C4BP(ß-)-mediated immunomodulation attenuates inflammation in DSS-induced murine colitis and in myeloid cells from IBD patients.

The most recent and promising therapeutic strategies for inflammatory bowel disease (IBD) have engaged biologics targeting single effector components involved in major steps of the immune-inflammatory processes, such as tumor necrosis factor, interleukins or integrins. Nevertheless, these molecules have not yet met expectations regarding efficacy and safety, resulting in a significant percentage of refractory or relapsing patients. Thus, novel treatment options are urgently needed. The minor isoform of the complement inhibitor C4b-binding protein, C4BP(ß-), has been shown to confer a robust anti-inflammatory and immunomodulatory phenotype over inflammatory myeloid cells. Here we show that C4BP(ß-)-mediated immunomodulation can significantly attenuate the histopathological traits and preserve the intestinal epithelial integrity in dextran sulfate sodium (DSS)-induced murine colitis. C4BP(ß-) downregulated inflammatory transcripts, notably those related to neutrophil activity, mitigated circulating inflammatory effector cytokines and chemokines such as CXCL13, key in generating ectopic lymphoid structures, and, overall, prevented inflammatory immune cell infiltration in the colon of colitic mice. PRP6-HO7, a recombinant curtailed analogue with only immunomodulatory activity, achieved a similar outcome as C4BP(ß-), indicating that the therapeutic effect is not due to the complement inhibitory activity. Furthermore, both C4BP(ß-) and PRP6-HO7 significantly reduced, with comparable efficacy, the intrinsic and TLR-induced inflammatory markers in myeloid cells from both ulcerative colitis and Crohn's disease patients, regardless of their medication. Thus, the pleiotropic anti-inflammatory and immunomodulatory activity of PRP6-HO7, able to "reprogram" myeloid cells from the complex inflammatory bowel environment and to restore immune homeostasis, might constitute a promising therapeutic option for IBD.

Complement components as promoters of immunological tolerance in dendritic cells.

Complement and dendritic cells (DCs) share many functional features that drive the outcome of immune-inflammatory processes. Both have a sentinel function, acting as danger sensors specialized for a rapid, comprehensive and selective action against potential threats without damaging the healthy host cells. But while complement has been considered as a "master alarm" system poised for direct pathogen killing, DCs are regarded as "master regulators" or orchestrators of a vast range of effector immune cells for an effective immune response against threatening insults. The original definition of the complement system, coined to denote its auxiliary function to enhance or assist in the role of antibodies or phagocytes to clear microbes or damaged cells, envisaged an important crosstalk between the complement and the mononuclear phagocyte systems. More recent studies have shown that, depending on the microenvironmental conditions, several complement effectors are competent to influence the differentiation and/or function of different DC subsets toward immunogenicity or tolerance. In this review we will infer about the capability of complement activators and inhibitors to "condition" a tolerogenic and anti-inflammatory immune response by direct interaction with DC surface receptors, and about the implications of this knowledge to devise new complement-based therapeutic approaches for autoimmune pathologies.

ncRNAs in Therapeutics: Challenges and Limitations in Nucleic Acid-Based Drug Delivery.

Non-coding RNAs (ncRNAs) are emerging therapeutic tools but there are barriers to their translation to clinical practice. Key issues concern the specificity of the targets, the delivery of the molecules, and their stability, while avoiding "on-target" and "off-target" side effects. In this "ncRNA in therapeutics" issue, we collect several studies of the differential expression of ncRNAs in cardiovascular diseases, bone metabolism-related disorders, neurology, and oncology, and their potential to be used as biomarkers or therapeutic targets. Moreover, we review recent advances in the use of antisense ncRNAs in targeted therapies with a particular emphasis on their basic biological mechanisms, their translational potential, and future trends.

Noncanonical immunomodulatory activity of complement regulator C4BP(ß-) limits the development of lupus nephritis.

Lupus nephritis is a chronic autoimmune-inflammatory condition that can lead to end-stage kidney disease. Presently available immunosuppressive treatments for lupus nephritis are suboptimal and can induce significant side effects. Recently, we characterized a novel immunomodulatory activity of the minor isoform of the classical pathway complement inhibitor, C4BP(ß-). We show here that C4BP(ß-) treatment prevented the development of proteinuria and albuminuria, decreased significantly the formation of anti-dsDNA antibodies and, locally, mitigated renal glomerular IgG and C3 deposition and generation of apoptotic cells. There was a consequent histological improvement and increased survival in lupus-prone mice. The therapeutic efficacy of C4BP(ß-) was analogous to that of the broad-acting immunosuppressant cyclophosphamide. Remarkably, a comparative transcriptional profiling analysis revealed that the kidney gene expression signature resulting from C4BP(ß-) treatment turned out to be 10 times smaller than that induced by cyclophosphamide treatment. C4BP(ß-) immunomodulation induced significant downregulation of transcripts relevant to lupus nephritis indicating immunopathogenic cell infiltration, including activated T cells (Lat), B cells (Cd19, Ms4a1, Tnfrsf13c), inflammatory phagocytes (Irf7) and neutrophils (Prtn3, S100a8, S100a9). Furthermore, cytokine profiling and immunohistochemistry confirmed that C4BP(ß-), through systemic and local CXCL13 downregulation, was able to prevent ectopic lymphoid structures neogenesis in aged mice with lupus nephritis. Thus, due to its anti-inflammatory and immunomodulatory activities and high specificity, C4BP(ß-) could be considered for further clinical development in patients with systemic lupus erythematosus.

Airway Redox Homeostasis and Inflammation Gone Awry: From Molecular Pathogenesis to Emerging Therapeutics in Respiratory Pathology.

As aerobic organisms, we are continuously and throughout our lifetime subjected to an oxidizing atmosphere and, most often, to environmental threats. The lung is the internal organ most highly exposed to this milieu. Therefore, it has evolved to confront both oxidative stress induced by reactive oxygen species (ROS) and a variety of pollutants, pathogens, and allergens that promote inflammation and can harm the airways to different degrees. Indeed, an excess of ROS, generated intrinsically or from external sources, can imprint direct damage to key structural cell components (nucleic acids, sugars, lipids, and proteins) and indirectly perturb ROS-mediated signaling in lung epithelia, impairing its homeostasis. These early events complemented with efficient recognition of pathogen- or damage-associated recognition patterns by the airway resident cells alert the immune system, which mounts an inflammatory response to remove the hazards, including collateral dead cells and cellular debris, in an attempt to return to homeostatic conditions. Thus, any major or chronic dysregulation of the redox balance, the air-liquid interface, or defects in epithelial proteins impairing mucociliary clearance or other defense systems may lead to airway damage. Here, we review our understanding of the key role of oxidative stress and inflammation in respiratory pathology, and extensively report current and future trends in antioxidant and anti-inflammatory treatments focusing on the following major acute and chronic lung diseases: acute lung injury/respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and cystic fibrosis.

The Complement Inhibitor Factor H Generates an Anti-Inflammatory and Tolerogenic State in Monocyte-Derived Dendritic Cells.

The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury, although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation, respectively, attenuating the activity of the C3/C5 convertases and, consequently, avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the ß-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study, we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly, FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture, resembling immature MoDCs, lower expression of the maturation marker CD83 and the costimulatory molecules CD40, CD80, and CD86, decreased production of key proinflammatory Th1-cytokines (IL-12, TNF-α, IFN-γ, IL-6, and IL-8), and preferential production of immunomodulatory mediators (IL-10 and TGF-ß). Moreover, FH-treated MoDCs show low Ag uptake and, when challenged with LPS, display reduced CCR7 expression and chemotactic migration, impaired CD4(+) T cell alloproliferation, inhibition of IFN-γ secretion by the allostimulated T cells, and, conversely, induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus, this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity, transplantation, and autoimmunity.

CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury.

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte-endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation.

The α7ß0 isoform of the complement regulator C4b-binding protein induces a semimature, anti-inflammatory state in dendritic cells.

The classical pathway complement regulator C4b-binding protein (C4BP) is composed of two polypeptides (α- and ß-chains), which form three plasma oligomers with different subunit compositions (α7ß1, α7ß0, and α6ß1). We show in this article that the C4BP α7ß0 isoform (hereafter called C4BP[ß(-)] [C4BP lacking the ß-chain]), overexpressed under acute-phase conditions, induces a semimature, tolerogenic state on human monocyte-derived dendritic cells (DCs) activated by a proinflammatory stimulus. C4BP isoforms containing ß-chain (α7ß1 and α6ß1; C4BP[ß(+)]) neither interfered with the normal maturation of DCs nor competed with C4BP(ß(-)) activity on these cells. Immature DCs (iDCs) treated with C4BP(ß(-)) retained high endocytic activity, but, upon LPS treatment, they did not upregulate surface expression of CD83, CD80, and CD86. Transcriptional profiling of these semimature DCs revealed that treatment with C4BP(ß(-)) prevented the induction of IDO and BIC-1, whereas TGF-ß1 expression was maintained to the level of iDCs. C4BP(ß(-))-treated DCs were also unable to release proinflammatory Th1 cytokines (IL-12, TNF-α, IFN-γ, IL-6, IL-8) and, conversely, increased IL-10 secretion. They prevented surface CCR7 overexpression and, accordingly, displayed reduced chemotaxis, being morphologically indistinguishable from iDCs. Moreover, C4BP(ß(-))-treated DCs failed to enhance allogeneic T cell proliferation, impairing IFN-γ production in these cells and, conversely, promoting CD4(+)CD127(low/neg)CD25(high)Foxp3(+) T cells. Deletion mutant analysis revealed that the complement control protein-6 domain of the α-chain is necessary for the tolerogenic activity of C4BP(ß(-)). Our data demonstrate a novel anti-inflammatory and immunomodulatory function of the complement regulator C4BP, suggesting a relevant role of the acute-phase C4BP(ß(-)) isoform in a number of pathophysiological conditions and potential applications in autoimmunity and transplantation.

TNFRSF1B Signaling Blockade Protects Airway Epithelial Cells from Oxidative Stress.

Progressive respiratory airway destruction due to unresolved inflammation induced by periodic infectious exacerbation episodes is a hallmark of cystic fibrosis (CF) lung pathology. To clear bacteria, neutrophils release high amounts of reactive oxygen species (ROS), which inflict collateral damage to the neighboring epithelial cells causing oxidative stress. A former genome-wide small interfering RNA (siRNA) screening in CF submucosal gland cells, instrumental for mucociliary clearance, proposed tumor necrosis factor receptor superfamily member 1B (TNFRSF1B; TNFR2) as a potential hit involved in oxidative stress susceptibility. Here, we demonstrate the relevance of TNFRSF1B transcript knock-down for epithelial cell protection under strong oxidative stress conditions. Moreover, a blockade of TNFR signaling through its ligand lymphotoxin-α (LTA), overexpressed in airway epithelial cells under oxidative stress conditions, using the anti-tumor necrosis factor (TNF) biologic etanercept significantly increased the viability of these cells from a toxic oxidizing agent. Furthermore, bioinformatic analyses considering our previous RNA interference (RNAi) screening output highlight the relevance of TNFRSF1B and of other genes within the TNF pathway leading to epithelial cell death. Thus, the inhibition of the LTα3-TNFR2 axis could represent a useful therapeutic strategy to protect the respiratory airway epithelial lining from the oxidative stress challenge because of recurrent infection/inflammation cycles faced by CF patients.

Human mesenchymal stem cells overexpressing the IL-33 antagonist soluble IL-1 receptor-like-1 attenuate endotoxin-induced acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by pulmonary edema attributable to alveolar epithelial-interstitial-endothelial injury, associated with profound inflammation and respiratory dysfunction. The IL-33/IL-1 receptor-like-1 (ST2) axis plays a key role in the development of immune-inflammatory responses in the lung. Cell-based therapy has been recently proposed as an effective alternative for the treatment of ALI and ARDS. Here, we engineered human adipose tissue-derived mesenchymal stem cells (hASCs) overexpressing soluble IL-1 receptor-like-1 (sST2), a decoy receptor for IL-33, in order to enhance their immunoregulatory and anti-inflammatory properties when applied in a murine ALI model. We administered both hASCs and hASC-sST2 systemically at 6 hours after intranasal LPS instillation, when pathological changes had already occurred. Bioluminescence imaging, immunohistochemistry, and focused transcriptional profiling confirmed the increased presence of hASCs in the injured lungs and the activation of an immunoregulatory program (CXCR-4, tumor necrosis factor-stimulated gene 6 protein, and indoleamine 2,3-dioxygenase up-regulation) in these cells, 48 hours after endotoxin challenge. A comparative evaluation of hASCs and the actions of hASC-sST2 revealed that local sST2 overproduction by hASC-sST2 further prevented IL-33, Toll-like receptor-4, IL-1ß, and IFN-γ induction, but increased IL-10 expression in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in protein content, differential neutrophil counts, and proinflammatory cytokine (TNF-α, IL-6, and macrophage inflammatory protein 2) concentrations in bronchoalveolar lavage fluid. In addition, hASC-sST2-treated ALI lungs showed preserved alveolar architecture, an absence of apoptosis, and minimal inflammatory cell infiltration. These results suggest that hASCs genetically engineered to produce sST2 could become a promising therapeutic strategy for ALI/ARDS management.

The Hidden Side of Complement Regulator C4BP: Dissection and Evaluation of Its Immunomodulatory Activity.

C4b-binding protein (C4BP) is a well-known regulator of the complement system that holds additional and important activities unrelated to complement inhibition. Recently, we have described a novel immunomodulatory activity in the minor C4BP(ß-) isoform directly acting over inflammatory phagocytes. Here we show that incorporation of the ß-chain to the C4BP α-chain oligomer interferes with this immunomodulatory activity of C4BP. Moreover, an oligomeric form including only the complement control protein 6 (CCP6) domain of the C4BP α-chain (PRP6-HO7) is sufficient to "reprogram" monocyte-derived DCs (Mo-DCs) from a pro-inflammatory and immunogenic phenotype to an anti-inflammatory and tolerogenic state. PRP6-HO7 lacks complement regulatory activity but retains full immunomodulatory activity over inflammatory Mo-DCs induced by TLRs, characterized by downregulation of relevant surface markers such as CD83, HLA-DR, co-stimulatory molecules such as CD86, CD80 and CD40, and pro-inflammatory cytokines such as IL-12 and TNF-α. Furthermore, PRP6-HO7-treated Mo-DCs shows increased endocytosis, significantly reduced CCR7 expression and CCL21-mediated chemotaxis, and prevents T cell alloproliferation. Finally, PRP6-HO7 shows also full immunomodulatory activity over Mo-DCs isolated from lupus nephritis patients with active disease, even without further pro-inflammatory stimulation. Therefore PRP6-HO7, retaining the immunomodulatory activity of C4BP(ß-) and lacking its complement regulatory activity, might represent a promising and novel alternative to treat autoimmune diseases.

ABCG2 is required to control the sonic hedgehog pathway in side population cells with stem-like properties.

The Sonic Hedgehog (Hh) pathway has been implicated in the maintenance of stem or progenitor cells in many adult tissues. Importantly, abnormal Hh pathway activation is also associated with initiation of neoplasia, but its role in tumor growth is still unclear. Here, we demonstrate that cyclopamine, a plant-derived alkaloid product used to inhibit the Hh signaling pathway, reduces the Side Population (SP) obtained by Hoechst 33342 (Ho342) dye measurements. In addition, cyclopamine is able to modulate, along with oxysterols and other products, the ABCG2 transporter by increasing Ho342 and mitoxantrone uptake. Therefore, if the SP is solely measured as a Ho342 dye extruding fraction, this may be significantly modulated by the inhibition of ABCG2 transport fraction, independently from the action of cyclopamine on the Hh pathway. Our results indicate that ABCG2 may act in the upstream regulation of the Hh signaling pathway to protect the stemness of the SP compartment, giving support to the cancer stem cell hypothesis and suggesting that ABCG2 is not only critical for increased resistance to anticancer agents.

Genome-Wide RNAi Screening Identifies Novel Pathways/Genes Involved in Oxidative Stress and Repurposable Drugs to Preserve Cystic Fibrosis Airway Epithelial Cell Integrity.

Recurrent infection-inflammation cycles in cystic fibrosis (CF) patients generate a highly oxidative environment, leading to progressive destruction of the airway epithelia. The identification of novel modifier genes involved in oxidative stress susceptibility in the CF airways might contribute to devise new therapeutic approaches. We performed an unbiased genome-wide RNAi screen using a randomized siRNA library to identify oxidative stress modulators in CF airway epithelial cells. We monitored changes in cell viability after a lethal dose of hydrogen peroxide. Local similarity and protein-protein interaction network analyses uncovered siRNA target genes/pathways involved in oxidative stress. Further mining against public drug databases allowed identifying and validating commercially available drugs conferring oxidative stress resistance. Accordingly, a catalog of 167 siRNAs able to confer oxidative stress resistance in CF submucosal gland cells targeted 444 host genes and multiple circuitries involved in oxidative stress. The most significant processes were related to alternative splicing and cell communication, motility, and remodeling (impacting cilia structure/function, and cell guidance complexes). Other relevant pathways included DNA repair and PI3K/AKT/mTOR signaling. The mTOR inhibitor everolimus, the α1-adrenergic receptor antagonist doxazosin, and the Syk inhibitor fostamatinib significantly increased the viability of CF submucosal gland cells under strong oxidative stress pressure. Thus, novel therapeutic strategies to preserve airway cell integrity from the harsh oxidative milieu of CF airways could stem from a deep understanding of the complex consequences of oxidative stress at the molecular level, followed by a rational repurposing of existing "protective" drugs. This approach could also prove useful to other respiratory pathologies.

Glycolytic pyruvate regulates P-Glycoprotein expression in multicellular tumor spheroids via modulation of the intracellular redox state.

UNLABELLED: ABC transporters like P-glycoprotein (P-gp/ABCB1) are membrane proteins responsible for the transport of toxic compounds out of non-malignant cells and tumor tissue. AIM: To investigate the effect of glycolysis and the tissue redox state on P-gp expression in multicellular tumor spheroids derived from prostate adenocarcinoma cells (DU-145), glioma cells (Gli36), and the human cervix carcinoma cell line KB-3-1 transfected with a P-gp-EGFP fusion gene that allows monitoring of P-gp expression in living cells. During cell culture of DU-145, Gli36, and KB-3-1 tumor spheroids P-gp expression was observed as well as increased lactate and decreased pyruvate levels and expression of glycolytic enzymes. Inhibition of glycolysis for 24 h by either iodoacetate (IA) or 2-deoxy-D-glucose (2-DDG) downregulated P-gp expression which was reversed upon coincubation with the radical scavenger ebselen as shown by semi-quantitative immunohistochemisty in DU-145 and Gli36 tumor spheroids, and by EGFP fluorescence in KB-3-1 tumor spheroids. Consequently endogenous ROS generation in DU-145 tumor spheroids was increased in the presence of either IA or 2-DDG, which was abolished upon coincubation with ebselen. Exogenous addition of pyruvate significantly reduced ROS generation, increased P-gp expression as well as efflux of the P-gp substrate doxorubicin. Doxorubicin transport was significantly blunted by 2-DDG and IA, indicating that inhibition of glycolysis reversed the multidrug resistance phenotype. In summary our data demonstrate that P-gp expression in tumor spheroids is closely related to the glycolytic metabolism of tumor cells and can be downregulated by glycolysis inhibitors via mechanisms that involve changes in the cellular redox state.

CD40: an upstream master switch for endothelial cell activation uncovered by RNAi-coupled transcriptional profiling.

The CD40-CD154 dyad seems to play a prominent role fostering the immune-inflammatory response triggered by endothelial cell (EC)-T-cell communication. To delineate comprehensively the involvement of CD40 (TNFRSF5) in EC activation, we combined RNAi-mediated CD40 knockdown with comparative genome-wide transcriptional profiling of ECs interacting with (CD154+) T cells. We report the initiation of a profound stress response in ECs upon CD40-CD154 engagement through early up-regulation of, among others, the major proinflammatory NF-kappaB and MAPK/SAPK pathways and their associated transcription factors. Moreover, we have identified novel genes regulated through the CD40-CD154 interaction, and pathways previously unrecognized to be induced by CD40 signaling in ECs. Thus, we document a significant down-regulation of endothelial APLN by CD40-CD154 interaction, TNFalpha/IFNgamma exposure, and in immune-inflammatory pathologies, which could lead to hemodynamic dysfunction. Conversely, CD40-mediated up-regulation of the viral immune surveillance system, notably TLR3, IFIH1, RIG-I, and RNASEL, establishes a reverse link from adaptive to innate immunity in ECs. Moreover, systematic enrichment analysis substantiates endothelial CD40 involvement in the transcriptional regulation of gene networks associated with adhesion and motility, immunity, cell fate control, hemostasis, and metabolism. Our study also highlights the anti-inflammatory potential of RNAi-mediated CD40 inhibition, and the relevance of CD40 signaling for therapeutic intervention.

Reactive Oxygen Species: Drivers of Physiological and Pathological Processes.

Since the Great Oxidation Event, about 2.4 billion years ago, the Earth is immersed in an oxidizing atmosphere. Thus, it has been proposed that excess oxygen, originally a waste product of photosynthetic cyanobacteria, induced oxidative stress and the production of reactive oxygen species (ROS), which have since acted as fundamental drivers of biologic evolution and eukaryogenesis. Indeed, throughout an organism's lifespan, ROS affect directly (as mutagens) or indirectly (as messengers and regulators) all structural and functional components of cells, and many aspects of cell biology. Whether left unchecked by protective antioxidant systems, excess ROS not only cause genomic mutations but also induce irreversible oxidative modification of proteins (protein oxidation and peroxidation), lipids and glycans (advanced lipoxidation and glycation end products), impairing their function and promoting disease or cell death. Conversely, low-level local ROS play an important role both as redox-signaling molecules in a wide spectrum of pathways involved in the maintenance of cellular homeostasis (MAPK/ERK, PTK/PTP, PI3K-AKT-mTOR), and regulating key transcription factors (NFκB/IκB, Nrf2/KEAP1, AP-1, p53, HIF-1). Consequently, ROS can shape a variety of cellular functions, including proliferation, differentiation, migration and apoptosis. In this review, we will give a brief overview of the relevance of ROS in both physiological and pathological processes, particularly inflammation and aging. In-depth knowledge of the molecular mechanisms of ROS actuation and their influence under steady-state and stressful conditions will pave the way for the development of novel therapeutic interventions. This will mitigate the harmful outcomes of ROS in the onset and progression of a variety of chronic inflammatory and age-related diseases.

Boundary sequences stabilize transgene expression from subtle position effects in retroviral vectors.

Transgene expression shut-down, attenuation and/or variability from integrated retroviral vectors pose a major obstacle to gene therapy trials involving hematopoietic cells. We have undertaken a systematic assessment of the behavior of different configurations containing IFN-beta SAR and/or 5'HS4 beta-globin insulator sequences within a gammaretroviral vector optimized for high-level expression, focusing on the long-term achievement of stable, homogeneous transgene expression in the successfully transduced cells. Introduction of these cis regulatory elements did not perturb virus production and stability. Conversely, the SAR/5'HS4 insulator combination appeared to increase the homogeneity of EGFP expression in mass cultures. Furthermore, a clonal analysis of the dispersion of EGFP expression revealed that the IFN-SAR/5'HS4 insulator dyad was particularly effective in reducing the variability of transgene expression when both sequences were placed in opposite orientations within the retroviral backbone. These results may prove useful for the design of more stable retroviral expression cassettes able to counteract chromosomal position effects.

Restricted transgene persistence after lentiviral vector-mediated fetal gene transfer in the pregnant rabbit model.

BACKGROUND: Prenatal gene transfer may enable early causal intervention for the treatment or prevention of many devastating diseases. Nevertheless, permanent correction of most inherited disorders requires a sustained level of expression from the therapeutic transgene, which could theoretically be achieved with integrating vectors. METHODS: Rabbit fetuses received 8.5 x 10(6) HIV-based recombinant lentivirus particles containing the enhanced green fluorescent protein (EGFP) transgene by intrahepatic, intra-amniotic or intraperitoneal injection at 22 days of gestation. Provirus presence and transgene expression in rabbit tissues were evaluated at both 1.5 and 16 weeks post-in utero intervention by polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively. Moreover, we assessed persistence of EGFP by immunohistochemistry. Enzyme-linked immunosorbent assays confirmed the development of antibodies specific against both the viral vector and the reporter protein. RESULTS: Regardless of the route of administration employed, lentiviral vector-based in utero gene transfer was safe and reached 85% of the intervened fetuses at birth. However, the integrated provirus frequency was significantly reduced to 50% of that in young rabbits at 16 weeks post-treatment. In these animals, EGFP expression was evident in many tissues, including cytokeratin 5-rich basal cells from stratified and pseudostratified epithelia, suggesting that the lentiviral vector might have reached progenitor cells. Conversely, we identified the presence of immune-inflammatory infiltrates in several EGFP-expressing tissues. Moreover, almost 70% of the lentiviral vector-treated rabbits elicited a humoral immune response against the viral envelope and/or the EGFP. CONCLUSIONS: At two-thirds gestational age, the adaptive immune system of the rabbit appears a relevant factor limiting transgene persistence and expression following lentiviral vector-mediated in utero gene transfer.

Exploring the Immunomodulatory Moonlighting Activities of Acute Phase Proteins for Tolerogenic Dendritic Cell Generation.

The acute phase response is generated by an overwhelming immune-inflammatory process against infection or tissue damage, and represents the initial response of the organism in an attempt to return to homeostasis. It is mediated by acute phase proteins (APPs), an assortment of highly conserved plasma reactants of seemingly different functions that, however, share a common protective role from injury. Recent studies have suggested a crosstalk between several APPs and the mononuclear phagocyte system (MPS) in the resolution of inflammation, to restore tissue integrity and function. In fact, monocyte-derived dendritic cells (Mo-DCs), an integral component of the MPS, play a fundamental role both in the regulation of antigen-specific adaptive responses and in the development of immunologic memory and tolerance, particularly in inflammatory settings. Due to their high plasticity, Mo-DCs can be modeled in vitro toward a tolerogenic phenotype for the treatment of aberrant immune-inflammatory conditions such as autoimmune diseases and allotransplantation, with the phenotypic outcome of these cells depending on the immunomodulatory agent employed. Yet, recent immunotherapy trials have emphasized the drawbacks and challenges facing tolerogenic Mo-DC generation for clinical use, such as reduced therapeutic efficacy and limited in vivo stability of the tolerogenic activity. In this review, we will underline the potential relevance and advantages of APPs for tolerogenic DC production with respect to currently employed immunomodulatory/immunosuppressant compounds. A further understanding of the mechanisms of action underlying the moonlighting immunomodulatory activities exhibited by several APPs over DCs could lead to more efficacious, safe, and stable protocols for precision tolerogenic immunotherapy.

Association between low levels of serum miR-638 and atherosclerotic plaque vulnerability in patients with high-grade carotid stenosis.

OBJECTIVE: Carotid artery atherosclerosis is a major cause of ischemic stroke. However, reliable criteria to identify patients with high-risk carotid plaques beyond the severity of stenosis are still lacking. Circulating microRNAs (miRNAs) are being postulated as biomarkers for a variety of vascular immune-inflammatory diseases. The authors investigated whether cell-free circulating miR-638, highly expressed in vascular smooth muscle cells and implicated in proliferative vascular diseases, is associated with vulnerable atherosclerotic plaques in high-risk patients with advanced carotid artery stenosis undergoing carotid endarterectomy (CEA). METHODS: The authors conducted a prospective study in 22 consecutive symptomatic patients with high-grade carotid stenosis undergoing CEA and 36 age- and sex-matched patients without ischemic stroke history or carotid atherosclerosis (control group). In addition, they reviewed data from a historical group of 9 CEA patients who underwent long-term follow-up after revascularization. Total RNA was isolated from all serum samples, and relative miR-638 expression levels were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and compared among groups. A correlation analysis of serum miR-638 levels with vascular risk factors and treatments, and with plaque features, was performed. The ability of serum miR-638 to discriminate between the non-CEA control group and the different CEA groups was assessed by receiver operating characteristic evaluation. A logistic regression model was employed to examine the association between stratified CEA patients and serum miR-638 levels. RESULTS: Serum levels of miR-638 were significantly lower in symptomatic CEA patients (p = 0.009) and particularly in the subgroup of CEA patients who had experienced stroke (p = 0.0006) than in non-CEA controls. Discrimination of high-risk plaques was accurate (area under the curve [AUC] 0.66 for symptomatic CEA patients in general and 0.76 for those who had experienced stroke). When only patients with high cardiovascular risk were considered, the diagnostic value of serum miR-638 from symptomatic CEA patients and CEA patients who had experienced stroke improved (AUC 0.79 and 0.85). Moreover, serum miR-638 was negatively correlated with the occurrence of stroke, smoker status, presence of bilateral pathology, coronary artery disease, and cholesterol treatment; and with the high-risk fibroatheroma plaques extracted from CEA patients. Multivariate logistic regression analysis demonstrated that serum miR-638 was an independent predictor of plaque instability. Furthermore, serum miR-638 appeared to attain good discrimination for atherosclerotic stenosis in CEA patients based on analysis of blood samples obtained in the historical group before and 5 years after intervention (p = 0.04) (AUC = 0.79). CONCLUSIONS: According to this preliminary proof-of-concept study, serum miR-638 might constitute a promising noninvasive biomarker associated with plaque vulnerability and ischemic stroke, particularly in individuals with elevated cardiovascular risk.