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Abstract Background Urinary kidney injury molecule 1 is a recently discovered early biomarker for renal damage that has been proven to be correlated to urinary cadmium in rats. However, so far the association between urinary cadmium and kidney injury molecule 1 in humans after long-term, low-dose cadmium exposure has not been studied. Methods We collected urine and blood samples from 153 non-smoking men and women aged 60+, living in an area with moderate cadmium pollution from a non-ferrous metal plant for a significant period. Urinary cadmium and urinary kidney injury molecule 1 as well as other renal biomarkers (alpha1-microglobulin, beta2-microglobulin, blood urea nitrogen, urinary proteins and microalbumin) were assessed. Results Both before (r = 0.20; p = 0.01) and after (partial r = 0.32; p < 0.0001) adjustment for creatinine, age, sex, past smoking, socio-economic status and body mass index, urinary kidney injury molecule 1 correlated with urinary cadmium concentrations. No significant association was found between the other studied renal biomarkers and urinary cadmium. Conclusions We showed that urinary kidney injury molecule 1 levels are positively correlated with urinary cadmium concentration in an elderly population after long-term, low-dose exposure to cadmium, while other classical markers do not show an association. Therefore, urinary kidney injury molecule 1 might be considered as a biomarker for early-stage metal-induced kidney injury by cadmium.

The therapeutic potential of liposomes to deliver drugs into inflamed tissue is well documented. Liposomes are believed to largely transport drugs into inflamed joints by selective extravasation through endothelial gaps at the inflammatory sites, known as the enhanced permeation and retention effect. However, the potential of blood-circulating myeloid cells for the uptake and delivery of liposomes has been largely overlooked. Here we show that myeloid cells can transport liposomes to inflammatory sites in a collagen-induced arthritis model. It is shown that the selective depletion of the circulating myeloid cells reduces the accumulation of liposomes up to 50-60%, suggesting that myeloid-cell-mediated transport accounts for more than half of liposomal accumulation in inflamed regions. Although it is widely believed that PEGylation inhibits premature liposome clearance by the mononuclear phagocytic system, our data show that the long blood circulation times of PEGylated liposomes rather favours uptake by myeloid cells. This challenges the prevailing theory that synovial liposomal accumulation is primarily due to the enhanced permeation and retention effect and highlights the potential for other pathways of delivery in inflammatory diseases., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Rationale: Although promising responses are obtained in patients treated with immune checkpoint inhibitors targeting programmed death ligand 1 (PD-L1) and its receptor programmed death-1 (PD-1), only a fraction of patients benefits from this immunotherapy. Cancer vaccination may be an effective approach to improve the response to immune checkpoint inhibitors anti-PD-L1/PD-1 therapy. However, there is a lack of research on the dynamics of PD-L1 expression in response to cancer vaccination. Methods: We performed non-invasive whole-body imaging to visualize PD-L1 expression at different timepoints after vaccination of melanoma-bearing mice. Mice bearing ovalbumin (OVA) expressing B16 tumors were i.v. injected with the Galsome mRNA vaccine: OVA encoding mRNA lipoplexes co-encapsulating a low or a high dose of the atypical adjuvant α-galactosylceramide (αGC) to activate invariant natural killer T (iNKT) cells. Serial non-invasive whole-body immune imaging was performed using a technetium-99m ( 99m Tc)-labeled anti-PD-L1 nanobody, single-photon emission computerized tomography (SPECT) and X-ray computed tomography (CT) images were quantified. Additionally, cellular expression of PD-L1 was evaluated with flow cytometry. Results: SPECT/CT-imaging showed a rapid and systemic upregulation of PD-L1 after vaccination. PD-L1 expression could not be correlated to the αGC-dose, although we observed a dose-dependent iNKT cell activation. Dynamics of PD-L1 expression were organ-dependent and most pronounced in lungs and liver, organs to which the vaccine was distributed. PD-L1 expression in lungs increased immediately after vaccination and gradually decreased over time, whereas in liver, vaccination-induced PD-L1 upregulation was short-lived. Flow cytometric analysis of these organs further showed myeloid cells as well as non-immune cells with elevated PD-L1 expression in response to vaccination. SPECT/CT imaging of the tumor demonstrated that the expression of PD-L1 remained stable over time and was overall not affected by vaccination although flow cytometric analysis at the cellular level demonstrated changes in PD-L1 expression in various immune cell populations following vaccination. Conclusion: Repeated non-invasive whole-body imaging using 99m Tc-labeled anti-PD-L1 nanobodies allows to document the dynamic nature of PD-L1 expression upon vaccination. Galsome vaccination rapidly induced systemic upregulation of PD-L1 expression with the most pronounced upregulation in lungs and liver while flow cytometry analysis showed upregulation of PD-L1 in the tumor microenvironment. This study shows that imaging using nanobodies may be useful for monitoring vaccine-mediated PD-L1 modulation in patients and could provide a rationale for combination therapy. To the best of our knowledge, this is the first report that visualizes PD-L1 expression upon cancer vaccination., Competing Interests: Competing Interests: Nick Devoogdt and Marleen Keyaerts are founders and shareholders in Abscint. Nick Devoogdt is a consultant for Precirix and reports grants, personal fees, and non-financial support from Precirix, and non-financial support from Abscint. Marleen Keyaerts and Karine Breckpot have ongoing collaborations with Precerix for which they receive non-financial support. Karine Breckpot, Katrijn Broos, Nick Devoogdt and Marleen Keyaerts hold a patent on 'Human pd-l1-binding immunoglobulins' (WO2019166622A1). Ine Lentacker, Stefaan De Smedt, Rein Verbeke and Heleen Dewitte have a patent application on 'Therapeutic nanoparticles and methods of use thereof”. (WO2020/058239)., (© The author(s).)

Objectives: We aimed to investigate whether the use of an e-health tool, guided by a healthcare provider, can improve health literacy (HL) in primary care., Methods: We set up a longitudinal prospective cohort study in a primary care clinic in Brussels. Diabetes patients were invited to participate in two study consultations with a trained healthcare provider, in which an e-health tool was introduced. The Health Literacy Questionnaire (HLQ) was used to evaluate HL before (n = 59) and after intervention (n = 41). The data were analysed within SPSS, Version 26. Additionally, impressions and experiences of both patients and healthcare providers were collected throughout the different phases of the study., Results: Patients feel significantly stronger in finding good health information after intervention (p = 0.041), with relatively stronger progress for the subgroup with weaker digital skills (p = 0.029). Participants also declare understanding health information better after intervention (p = 0.050). Specifically, the lower educated participants feel reinforced to correctly evaluate and assess health information and come closer to the skill level of the higher educated patients after intervention. The relationship with the healthcare provider was also more markedly enhanced within the group of the lower educated (p = 0.008; difference between higher and lower educated), which could strengthen self-management in the long run., Conclusions: The guided use of an e-health tool in primary care strengthens various patient HL skills. Most particularly the skills "the ability to find good health information" and "understand health information well enough to know what to do" are reinforced. Moreover, patient populations with lower HL, such as the lower educated and lower digitally skilled, show a greater learning potential., Innovation: Our results offer further proof for the learnable and flexible nature of HL, and show that even a small e-health intervention, in a very diverse patient population, can produce significant, positive effects on HL. These results need to be considered as promising, and a motivation for further investments in more widely accessible e-health tools to further improve HL at population level and to bridge health differences., Competing Interests: The authors have no conflict of interest., (© 2022 The Authors.)

Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T 1 -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines., (© 2021 Wiley-VCH GmbH.)

In less than one year since the outbreak of the COVID-19 pandemic, two mRNA-based vaccines, BNT162b2 and mRNA-1273, were granted the first historic authorization for emergency use, while another mRNA vaccine, CVnCoV, progressed to phase 3 clinical testing. The COVID-19 mRNA vaccines represent a new class of vaccine products, which consist of synthetic mRNA strands encoding the SARS-CoV-2 Spike glycoprotein, packaged in lipid nanoparticles to deliver mRNA to cells. This review digs deeper into the scientific breakthroughs of the last decades that laid the foundations for the rapid rise of mRNA vaccines during the COVID-19 pandemic. As well as providing momentum for mRNA vaccines, SARS-CoV-2 represents an ideal case study allowing to compare design-activity differences between the different mRNA vaccine candidates. Therefore, a detailed overview of the composition and (pre)clinical performance of the three most advanced mRNA vaccines is provided and the influence of choices in their structural design on to their immunogenicity and reactogenicity profile is discussed in depth. In addition to the new fundamental insights in the mRNA vaccines' mode of action highlighted here, we also point out which unknowns remain that require further investigation and possibly, optimization in future mRNA vaccine development., (Copyright © 2021 Elsevier B.V. All rights reserved.)

mRNA therapeutics have become the focus of molecular medicine research. Various mRNA applications have reached major milestones at high speed in the immuno-oncology field. This can be attributed to the knowledge that mRNA is one of nature's core building blocks carrying important information and can be considered as a powerful vector for delivery of therapeutic proteins to the patient.For a long time, the major focus in the use of in vitro transcribed mRNA was on development of cancer vaccines, using mRNA encoding tumor antigens to modify dendritic cells ex vivo. However, the versatility of mRNA and its many advantages have paved the path beyond this application. In addition, due to smart design of both the structural properties of the mRNA molecule as well as pharmaceutical formulations that improve its in vivo stability and selective targeting, the therapeutic potential of mRNA can be considered as endless.As a consequence, many novel immunotherapeutic strategies focus on the use of mRNA beyond its use as the source of tumor antigens. This review aims to summarize the state-of-the-art on these applications and to provide a rationale for their clinical application.

To date, mRNA-based biologics have mainly been developed for prophylactic and therapeutic vaccination to combat infectious diseases or cancer. In the past years, optimization of the characteristics of in vitro transcribed mRNA has led to significant reduction of the inflammatory responses. Thanks to this, mRNA therapeutics have entered the field of passive immunization. Here, we established an mRNA treatment that is based on mRNA that codes for a bispecific single-domain antibody construct that can selectively recruit innate immune cells to cells infected with influenza A virus. The constructs consist of a single-domain antibody that binds to the ectodomain of the conserved influenza A matrix protein 2, while the other single-domain antibody binds to the activating mouse Fcγ receptor IV. Formulating the mRNA into DOTAP (1,2-dioleoyl-3-trimethylammonium-propane)/cholesterol nanoparticles and delivering these intratracheally to mice allowed the production of the bispecific single-domain antibody in the lungs, and administration of these mRNA-particles prior to influenza A virus infection was associated with a significant reduction in viral titers and a reduced morbidity in mice. Overall, our data provide evidence that the local delivery of mRNA encoding a bispecific single-domain antibody format in the lungs could be a promising pulmonary antiviral prophylactic treatment., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Therapeutic ultrasound strategies that harness the mechanical activity of cavitation nuclei for beneficial tissue bio-effects are actively under development. The mechanical oscillations of circulating microbubbles, the most widely investigated cavitation nuclei, which may also encapsulate or shield a therapeutic agent in the bloodstream, trigger and promote localized uptake. Oscillating microbubbles can create stresses either on nearby tissue or in surrounding fluid to enhance drug penetration and efficacy in the brain, spinal cord, vasculature, immune system, biofilm or tumors. This review summarizes recent investigations that have elucidated interactions of ultrasound and cavitation nuclei with cells, the treatment of tumors, immunotherapy, the blood-brain and blood-spinal cord barriers, sonothrombolysis, cardiovascular drug delivery and sonobactericide. In particular, an overview of salient ultrasound features, drug delivery vehicles, therapeutic transport routes and pre-clinical and clinical studies is provided. Successful implementation of ultrasound and cavitation nuclei-mediated drug delivery has the potential to change the way drugs are administered systemically, resulting in more effective therapeutics and less-invasive treatments., Competing Interests: Conflict of interest disclosure The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a low response to treatment and a five-year survival rate below 5%. The ineffectiveness of treatment is partly because of an immunosuppressive tumor microenvironment, which comprises tumor-supportive pancreatic stellate cells (PSCs). Therefore, new therapeutic strategies are needed to tackle both the immunosuppressive PSC and pancreatic cancer cells (PCCs). Recently, physical cold atmospheric plasma consisting of reactive oxygen and nitrogen species has emerged as a novel treatment option for cancer. In this study, we investigated the cytotoxicity of plasma-treated phosphate-buffered saline (pPBS) using three PSC lines and four PCC lines and examined the immunogenicity of the induced cell death. We observed a decrease in the viability of PSC and PCC after pPBS treatment, with a higher efficacy in the latter. Two PCC lines expressed and released damage-associated molecular patterns characteristic of the induction of immunogenic cell death (ICD). In addition, pPBS-treated PCC were highly phagocytosed by dendritic cells (DCs), resulting in the maturation of DC. This indicates the high potential of pPBS to trigger ICD. In contrast, pPBS induced no ICD in PSC. In general, pPBS treatment of PCCs and PSCs created a more immunostimulatory secretion profile (higher TNF-α and IFN-γ, lower TGF-β) in coculture with DC. Altogether, these data show that plasma treatment via pPBS has the potential to induce ICD in PCCs and to reduce the immunosuppressive tumor microenvironment created by PSCs. Therefore, these data provide a strong experimental basis for further in vivo validation, which might potentially open the way for more successful combination strategies with immunotherapy for PDAC.

Ultrasound-triggered microbubble-assisted drug delivery is a promising tool for localized therapy. Several studies have shown the potential of nanoparticle-loaded microbubbles to effectively enhance the delivery of therapeutic agents to target tissue. We recently discovered that nanoparticle-carrying microbubbles can deposit the nanoparticles in patches onto cell membranes, a process which we termed 'sonoprinting'. However, the biophysical mechanisms behind sonoprinting are not entirely clear. In addition, the question remains how the ultrasound parameters, such as acoustic pressure and pulse duration, influence sonoprinting. Aiming for a better understanding of sonoprinting, this report investigates the behavior of nanoparticle-loaded microbubbles under ultrasound exposure, making use of three advanced optical imaging techniques with frame rates ranging from 5 frames per second to 10 million frames per second, to capture the biophysical cell-bubble interactions that occur on a multitude of timescales. We observed that non-spherically oscillating microbubbles release their nanoparticle payload in the first few cycles of ultrasound insonation. At low acoustic pressures, the released nanoparticles are transported away from the cells by microstreaming, which does not favor uptake of the nanoparticles by the cells. However, higher acoustic pressures (>300 kPa) and longer ultrasound pulses (>100 cycles) lead to rapid translation of the microbubbles, due to acoustic radiation forces. As a result, the released nanoparticles are transported along in the wake of the microbubbles, which eventually leads to the deposition of nanoparticles in elongated patches on the cell membrane, i.e. sonoprinting. We conclude that a sufficiently high acoustic pressure and long pulses are needed for sonoprinting of nanoparticles on cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

mRNA therapeutics have recently experienced a new wave of interest, mainly due to the discovery that chemical modifications to mRNA's molecular structure could drastically reduce its inherent immunogenicity and perceived instability. On this basis, we aimed to explore the potential of chemically stabilized mRNA for ocular applications. More specifically, we investigated the behavior of mRNA-loaded lipid-based carriers in human retinal cells (in vitro), in bovine retinal explants (ex vivo) and in mouse retinas (in vivo). We demonstrate a clear superiority of mRNA over pDNA to induce protein expression in different retinal cell types, which was further enhanced by chemical modification of the mRNA, providing up to ~1800-fold higher reporter gene expression compared to pDNA. Moreover, transgene expression could be detected for at least 20 days after a single administration of chemically modified mRNA in vitro. We furthermore determined the localization and extent of mRNA expression depending on the administration route. After subretinal (SR) administration, mRNA expression was observed in vivo and ex vivo. By contrast, intravitreal (IVT) administration resulted in limited expression in vivo. Using ex vivo bovine explants with an intact vitreoretinal (VR) interface we could attribute this to the inner limiting membrane (ILM), which presents a large barrier for non-viral delivery of mRNA, trapping mRNA complexes at the vitreal side. When the vitreous was removed, which compromises the ILM, mRNA expression was apparent and seemed to colocalize with Müller cells or photoreceptors after respectively IVT or SR administration. Taken together, this study represents a first step towards mRNA-mediated therapy for retinal diseases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Considerable research over the last few years has revealed dysregulation of growth factors in various retinal diseases, such as glaucoma, diabetic retinopathy and photoreceptor degenerations. The use of messengerRNA (mRNA) to transiently overexpress a specific factor could compensate for this imbalance. However, a critical challenge of this approach lies in the ability to efficiently deliver mRNA molecules to the retinal target cells. In this study we found that intravitreal (IVT) injection is an attractive approach to deliver mRNA to the retina, providing two critical barriers can be overcome: the vitreous and the inner limiting membrane (ILM). We demonstrated that the vitreous is indeed a major hurdle in the delivery of the cationic mRNA-complexes to retinal cells, both in terms of vitreal mobility and cellular uptake. To improve their intravitreal mobility and avoid unwanted extracellular interactions, we evaluated the use of hyaluronic acid (HA) as an electrostatic coating strategy. This HA-coating provided the complexes with a negative surface charge, markedly enhancing their mobility in the vitreous humor, without reducing their cellular internalization and transfection efficiency. However, although this coating strategy allows the mRNA-complexes to successfully overcome the vitreal barrier, the majority of the particles accumulated at the ILM. This study therefore underscores the crucial barrier function of the ILM toward non-viral retinal gene delivery and the need to smartly design mRNA-carriers able to surmount the vitreous as well as the ILM., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Messenger RNA encoding tumor antigens has the potential to evoke effective antitumor immunity. This study reports on a nanoparticle platform, named mRNA Galsomes, that successfully co-delivers nucleoside-modified antigen-encoding mRNA and the glycolipid antigen and immunopotentiator α-galactosylceramide (α-GC) to antigen-presenting cells after intravenous administration. By co-formulating low doses of α-GC, mRNA Galsomes induce a pluripotent innate and adaptive tumor-specific immune response in mice, with invariant natural killer T cells (iNKT) as a driving force. In comparison, mRNA Galsomes exhibit advantages over the state-of-the-art cancer vaccines using unmodified ovalbumin (OVA)-encoding mRNA, as we observed up to seven times more tumor-infiltrating antigen-specific cytotoxic T cells, combined with a strong iNKT cell and NK cell activation. In addition, the presence of suppressive myeloid cells (myeloid-derived suppressor cells and tumor-associated macrophages) in the tumor microenvironment was significantly lowered. Owing to these antitumor effects, OVA mRNA Galsomes significantly reduced tumor growth in established E.G7-OVA lymphoma, with a complete tumor rejection in 40% of the animals. Moreover, therapeutic vaccination with mRNA Galsomes enhanced the responsiveness to treatment with a PD-L1 checkpoint inhibitor in B16-OVA melanoma, as evidenced by a synergistic reduction of tumor outgrowth and a significantly prolonged median survival. Taken together, these data show that intravenously administered mRNA Galsomes can provide controllable, multifaceted, and effective antitumor immunity, especially when combined with checkpoint inhibition.

For gene therapy to work in vivo, nucleic acids need to reach the target cells without causing major side effects to the patient. In many cases the gene only has to reach a subset of cells in the body. Therefore, targeted delivery of genes to the desired tissue is a major issue in gene delivery. Many different possibilities of targeted gene delivery have been studied. A physical approach to target nucleic acids and other drugs to specific regions in the body is the use of ultrasound and microbubbles. Microbubbles are gas filled spheres with a stabilizing lipid, protein, or polymer shell. When these microbubbles enter an ultrasonic field, they start to oscillate. The bubbles' expansion and compression are inversely related to the pressure phases in the ultrasonic field. When microbubbles are exposed to high-intensity ultrasound the microbubbles will eventually implode and fragment. This generates shockwaves and microjets which can temporarily permeate cell membranes and blood vessels. Nucleic acids or (non)viral vectors can as a result gain direct access to either the cytoplasm of neighboring cells, or extravasate to the surrounding tissue. The nucleic acids can either be mixed with the microbubbles or loaded on the microbubbles. Nucleic acid loaded microbubbles can be obtained by coupling nucleic acid-containing particles (i.e., lipoplexes) to the microbubbles. Upon ultrasound-mediated implosion of the microbubbles, the nucleic acid-containing particles will be released and will deliver their nucleic acids in the ultrasound-targeted region.

Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies.

This study reports on the design of mRNA and adjuvant-loaded lipid nanoparticles for therapeutic cancer vaccination. The use of nucleoside-modified mRNA has previously been shown to improve the translational capacity and safety of mRNA-therapeutics, as it prevents the induction of type I interferons (IFNs). However, type I IFNs were identified as the key molecules that trigger the activation of antigen presenting cells, and as such drive T cell immunity. We demonstrate that nucleoside-modified mRNA can be co-delivered with the clinically approved TLR agonist monophosphoryl lipid A (MPLA). As such, we simultaneously allow high antigen expression in vivo while substituting the type I IFN response by a more controllable adjuvant. This strategy shows promise to induce effective antigen-specific T cell immunity and may be useful to enhance the safety of mRNA vaccines., (Copyright © 2017. Published by Elsevier B.V.)

Background: Telomere length and mitochondrial DNA (mtDNA) content are markers of aging and aging-related diseases. There is inconclusive evidence concerning the mechanistic effects of airborne particulate matter (PM) exposure on biomolecular markers of aging., Objective: The present study examines the association between short- and long-term PM exposure with telomere length and mtDNA content in the elderly and investigates to what extent this association is mediated by expression of genes playing a role in the telomere-mitochondrial axis of aging., Methods: Among 166 nonsmoking elderly participants, we used qPCR to measure telomere length and mtDNA content in leukocytes and RNA from whole blood to measure expression of SIRT1, TP53, PPARGC1A, PPARGC1B, NRF1, and NFE2L2. Associations between PM exposure and markers of aging were estimated using multivariable linear regression models adjusted for sex, age, BMI, socioeconomic status, statin use, past smoking status, white blood cell count, and percentage of neutrophils. Mediation analysis was performed to explore the role of age-related markers between the association of PM exposure and outcome. Annual PM2.5 exposure was calculated for each participant's home address using a high-resolution spatial-temporal interpolation model., Results: Annual PM2.5 concentrations ranged from 15 to 23 μg/m3. A 5-μg/m3 increment in annual PM2.5 concentration was associated with a relative decrease of 16.8% (95% CI: -26.0%, -7.4%, p = 0.0005) in telomere length and a relative decrease of 25.7% (95% CI: -35.2%, -16.2%, p < 0.0001) in mtDNA content. Assuming causality, results of the mediation analysis indicated that SIRT1 mediated 19.5% and 22.5% of the estimated effect of PM2.5 exposure on telomere length and mtDNA content, respectively., Conclusions: Our findings suggest that the estimated effects of PM2.5 exposure on the telomere-mitochondrial axis of aging may play an important role in chronic health effects of PM2.5., Citation: Pieters N, Janssen BG, Dewitte H, Cox B, Cuypers A, Lefebvre W, Smeets K, Vanpoucke C, Plusquin M, Nawrot TS. 2016. Biomolecular markers within the core axis of aging and particulate air pollution exposure in the elderly: a cross-sectional study. Environ Health Perspect 124:943-950; http://dx.doi.org/10.1289/ehp.1509728.

Cancer vaccines based on mRNA are extensively studied. The fragile nature of mRNA has instigated research into carriers that can protect it from ribonucleases and as such enable its systemic use. However, carrier-mediated delivery of mRNA has been linked to production of type I interferon (IFN) that was reported to compromise the effectiveness of mRNA vaccines. In this study, we evaluated a cationic lipid for encapsulation of mRNA. The nanometer-sized, negatively charged lipid mRNA particles (LMPs) efficiently transfected dendritic cells and macrophages in vitro. Furthermore, i.v. delivery of LMPs resulted in rapid expression of the mRNA-encoded protein in spleen and liver, predominantly in CD11c(+) cells and to a minor extent in CD11b(+) cells. Intravenous immunization of mice with LMPs containing ovalbumin, human papilloma virus E7, and tyrosinase-related protein-2 mRNA, either combined or separately, elicited strong antigen-specific T-cell responses. We further showed the production of type I IFNs upon i.v. LMP delivery. Although this decreased the expression of the mRNA-encoded protein, it supported the induction of antigen-specific T-cell responses. These data question the current notion that type I IFNs hamper particle-mediated mRNA vaccines.

Following intravenous injection of anti-cancer nanomedicines, many barriers need to be overcome en route to the tumor. Cell-mediated delivery of nanoparticles (NPs) is promising in terms of overcoming several of these barriers based on the tumoritropic migratory properties of particular cell types. This guided transport aims to enhance the NP accumulation in the tumor and moreover enhance the infiltration of regions that are typically inaccessible for free NPs. Within this study, cytotoxic CD8(+) T cells were selected as carriers based on both their ability to migrate to the tumor and their intrinsic cytolytic activity against tumor cells. Many anti-cancer nanomedicines require tumor cell internalization to mediate cytosolic drug delivery and enhance the anti-cancer effect. This proof-of-concept therefore reports on the reversible attachment of liposomes to the surface of cytotoxic T lymphocytes via a reduction sensitive coupling. The activation status of the T cells and the liposome composition are shown to strongly influence the loading efficiency. Loading the cells with liposomes does not compromise T cell functionalities like proliferation and cytolytic function. Additionally, the triggered liposome release is demonstrated upon the addition of glutathione. Based on this optimization using liposomes as model NPs, a small interfering RNA (siRNA)-loaded NP was developed that can be coupled to the surface of CD8(+) T cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the field of nonviral gene therapy, in vitro transcribed (IVT) mRNA has emerged as a promising tool for the delivery of genetic information. Over the past few years it has become widely known that the introduction of IVT mRNA into mammalian cells elicits an innate immune response that has favored mRNA use toward immunotherapeutic vaccination strategies. However, for non-immunotherapy-related applications this intrinsic immune-stimulatory activity directly interferes with the aimed therapeutic outcome, because it can seriously compromise the expression of the desired protein. This review presents an overview of the immune-related obstacles that limit mRNA advance for non-immunotherapy-related applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The term "immunogenic cell death" (ICD) is commonly employed to indicate a peculiar instance of regulated cell death (RCD) that engages the adaptive arm of the immune system. The inoculation of cancer cells undergoing ICD into immunocompetent animals elicits a specific immune response associated with the establishment of immunological memory. Only a few agents are intrinsically endowed with the ability to trigger ICD. These include a few chemotherapeutics that are routinely employed in the clinic, like doxorubicin, mitoxantrone, oxaliplatin, and cyclophosphamide, as well as some agents that have not yet been approved for use in humans. Accumulating clinical data indicate that the activation of adaptive immune responses against dying cancer cells is associated with improved disease outcome in patients affected by various neoplasms. Thus, novel therapeutic regimens that trigger ICD are urgently awaited. Here, we discuss current combinatorial approaches to convert otherwise non-immunogenic instances of RCD into bona fide ICD.

About 25 years ago, mRNA became a tool of interest in anticancer vaccination approaches. However, due to its rapid degradation in situ, direct application of mRNA was confronted with considerable skepticism during its early use. Consequently, mRNA was for a long time mainly used for the ex vivo transfection of dendritic cells, professional antigen-presenting cells known to stimulate immunity. The interest in direct application of mRNA experienced a revival, as researchers became aware of the many advantages mRNA offers. Today, mRNA is considered to be an ideal vehicle for the induction of strong immune responses against cancer. The growing numbers of preclinical trials and as a consequence the increasing clinical application of mRNA as an off-the-shelf anticancer vaccine signifies a renaissance for transcript-based antitumor therapy. In this review, we highlight this renaissance using a timeline providing all milestones in the application of mRNA for anticancer vaccination.

Experimental evidence shows that telomere shortening induces mitochondrial damage but so far studies in humans are scarce. Here, we investigated the association between leukocyte telomere length (LTL) and mitochondrial DNA (mtDNA) content in elderly and explored possible intermediate mechanisms by determining the gene expression profile of candidate genes in the telomere-mitochondrial axis of ageing. Among 166 non-smoking elderly, LTL, leukocyte mtDNA content and expression of candidate genes: sirtuin1 (SIRT1), tumor protein p53 (TP53), peroxisome proliferator-activated receptor γ-coactivator1α (PGC-1α), peroxisome proliferator-activated receptor γ-coactivator1β (PGC-1β), nuclear respiratory factor 1 (NRF1) and nuclear factor, erythroid 2 like 2 (NRF2), using a quantitave real time polymerase chain assay (qPCR). Statistical mediation analysis was used to study intermediate mechanisms of the telomere-mitochondrial axis of ageing. LTL correlated with leukocyte mtDNA content in our studied elderly (r = 0.23, p = 0.0047). SIRT1 gene expression correlated positively with LTL (r = 0.26, p = 0.0094) and leukocyte mtDNA content (r = 0.43, p < 0.0001). The other studied candidates showed significant correlations in the telomere-mitochondrial interactome but not independent from SIRT1. SIRT1 gene expression was estimated to mediate 40% of the positive association between LTL and leukocyte mtDNA content. The key finding of our study was that SIRT1 expression plays a pivotal role in the telomere-mitochondrial interactome., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Microbubbles have shown potential as intralymphatic ultrasound contrast agents while nanoparticle-loaded microbubbles are increasingly investigated for ultrasound-triggered drug and gene delivery. To explore whether mRNA-nanoparticle loaded microbubbles could serve as theranostics for detection of and mRNA transfer to the lymph nodes, we investigate the behavior of unloaded and mRNA-loaded microbubbles using contrast-enhanced ultrasound imaging after subcutaneous injection in dogs. Our results indicate that both types of microbubbles are equally capable of rapidly entering the lymph vessels and nodes upon injection, and novel, valuable and detailed information on the lymphatic structure in the animals could be obtained. Furthermore, additional observations were made regarding the dynamics of microbubble lymph node uptake. Importantly, neither the microbubble migration distance within the lymphatics, nor the observed contrast signal intensity was influenced by mRNA-loading. Although further optimization of acoustic parameters will be needed, this could represent a first step towards ultrasound-guided, ultrasound-triggered intranodal mRNA delivery using these theranostic microbubbles.

Dendritic cell (DC)-based cancer vaccination has shown great potential in cancer immunotherapy. As a result, novel nanoparticles aiming to load DCs with tumor antigens are being developed and evaluated in vitro. For this, murine bone marrow-derived DCs (BM-DCs) are most commonly used as model DCs. However, many different protocols exist to generate these cells. Therefore, we investigated to what extent different BM-DC culture protocols impact on the immunobiology of the cells, as well as their response to particulate antigens. We evaluated 4 different BM-DC protocols with 2 main variables: bovine serum and cytokine combinations. Our results show distinct differences in yield, phenotypical maturation status and the production of immune stimulatory and immune suppressive cytokines by the different BM-DCs. Importantly, we demonstrate that the antigen-loading of these different BM-DCs via transfection with mRNA lipoplexes results in large differences in transfection efficiency as well as in the capacity of mRNA-transfected BM-DCs to stimulate antigen-specific T cells. Thus, it is clear that the BM-DC model can have significant confounding effects on the evaluation of novel nanoparticulate vaccines. To take this into account when testing novel particulate antigen-delivery systems in BM-DC models, we propose to (1) perform a thorough immunological characterization of the BM-DCs and to (2) not only judge a particle's potential for cancer vaccination based on transfection efficiency, but also to include an evaluation of functional end-points such as T cell activation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Dendritic cell (DC)-based cancer vaccines, where the patient's own immune system is harnessed to target and destroy tumor tissue, have emerged as a potent therapeutic strategy. In the development of such DC vaccines, it is crucial to load the DCs with tumor antigens, and to simultaneously activate them to become more potent antigen-presenting cells. For this, we report on microbubbles, loaded with both antigen mRNA as well as immunomodulating TriMix mRNA, which can be used for the ultrasound-triggered transfection of DCs. In vivo experiments with in vitro sonoporated DCs show the effective induction of antigen-specific T cells, resulting in specific lysis of antigen-expressing cells. Especially in a therapeutic setting, sonoporation with TriMix has an important added value, resulting in a significant reduction of tumor outgrowth and a marked increase in overall survival. What is more, complete tumor regression was observed in 30% of the antigen+TriMix DC vaccinated animals, which also displayed long-term antigen-specific immunological memory. As a result, DC sonoporation using microbubbles loaded with a combination of antigen and TriMix mRNA can elicit powerful immune responses in vivo, and might serve as a potential tool for further in vivo DC vaccination applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Intego is the only operational computerized morbidity registration network in Belgium based on general practice data. Intego collects data from over 90 general practitioners. All the information is routinely collected in the electronic health record during daily practice., Methods: In this article we describe the design and methods used within the Intego network together with some of its basic results. The collected data, the quality control procedures, the ethical-legal aspects and the statistical procedures are discussed., Results: Intego contains longitudinal information on 285 357 different patients, corresponding to over 2.3% of the Flemish population representative in terms of age and sex. More than 3 million diagnoses, 12 million drug prescriptions and 29 million laboratory tests have been recorded., Conclusions: Intego enables us to present and compare data on health parameters, incidence and prevalence rates, laboratory results, and prescribed drugs for all relevant subgroups on a routine basis and is unique in Belgium.

Background: Exhaled breath temperature has been suggested as a new method to detect and monitor pathological processes in the respiratory system. The putative mechanism of this approach is based upon changes in the blood flow. So far potential factors that influence breath temperature have not been studied in the general population., Methods: The exhaled breath temperature was measured in 151 healthy non-smoking elderly (aged: 60-80 years) at room temperature with the X-halo device with an accuracy of 0.03°C. We related exhaled breath temperature by use of regression models with potential predictors including: host factors (sex, age) and environmental factors (BMI, physical activity, and traffic indicators)., Results: Exhaled breath temperature was lower in women than in men and was inversely associated with age, physical activity. BMI and daily average ambient temperature were positively associated with exhaled breath temperature. Independent of the aforementioned covariates, exhaled breath temperature was significantly associated with several traffic indicators. Residential proximity to major road was inversely associated with exhaled breath temperature: doubling the distance to the nearest major intense road was observed a decrease of 0.17°C (95% CI: -0.33 to -0.01; p=0.036)., Conclusions: Exhaled breath temperature has been suggested as a noninvasive method for the evaluation of airway inflammation. We provide evidence that several factors known to be involved in proinflammatory conditions including BMI, physical activity and residential proximity to traffic affect exhaled breath temperature. In addition, we identified potential confounders that should be taken into account in clinical and epidemiological studies on exhaled breath temperature including sex, age, and ambient temperature.

Perfluorocarbon (PFC) particles are currently on the rise as cell labeling agents for ¹⁹F-MRI tracking of dendritic cell (DC)-based vaccines. In this work, we design theranostic PFC particles for single-step loading of DCs with both antigenic protein and with a liquid PFC for ¹⁹F-MRI detection of the antigen-loaded cells. Upon addition to DCs in vitro, the antigen-loaded PFC particles are efficiently internalized, resulting in intracellular presence of up to 40 pmol ¹⁹F atoms per cell. At the same time, the DCs become loaded with antigenic proteins, that can be efficiently processed, without important effects on cell viability or altering the DC's phenotype and the cell's capacity to respond to danger signals. In addition, antigen-loaded PFC particle containing DCs are capable of inducing extensive proliferation of antigen-specific CD8⁺ T cells in vitro. Importantly, the antigen-coated PFC particles allow in vitro ¹⁹F-MRI-based detection of the antigen-containing DCs with detection limits as low as 10³ cells μl⁻¹. The dual-modality characteristics of the designed particles could assure that only those DCs that have taken up the antigen, and hence are responsible for an immune response, are traceable via ¹⁹F-MRI. Taken together, these novel dual-modality particles represent an interesting strategy in the development of a traceable DC vaccine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Time and space controlled drug delivery still remains a huge challenge in medicine. A novel approach that could offer a solution is ultrasound guided drug delivery. “Ultrasonic drug delivery” is often based on the use of small gas bubbles (so-called microbubbles) that oscillate and cavitate upon exposure to ultrasound waves. Some microbubbles are FDA approved contrast agents for ultrasound imaging and are nowadays widely investigated as promising drug carriers. Indeed, it has been observed that upon exposure to ultrasound waves, microbubbles may (a) release the encapsulated drugs and (b) simultaneously change the structure of the cell membranes in contact with the microbubbles which may facilitate drug entrance into cells. This review aims to highlight (a) major factors known so far which affect ultrasonic drug delivery (like the structure of the microbubbles, acoustic settings, etc.) and (b) summarizes the recent preclinical progress in this field together with a number of promising new concepts and applications.

In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patient's body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The European Union claims that the defense of its welfare state is one of today's most important challenges. This article analyzes whether the European governments and the European Union really pursue a policy that strengthens their health and social security systems, or one that is in itself a threat to health and social security. After a summary of the origin and evolution of the European health systems, the authors pinpoint underlying reasons for reform and demonstrate how, since the 1990s, the European Union has built a strict financial and political straitjacket, forcing these systems to carry out privatization and cutbacks. Reform measures can be divided into three interdependent categories: (1) the increasing influence of governments on health care organization, to enable restructuring; (2) measures aimed at reducing public expenses, including higher financial contributions by patients and restrictions on the range of services provided; and (3) measures that establish competition and hidden or open privatization of services and insurance systems. Through these mechanisms public expenses are reduced while private health care expenses (and private profits) rise freely. Ongoing European health care reforms thus struggle with the contradictions between responding to growing collective needs and securing or increasing private profits.