innate immune defense mechanisms, in response to the release of endogenous molecules by necrotic cells and the extracellular matrix. These danger signals are sensed by cellular receptors normally involved in antimicrobial defenses, including toll-like receptors and a subset of NOD-like receptors, which promote intracellular signaling dependent on nuclear factor kappaB and on the formation of the inflammasome. These mechanisms stimulate the expression of multiple inflammatory mediators and growth factors, sequentially inducing the recruitment of inflammatory cells, the clearance of injured tissue, angiogenesis, and the proliferation of fibroblasts, eventually resulting in scar formation and infarct healing. Dysregulation of these responses may result in continued cardiomyocyte loss, fibrosis beyond the limits of the infarcted area, reactive hypertrophy and chamber dilatation, a process termed adverse cardiac remodeling, leading to functional compromise and heart failure. This review presents the current state of knowledge on the process of immune activation within the infarcted myocardium and its consequences.

RNA interference screening identifies a novel role for autocrine fibroblast growth factor signaling in neuroblastoma chemoresistance.

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLXL. Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.

PAR2 absence completely rescues inflammation and ichthyosis caused by altered CAP1/Prss8 expression in mouse skin.

Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3(PAR2/+)), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2-null background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases.