Search

In the last decades, several approaches were developed to design drug delivery systems to address the multiple biological barriers encountered after administration while safely delivering a payload. In this scenario, bio-inspired and bio-mimetic approaches have emerged as promising solutions to evade the mononuclear phagocytic system while simultaneously negotiating the sequential transport across the various biological barriers. Leukocytes freely circulate in the bloodstream and selectively target the inflamed vasculature in response to injury, infection, and cancer. Recently we have shown the use of biomimetic nanovesicles, called leukosomes, which combine both the physical and biological properties of liposomes and leukocytes, respectively, to selectively deliver drugs to the inflamed vasculature. Here we report the use of leukosomes to target and deliver doxorubicin, a model chemotherapeutic, to tumors in syngeneic murine models of breast cancer and melanoma. Exploiting the inflammatory pathway responsible for recruiting immune cells to the site of injury, leukosomes exhibited increased targeting of cancer vasculature and stroma. Furthermore, delivery of doxorubicin with leukosomes enabled significant tumor growth inhibition compared with free doxorubicin in both breast and melanoma tumors. This study demonstrates the promise of using biomimetic nanovesicles for effective cancer management in solid tumors.

Despite numerous advances in medical treatment, sepsis remains one of the leading causes of death worldwide. Sepsis is characterized by the involvement of all organs and tissues as a consequence of blood poisoning, resulting in organ failure and eventually death. Effective treatment remains an unmet need and novel approaches are urgently needed. The growing evidence of clinical and biological heterogeneity of sepsis suggests precision medicine as a possible key for achieving therapeutic breakthroughs. In this scenario, biomimetic nanomedicine represents a promising avenue for the treatment of inflammatory diseases, including sepsis. We investigated the role of macrophage-derived biomimetic nanoparticles, namely leukosomes, in a lipopolysaccharide-induced murine model of sepsis. We observed that treatment with leukosomes was associated with significantly prolonged survival. In vitro studies elucidated the potential mechanism of action of these biomimetic vesicles. The direct treatment of endothelial cells (ECs) with leukosomes did not alter the gene expression profile of EC-associated cell adhesion molecules. In contrast, the interaction of leukosomes with macrophages induced a decrease of pro-inflammatory genes (IL-6, IL-1b, and TNF-α), an increase of anti-inflammatory ones (IL-10 and TGF-β), and indirectly an anti-inflammatory response on ECs. Taken together, these results showed the ability of leukosomes to regulate the inflammatory response in target cells, acting as a bioactive nanotherapeutic.

Aims Recent evidence suggests that ‘vulnerable plaques’, which have received intense attention as underlying mechanism of acute coronary syndromes over the decades, actually rarely rupture and cause clinical events. Superficial plaque erosion has emerged as a growing cause of residual thrombotic complications of atherosclerosis in an era of increased preventive measures including lipid lowering, antihypertensive therapy, and smoking cessation. The mechanisms of plaque erosion remain poorly understood, and we currently lack validated effective diagnostics or therapeutics for superficial erosion. Eroded plaques have a rich extracellular matrix, an intact fibrous cap, sparse lipid, and few mononuclear cells, but do harbour neutrophil extracellular traps (NETs). We recently reported that NETs amplify and propagate the endothelial damage at the site of arterial lesions that recapitulate superficial erosion in mice. We showed that genetic loss of protein arginine deiminase (PAD)-4 function inhibited NETosis and preserved endothelial integrity. The current study used systemic administration of targeted nanoparticles to deliver an agent that limits NETs formation to probe mechanisms of and demonstrate a novel therapeutic approach to plaque erosion that limits endothelial damage. Methods and results We developed Collagen IV-targeted nanoparticles (Col IV NP) to deliver PAD4 inhibitors selectively to regions of endothelial cell sloughing and collagen IV-rich basement membrane exposure. We assessed the binding capability of the targeting ligand in vitro and evaluated Col IV NP targeting to areas of denuded endothelium in vivo in a mouse preparation that recapitulates features of superficial erosion. Delivery of the PAD4 inhibitor GSK484 reduced NET accumulation at sites of intimal injury and preserved endothelial continuity. Conclusions NPs directed to Col IV show selective uptake and delivery of their payload to experimentally eroded regions, illustrating their translational potential. Our results further support the role of PAD4 and NETs in superficial erosion.

Introduction: Superficial plaque erosion has emerged as a growing cause of residual thrombotic complications of atherosclerosis in an era of effective lipid lowering, anti-hypertensive therapy, and smoking cessation. The mechanisms of plaque erosion remain poorly understood, and we currently lack validated effective diagnostics or therapeutics for superficial erosion. Lesions complicated by erosion have a rich extracellular matrix, sparse lipid, and few mononuclear cells, but do harbor neutrophil extracellular traps (NETs). We recently reported that NETs amplify and propagate the endothelial damage at the site of arterial lesions that recapitulate in mice elements of superficial erosion. We previously showed that genetic loss of protein arginine deiminase (PAD)-4 function inhibited NETosis and preserved endothelial integrity. Hypothesis: We hypothesized that Col IV-targeted NPs (Col IV-NPs) can deliver PAD4 inhibitors selectively to regions of EC sloughing and collagen IV-rich basement membrane exposure, and attenuate the amplification, persistence, and propagation of superficial erosion and consequent thrombosis. Methods: We developed Col IV-NPs to deliver PAD4 inhibitors selectively to regions of endothelial cell sloughing and collagen IV-rich basement membrane exposure using our validated experimental preparation that reproduces in mice some of the conditions ascribed to lesions associated with superficial erosion. Results and Conclusions: We first demonstrated the binding of the targeting ligand in vitro and evaluated Col IV NP targeting towards areas of denuded endothelium in vivo in this mouse preparation. Delivery of the PAD4 inhibitor GSK484 as cargo in these nanoparticles reduced NETs release at sites of eroded areas. Compared to non-targeted NPs, Col IV NPs showed a 1.8-fold increased accumulation at regions of endothelial injury (p

As the population affected by Alzheimer's disease (AD) grows, so does the need for a noninvasive and accurate diagnostic tool. Current research reveals that AD pathogenesis begins as early as decades before clinical symptoms. The unique properties of nanoparticles (NPs) may be exploited to develop noninvasive diagnostics for early detection of AD. After exposure of NPs to biological fluids, the NP surface is altered by an unbiased but selective and reproducible adsorption of biomolecules commonly referred to as the biomolecular corona or protein corona (PC). The discovery that the plasma proteome may be differentially altered during health and disease leads to the concept of disease‐specific PCs. Herein, the disease‐specific PCs formed around NPs in a multi‐NPs platform are employed to successfully identify subtle changes in plasma protein patterns and detect AD (>92% specificity and ≈100% sensitivity). Similar discrimination power is achieved using banked plasma samples from a cohort of patients several years prior to their diagnosis with AD. With the nanoplatform's analytic ability to analyze pathological proteomic changes into a disease‐specific identifier, this promising, noninvasive technology with implications for early detection and intervention could benefit not only patients with AD but other diseases as well.

Background:This prospective, multicentre, observational INVIDIa-2 study is investigating the clinical efficacy of influenza vaccination in advanced-cancer patients receiving immune-checkpoint inhibitors (ICIs), enrolled in 82 Italian centres, from October 2019 to January 2020. The primary endpoint was the incidence of influenza-like illness (ILI) until 30 April 2020. All the ILI episodes, laboratory tests, complications, hospitalizations and pneumonitis were recorded. Therefore, the study prospectively recorded all the COVID-19 ILI events.Patients and methods:Patients were included in this non-prespecified COVID-19 analysis, if alive on 31 January 2020, when the Italian government declared the national emergency. The prevalence of confirmed COVID-19 cases was detected as ILI episode with laboratory confirmation of SARS-CoV-2. Cases with clinical-radiological diagnosis of COVID-19 (COVID-like ILIs), were also reported.Results:Out of 1257 enrolled patients, 955 matched the inclusion criteria for this unplanned analysis. From 31 January to 30 April 2020, 66 patients had ILI: 9 of 955 cases were confirmed COVID-19 ILIs, with prevalence of 0.9% [95% confidence interval (CI): 0.3–2.4], a hospitalization rate of 100% and a mortality rate of 77.8%. Including 5 COVID-like ILIs, the overall COVID-19 prevalence was 1.5% (95% CI: 0.5–3.1), with 100% hospitalization and 64% mortality. The presence of elderly, males and comorbidities was significantly higher among patients vaccinated against influenza versus unvaccinated ( p = 0.009, p Conclusion:COVID-19 has a meaningful clinical impact on the cancer-patient population receiving ICIs, with high prevalence, hospitalization and an alarming mortality rate among symptomatic cases. Influenza vaccination does not protect from SARS-CoV-2 infection.

Cancer treatment still represents a formidable challenge, despite substantial advancements in available therapies being made over the past decade. One major issue is poor therapeutic efficacy due to lack of specificity and low bioavailability. The progress of nanotechnology and the development of a variety of nanoplatforms have had a significant impact in improving the therapeutic outcome of chemotherapeutics. Nanoparticles can overcome various biological barriers and localize at tumor site, while simultaneously protecting a therapeutic cargo and increasing its circulation time. Despite this, due to their synthetic origin, nanoparticles are often detected by the immune system and preferentially sequestered by filtering organs. Exosomes have recently been investigated as suitable substitutes for the shortcomings of nanoparticles due to their biological compatibility and particularly small size (i.e., 30-150 nm). In addition, exosomes have been found to play important roles in cell communication, acting as natural carriers of biological cargoes throughout the body. This review aims to highlight the use of exosomes as drug delivery vehicles for cancer and showcases the various attempts used to exploit exosomes with a focus on the delivery of chemotherapeutics and nucleic acids.

Recent studies on coronavirus infectious disease 2019 (COVID-19) pathophysiology indicated the cytokine release syndrome induced by the virus as the main cause of mortality. Patients with severe COVID-19 infection present a systemic hyper inflammation that can lead to lung and multi-organ injuries. Among the most recent treatments, corticosteroids have been identified to be effective in mitigating these catastrophic effects. Our group has recently developed leukocyte-derived nanovesicles, termed leukosomes, able to target in vivo the inflamed vasculature associated with pathological conditions including cancer, cardiovascular diseases, and sepsis. Herein, to gain insights on the anti-inflammatory properties of leukosomes, we investigated their ability to reduce uncontrolled inflammation in a lethal model of lipopolysaccharide (LPS)-induced endotoxemia, recapitulating the cytokine storm syndrome observed in COVID-19 infection after encapsulating dexamethasone. Treated animals showed a significant survival advantage and an improved immune response resolution, as demonstrated by a cytokine array analysis of pro- and anti-inflammatory cytokines, chemokines, and other immune-relevant markers. Our results showed that leukosomes enhance the therapeutic activity of dexamethasone and better control the inflammatory response compared to the free drug. Such an approach could be useful for the development of personalized therapies in the treatment of hyperinflammation related to infectious diseases, including the ones caused by COVID-19.

Cancer treatment still remains a challenge due to the several limitations of currently used chemotherapeutics, such as their poor pharmacokinetics, unfavorable chemical properties, as well as inability to discriminate between healthy and diseased tissue. Nanotechnology offered potent tools to overcome these limitations. Drug encapsulation within a delivery system permitted i) to protect the payload from enzymatic degradation/ inactivation in the blood stream, ii) to improve the physicochemical properties of poorly water-soluble drugs, like paclitaxel, and iii) to selectively deliver chemotherapeutics to the cancer lesions, thus reducing the off-target toxicity, and promoting the intracellular internalization. To accomplish this purpose, several strategies have been developed, based on biological and physical changes happening locally and systemically as a consequence of tumorigenesis. Here, we will discuss the role of inflammation in the different steps of tumor development and the strategies based on the use of nanoparticles that exploit the inflammatory pathways in order to selectively target the tumor-associated microenvironment for therapeutic and diagnostic purposes.

Background: Uncertainty exists about the optimal schedule of adjuvant treatment of breast cancer with aromatase inhibitors and, to our knowledge, no trial has directly compared the three aromatase inhibitors anastrozole, exemestane, and letrozole. We investigated the schedule and type of aromatase inhibitors to be used as adjuvant treatment for hormone receptor-positive early breast cancer. Methods: FATA-GIM3 is a multicentre, open-label, randomised, phase 3 trial of six different treatments in postmenopausal women with hormone receptor-positive early breast cancer. Eligible patients had histologically confirmed invasive hormone receptor-positive breast cancer that had been completely removed by surgery, any pathological tumour size, and axillary nodal status. Key exclusion criteria were hormone replacement therapy, recurrent or metastatic disease, previous treatment with tamoxifen, and another malignancy in the previous 10 years. Patients were randomly assigned in an equal ratio to one of six treatment groups: oral anastrozole (1 mg per day), exemestane (25 mg per day), or letrozole (2·5 mg per day) tablets upfront for 5 years (upfront strategy) or oral tamoxifen (20 mg per day) for 2 years followed by oral administration of one of the three aromatase inhibitors for 3 years (switch strategy). Randomisation was done by a computerised minimisation procedure stratified for oestrogen receptor, progesterone receptor, and HER2 status; previous chemotherapy; and pathological nodal status. Neither the patients nor the physicians were masked to treatment allocation. The primary endpoint was disease-free survival. The minimum cutoff to declare superiority of the upfront strategy over the switch strategy was assumed to be a 2% difference in disease-free survival at 5 years. Primary efficacy analyses were done by intention to treat; safety analyses included all patients for whom at least one safety case report form had been completed. Follow-up is ongoing. This trial is registered with the European Clinical Trials Database, number 2006-004018-42, and ClinicalTrials.gov, number NCT00541086. Findings: Between March 9, 2007, and July 31, 2012, 3697 patients were enrolled into the study. After a median follow-up of 60 months (IQR 46–72), 401 disease-free survival events were reported, including 211 (11%) of 1850 patients allocated to the switch strategy and 190 (10%) of 1847 patients allocated to upfront treatment. 5-year disease-free survival was 88·5% (95% CI 86·7–90·0) with the switch strategy and 89·8% (88·2–91·2) with upfront treatment (hazard ratio 0·89, 95% CI 0·73–1·08; p=0·23). 5-year disease-free survival was 90·0% (95% CI 87·9–91·7) with anastrozole (124 events), 88·0% (85·8–89·9) with exemestane (148 events), and 89·4% (87·3 to 91·1) with letrozole (129 events; p=0·24). No unexpected serious adverse reactions or treatment-related deaths occurred. Musculoskeletal side-effects were the most frequent grade 3–4 events, reported in 130 (7%) of 1761 patients who received the switch strategy and 128 (7%) of 1766 patients who received upfront treatment. Grade 1 musculoskeletal events were more frequent with the upfront schedule than with the switch schedule (924 [52%] of 1766 patients vs 745 [42%] of 1761 patients). All other grade 3–4 adverse events occurred in less than 2% of patients in either group. Interpretation: 5 years of treatment with aromatase inhibitors was not superior to 2 years of tamoxifen followed by 3 years of aromatase inhibitors. None of the three aromatase inhibitors was superior to the others in terms of efficacy. Therefore, patient preference, tolerability, and financial constraints should be considered when deciding the optimal treatment approach in this setting. Funding: Italian Drug Agency.

About one million people/year develop colorectal cancer (CRC) and approximately half of them die. The extent of the disease (i.e. local invasion at the time of diagnosis) is a key prognostic factor. The 5-year survival rate is almost 90% in the case of delimited CRC and 10% in the case of metastasized CRC. Hence, one of the great challenges in the battle against CRC is to improve early diagnosis strategies. Large-scale proteomic approaches are widely used in cancer research to search for novel biomarkers. Such biomarkers can help in improving the accuracy of the diagnosis and in the optimization of personalized therapy. Herein, we provide an overview of studies published in the last 5 years on CRC that led to the identification of protein biomarkers suitable for clinical application by using proteomic approaches. We discussed these findings according to biomarker application, including also the role of protein phosphorylation and cancer stem cells in biomarker discovery. Our review provides a cross section of scientific approaches and can furnish suggestions for future experimental strategies to be used as reference by scientists, clinicians and researchers interested in proteomics for biomarker discovery. This article is protected by copyright. All rights reserved

Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is a chronic inflammatory condition of the gastrointestinal (GI) tract. Currently, it is treated with immunosuppressant or biologics that often induce severe adverse effects. Thus, there is an urgent clinical need for more specific treatments. To provide a valid therapeutic tool for IBD therapy, in this work we developed biomimetic nanovesicles by manipulating leukocyte membranes to exploit mechanisms of T-cell recruitment during inflammation. A subset of T-lymphocytes participates in homing to inflamed tissue in the gastrointestinal tract by overexpressing the α4β7 integrin, which is responsible for binding to its receptor on the endothelial membrane, the mucosal addressin cell adhesion molecule 1. Based on this principle, we engineered biomimetic vesicles, referred to as specialized leukosomes (SLKs), which are leukocyte-like carriers 'doped' with the α4β7 integrin over-induced in purified immune cells. We tested SLKs in an in vivo murine model of IBD induced by treatment with dextran sulfate sodium. Notably, treatment of IBD mice with SLKs allowed us to observe a reduction of inflammation (favorable modulation of both pro- and anti-inflammatory genes, as well as reduction of immune cells infiltration into the colon tissue), and a consequent enhanced intestinal repair (low epithelial damage). In this study, we demonstrate that biological-derived nanoparticles can be used not only as naturally targeted drug delivery systems, but also as nano-therapeutics endowed with intrinsic anti-inflammatory properties.

Breast cancer (BC) is the most common cancer in women. Although therapeutic armamentarium like chemotherapy, endocrine and target agents have increased survival, cardiovascular side effects have been observed. A comprehensive risk assessment, early detection and management of cardiac adverse events is therefore needed.In this review we focus on cardiotoxicity data deriving from Phase III randomized trials, systematic reviews and meta-analysis in BC patients. We provide insight into advances that have been made in the molecular mechanisms, clinical presentation and management of such adverse event.Despite the large number of data from Phase III trials about cardiac events incidence, there are poor evidences for detection, monitoring and management of cardiotoxicity during BC treatment. Future cardiotoxicity-oriented clinical cancer research can help to predict the risk of cardiac adverse events and improve patients' outcome. Multidisciplinary approach as well as integration of blood biomarkers with imaging will be desirable.

Despite an improved understanding of its pathophysiology and a wide range of new treatments, cardiovascular disease (CVD) remains a serious public health issue and the number one cause of mortality in the United States. Conditions that promote chronic systemic inflammation, such as obesity, cancer, and autoimmune and infectious diseases, are now known to play an important role in promoting CVD by inducing the expression of endothelial adhesion molecules and chemokines; these in turn promote leukocyte adherence and infiltration, which initiates and spurs the progression of CVD. In response to this new understanding, researchers are evaluating the potential cardiovascular benefits of new-generation therapies based on endogenous molecules with anti-inflammatory properties. Similarly, targeted approaches that leverage the phenotypic differences between non-inflamed and inflamed endothelia have the potential to selectively deliver therapeutics and decrease the morbidity and mortality of CVD patients. In this review, we discuss the role of inflammation in CVD and explore the therapeutic potential of targeting inflamed vasculature through conventional and biomimetic approaches.

BACKGROUND: Despite the well recognized expression of the cell surface markers cluster of differentiation 44 (homing cell adhesion molecule) and CD133 (Prominin 1) on human colorectal cancer stem cells (CCSCs), these molecules do not appear to be effective targets for stem cell-directed therapies. Because the surface marker CD66c (also known as carcinoembryonic antigen-related cell adhesion molecule 6) has demonstrated promise as a therapeutic target in pancreatic malignancy, the authors evaluated its potential as a target for stem cell-directed treatment of colorectal cancer. METHODS: First, the authors characterized CD66c expression by flow cytometry and immunohistochemistry in colon cancer samples and in normal colon tissues. Then, the coexpression of CD66c and CD133 was evaluated on putative CCSCs. CD66c expression also was measured in stem cell-enriched colon spheres. Finally, the effects of small-interfering RNA-mediated CD66c silencing on the in vitro and in vivo growth of Caco2 colon cancer cells were evaluated. RESULTS: CD66c expression was significantly higher in colon cancers than in contiguous normal colon tissues and paralleled cancer stage. CD66c was absent in CD133-positive cells that were isolated from normal colon, whereas its expression was brightest (CD66c bright ) in CD133-positive cells from colon cancer samples. In vitro experiments demonstrated that colon spheres were considerably enriched in a CD66c bright population in a fashion comparable to the enrichment observed in fresh liver metastases. In vitro proliferation and clonogenic potential were hampered when CD66c was silenced in Caco2 cells. Finally, in vivo xenograft experiments demonstrated that CD66c silencing almost completely abrogated the tumorigenic potential of Caco2 cells. CONCLUSIONS: CD66c bright expression was associated with colon cancer stem cells and CD66c silencing blocked tumor growth, thereby opening the way to a potential new treatment for colon cancer. Cancer 2012;000:000–000. V C 2012 American Cancer Society.

Each year, nearly one million people develop colorectal cancer, and 50 % of them are expected to die because of cancer within 5 years of diagnosis. Over the last two decades, significant advances in screening, surgery, adjuvant chemotherapy, and patient monitoring have improved the 5-year survival rate. Colorectal cancer is a molecularly heterogeneous disease, and the analysis of its molecular signatures and the identification of colorectal cancer biomarkers will lead to better screening approaches and personalized therapeutic plans. A biomarker is a substance that can be measured and used as an indicator of a biological and/or pathological state. In cancer pathology, biomarkers can be used to detect the disease, to predict prognosis or response to therapy, and to evaluate the efficacy of therapy thereby improving the patient’s life expectancy and quality of life. The carcinoembryonic antigen (CEA) is the biomarker most frequently used in colorectal cancer. It is effective in the surveillance of colorectal cancer patients and in monitoring the efficacy of therapy, whereas it is less useful in colorectal screening. Recent studies indicate that also CEA-related proteins (CEACAMs) are promising potential colorectal cancer biomarkers. CEA and CEACAM proteins play important roles in cancer pathology, and the analysis of their functions will be useful for the identification of diagnostic and/or prognostic factors and therapeutic targets in colorectal and other cancers. List of Abbreviations CEA Carcinoembyonic Antigen CEACAM Carcinoembryonic Antigen-Family Cell Adhesion Molecule FOBT Fecal Occult Blood Test GPI Glycosylphosphatidylinositol Ig Immunoglobulin IgCAM Ig-like Cell Adhesion Molecule PCR Polymerase Chain Reaction TNM Tumor Nodes Metastases Key Facts of Colorectal Cancer • The intestine is the main site of nutrient absorption; it is subdivided in small and large intestine. The latter comprises the right colon, transverse colon, left colon, sigma, and rectum. • Colorectal cancer develops from uncontrolled growth of colorectal mucosa cells. • Colorectal cancer is an epithelial cancer arising in almost 80 % of cases from nonmalignant lesions that turned into cancer within 5–10 years of their appearance. 686 M. Gemei et al.

Colorectal cancer (CRC) is one of the most common types of cancer; it is diagnosed in more than one million people each year and causes the death of about half of them. Early detection can help to decrease CRC-related mortality. In fact, the 5-year survival rate exceeds 90 % when the disease is localized and is only 10 % in case of metastases. Consequently, it is imperative to improve methods for the early detection of the disease. In this context, genomic approaches have resulted in new CRC biomarkers and have helped to shed light on the genetic basis of cancer as a whole. However, the proteome provides a more dynamic and faithful image of the genetic program of a cell. Hence, given the importance of proteins as effectors of cellular behavior, proteomic analysis has the potential to identify biomarkers that can help to classify and predict CRC. Several proteomic technique, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), quantitative isotopic protein labeling (SILAC, ICAT, and iTRAQ), and two-dimensional gel electrophoresis (2D-PAGE and 2D-DIGE), are being used in cancer research and can improve the diagnosis of patients; they are also helping to optimize personalized therapy. In this chapter, the main proteomic approaches used in biomarker research will be discussed, together with their impact and potential in the clinical setting. The proteomic biomarkers currently used for CRC diagnosis and/or therapy monitoring will be also described. List of Abbreviations 2D-DIGE Two-Dimensional Differential In-gel Electrophoresis 2D-PAGE Two-Dimensional Polyacrylamide Gel Electrophoresis APC Adenomatous Polyposis Coli CEA Carcinoembryonic Antigen CRC Colorectal Cancer Cy2 Cyanine 2 Fluorescent Dye Cy3 Cyanine 3 Fluorescent Dye Cy5 Cyanine 5 Fluorescent Dye DCBE Double-Contrast Barium Enema ESI Electrospray Ionization FIT Fecal Immunological Test FOBT Fecal Occult Blood Test *Email: salvator@unina.it Biomarkers in Cancer DOI 10.1007/978-94-007-7744-6_16-1 # Springer Science+Business Media Dordrecht 2014