Nanoparticle-formulated siRNA targeting integrins inhibits hepatocellular carcinoma progression in mice

Integrins play an important role during development, regulating cell differentiation, proliferation and survival. Here we show that knockdown of integrin subunits slows down the progression of hepatocellular carcinoma (HCC). Using nanoparticulate delivery of short interfering RNAs targeting β1 and αv integrin subunits we downregulate all integrin receptors in hepatocytes. Short-term integrin knockdown (two weeks) does not cause apparent structural or functional perturbations of normal liver tissue. Alterations in liver morphology accumulate upon sustained integrin downregulation (seven weeks). The integrin knockdown leads to significant retardation of HCC progression, reducing proliferation and increasing tumour cell death. This tumour retardation is accompanied by reduced activation of MET oncogene as well as expression of its mature form on the cell surface. Our data suggest that transformed proliferating cells from HCC are more sensitive to knockdown of integrins than normal quiescent hepatocytes, highlighting the potential of siRNA-mediated inhibition of integrins as an anti-cancer therapeutic approach

The Roles of Individual Mammalian Argonautes in RNA Interference In Vivo

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3′ untranslated region (3′UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3′UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3′UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3′UTR targeting as to harness the activity of several Ago proteins.Alnylam Pharmaceuticals (Firm

Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination

Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1–2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higher-affinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.National Institute of Allergy and Infectious Diseases (U.S.) (Awards UM1AI100663)National Institute of Allergy and Infectious Diseases (U.S.) (Awards AI110657

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)[superscript +] macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE[superscript −/−] mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques

Caffeine Prevents Transcription Inhibition and P-TEFb/7SK Dissociation Following UV-Induced DNA Damage

Background: The mechanisms by which DNA damage triggers suppression of transcription of a large number of genes are poorly understood. DNA damage rapidly induces a release of the positive transcription elongation factor b (P-TEFb) from the large inactive multisubunit 7SK snRNP complex. P-TEFb is required for transcription of most class II genes through stimulation of RNA polymerase II elongation and cotranscriptional pre-mRNA processing. Methodology/Principal Findings: We show here that caffeine prevents UV-induced dissociation of P-TEFb as well as transcription inhibition. The caffeine-effect does not involve PI3-kinase-related protein kinases, because inhibition of phosphatidylinositol 3-kinase family members (ATM, ATR and DNA-PK) neither prevents P-TEFb dissociation nor transcription inhibition. Finally, caffeine prevention of transcription inhibition is independent from DNA damage. Conclusion/Significance: Pharmacological prevention of P-TEFb/7SK snRNP dissociation and transcription inhibitio

Disruptores endocrinos y desórdenes tiroideos: identificación y análisis de los principales disruptores dietarios y no dietarios

[eng] In 2012, the Endocrine Society defined an endocrine disruptor as "an exogenous chemical, or a mixture of chemicals, that can interfere with any aspect of hormonal action." The thyroid gland, on which this work focuses, is one of the many target organs of endocrine disruptors. Thus, a thyroid disruptor is a synthetic or natural bioactive chemical that interferes at any point along the hypothalamic-pituitary-thyroid axis: biosynthesis and secretion of thyroid hormones, blood and transmembrane transport, metabolism or local actions of thyroid hormones. A wide variety of chemicals, present ubiquitously, have been found to have disruptive properties of the thyroid and it is impossible to live without exposure to them. In this work, these disruptors have been identified, dividing into two main types, dietary and non-dietary. The mechanisms of action of thyroid disruptors are varied, so the mechanisms of action of the two most studied dietary and non-dietary disruptors will be explained: isoflavones and polyunsaturated fatty acids, in the case of dietary disruptors while explaining perchlorate and pesticides in the case of non-dietary disruptors. Experimental in vitro and animal studies indicate possible mechanisms of action for endocrine disruptors, but evidence from mammalian and human studies is often scant. Furthermore, the disruptor mixtures used in laboratory studies are very simple mixtures and therefore unrealistic compared to mixtures present in the real world. In dealing with a very novel and little-studied subject, the net effect of the mixing of hundreds of endocrine disruptors (each with diverse activity) it is still unpredictable on an individual level in humans.[spa] En 2012 la Endocrine Society definió a un disruptor endocrino como "una sustancia química exógena, o una mezcla de sustancias químicas, que puede interferir con cualquier aspecto de la acción hormonal”. La glándula tiroides, en la que se centra este trabajo, es uno de los muchos órganos diana de los disruptores endocrinos. Así pues, se conoce como disruptor tiroideo aquel producto químico bioactivo sintético o natural que interfiere en cualquier punto del eje hipotálamo-hipófisis-tiroides: biosíntesis y secreción de hormonas tiroideas, transporte sanguíneo y transmembrana, metabolismo o acciones locales de las hormonas tiroideas. Se ha demostrado que gran variedad de productos químicos, presentes de manera ubicua, tienen propiedades disruptivas de la tiroides y que es imposible vivir sin exposición a ellos. En este trabajo se han identificado dichos disruptores, dividiéndose en dos grandes tipos, dietarios y no dietarios. Los mecanismos de acción de los disruptores de la tiroides son variados, y por lo tanto se explicarán los mecanismos de acción de los dos disruptores dietarios y no dietarios más estudiados: isoflavonas y ácidos grasos poliinsaturados, en el caso de los disruptores dietarios mientras que se explicarán el perclorato y pesticidas en el caso de los disruptores no dietarios. Los estudios experimentales in vitro y en animales indican posibles mecanismos de acción para los disruptores endocrinos, pero la evidencia de los estudios en mamíferos y humanos a menudo es escasa. Además, las mezclas de disruptores utilizadas en estudios de laboratorio son mezclas muy simples y, por lo tanto, poco realistas en comparación con las mezclas presentes en el mundo real. Al tratarse de un tema muy novedoso y poco estudiado, el efecto neto de la mezcla de cientos de disruptores endocrinos (cada uno de ellos con actividad diversa) es aún impredecible a nivel individual en humanos