Dmd gene resulting in the loss of the protein dystrophin. Patients do not only experience skeletal muscle degeneration, but also develop severe cardiomyopathy by their second decade, one of the main causes of death. The absence of dystrophin in the heart renders cardiomyocytes more sensitive to stretch-induced damage. Moreover, it pathologically alters intracellular calcium (Ca2+) concentration, neuronal nitric oxide synthase (nNOS) localization and mitochondrial function and leads to inflammation and necrosis, all contributing to the development of cardiomyopathy. Current therapies only treat symptoms and therefore the need for targeting the genetic defect is immense. Several preclinical therapies are undergoing development, including utrophin up-regulation, stop codon read-through therapy, viral gene therapy, cell-based therapy and exon skipping. Some of these therapies are undergoing clinical trials, but these have predominantly focused on skeletal muscle correction. However, improving skeletal muscle function without addressing cardiac aspects of the disease may aggravate cardiomyopathy and therefore it is essential that preclinical and clinical focus include improving heart function. This review consolidates what is known regarding molecular pathology of the DMD heart, specifically focusing on intracellular Ca2+, nNOS and mitochondrial dysregulation. It briefly discusses the current treatment options and then elaborates on the preclinical therapeutic approaches currently under development to restore dystrophin thereby improving pathology, with a focus on the heart.Duchenne muscular dystrophy (DMD) is a genetic muscle disorder caused by mutations in the  DMD gene i) (see As of yet it is unclear how the increase in Ca+]i) see .2+ influx and improved left ventricular end diastolic volume in mdx mice i in ventricular cardiomyocytes and restored protein kinase C (PKC) and phospholipase C (PLC) signaling [+ channels [2+]i could be mediated by both direct entry of Ca2+ ions through SACs, and indirect entry through the Na+/Ca2+-exchanger (NCX) or voltage-gated Ca2+ channels (VGCC) that open due to Na+-induced membrane depolarization , though other reports have shown down-regulation i in the heart and loss of these molecules results in worsening of heart function, though further research is needed to elucidate its exact function.The function of nNOS in the heart is currently still being investigated. Several knock-out studies have implicated a role in LV systolic function and shortening of myocytes, but this remains elusive due to opposing results ,71,72. O2+ ,41,72,73fraction . Togethe2+ store, supplying and taking up Ca2+ to and from the cell i is thought to induce a sudden increase in mitochondrial membrane permeability through the mitochondrial permeability transition pore (PTP). The PTP is a voltage-sensitive channel and an increase in the mitochondrial Ca2+ concentration induces the opening of this channel, leading to swelling. Short-term opening of these channels is thought to be beneficial as the mitochondria are able to regulate intracellular Ca2+ and ROS balance [In addition to the perturbed energy metabolism exhibited by mitochondria in dystrophin-deficient cells, the increased intracellular Ca balance ,81. Howe balance . The rup balance .There is currently no cure for DMD, and therefore therapy is limited to the management of symptoms. The three predominant treatment approaches include corticosteroids in order to ameliorate skeletal muscle phenotype, mechanical respiratory support and cardiac assessment and treatment. Corticosteroids have been shown to markedly improve muscle strength and function . More reDeterioration in respiratory muscles results in respiratory insufficiency and pulmonary hypertension. An increase in pulmonary hypertension compounds cardiac function leading to DCM . Thus mep < 0.0001) and delayed the progression of cardiomyopathy [Lastly, the approaches designed to specifically target cardiac involvement include angiotensin-converting enzyme (ACE) inhibitors, beta-blockers (β-blockers) and diuretics . ACE inhmyopathy . Beta-Blockers prevent the binding of catecholamines by interfering with β-receptor binding, thus reducing sympathetic nervous system activity. They are therefore prescribed for arrhythmic patients, and those with symptomatic but stable systolic dysfunction. Clinicians often prescribe a cocktail of ACE inhibitors, β-blockers and diuretics which significantly improves cardiac function and survival in DMD patients ,92. Howei.e., patients with low LV EF, accompanied by ventricular dyssynchrony (QRS > 120 ms) [In DMD patients whom are unresponsive to pharmacological treatment, cardiac resynchronization therapy may be considered,  120 ms) . These p 120 ms) . In bothmdx mice and administration of sildenafil improved contractile performance, cardiac metabolism and sarcolemmal integrity [mdx hearts [mdx mice [Several drugs that are already commercially available may benefit DMD patients and have therefore been investigated in animal models of DMD to explore their therapeutic potential. The synthetic copolymer poloxamer P188, which stabilizes the sarcolemma, has been shown to improve the cardiac phenotype and even prevent isoproterenol/dobutamine-induced cardiomyopathy/death ,96. The ntegrity . Furtherx hearts , and impx hearts . Unfortux hearts ,101. It x hearts ,103. Phax hearts . In addimdx mice . A recenmdx mice . This prThese drugs have demonstrated encouraging results in animal models of DMD and it is hoped that they will infer some clinical benefit in patients. Furthermore, these drugs may present viable combinatory therapies which may be administered in conjunction with treatments specifically aimed at correcting/compensating for the dystrophin mutation.Given that current treatment strategies are limited to reducing the symptoms of the disease, it is vital that new therapies are developed that directly target the underlying mutation. Several very promising strategies currently under investigation for treating DMD also have potential for treating the heart. This review will discuss some of these most promising therapies, namely; utrophin up-regulation, ribosomal read-through therapy, stem cell therapy, viral gene therapy delivering mini/micro-dystrophin and exon skipping antisense oligonucleotides. Not all experiments regarding the different approaches are discussed in this review, but a summary of selected references are cited in Utrophin up-regulation was one of the first therapeutic strategies developed to compensate for the lack of dystrophin in the cell. In the adult cell, utrophin is mainly found in regenerating cells and at the neuromuscular and myotendinous junctions . It is tmdx/utrn-/−) mouse model. These animals exhibit worsened skeletal muscle and cardiac pathology and have a life span of only 2–3 months [mdx skeletal muscle, though it should be noted that the heart is not corrected [www.trivorsan.com).Consequently, up-regulation of utrophin to ameliorate DMD pathology and improve the functional deficit is being investigated. Evidence supporting this concept comes from the dystrophin/utrophin double knockout  thereby disrupting the open reading frame ,182. Antpatients .in vitro and in vivo studies [Subsequently, a high-throughput screen has identified PTC124, a small-molecule able to induce read-through of premature stop codons, which has shown significant promise in both  studies . A major studies , though  studies . Nonethe studies . Further studies ,122  than gentamicin, PTC124 and RTC14. In addition, muscle function was improved and serum CK levels were decreased in mdx mice treated with RTC13 [Other studies have aimed at identifying more effective drugs to induce read-through. A sensitive small-molecule screen was performed, which identified compounds RTC13 and RTC14. These compounds were subsequently administered intramuscularly and intravenously in th RTC13 . RTC13 pAnother therapeutic strategy undergoing development is the use of viral vectors to deliver a dystrophin transgene to the cell. Given dystrophin’s enormous size, multiple studies have endeavored to distinguish the integral components of the dystrophin gene to develop a shortened truncated version that may be inserted into the viral capsid (mini- or microdystrophin) . Severalmdx heart when using mini- or micro-dystrophin. rAAV6 microdystrophin was successful at transfection and induction of dystrophin in TA, diaphragm and heart in young mdx mice. Interestingly, however, heart function did not recover even though dystrophin was restored in the heart and heart mass was normal [mdx mice also produced positive results [mdx muscles had been significantly damaged by the disease, treatment was still capable of improving some muscle pathology [In order to safely apply the AAV virus, it has been modified to exclude all viral genes, but leave essential repeats for replication  . The AAVs normal . Injecti results , suggestathology .mdx mice also induced dystrophin expression and improved cardiac function [mdx mice. Lower titer levels of the virus resulted in almost 100% transfection, effective dystrophin expression in both skeletal muscle and heart and improved muscle function. Moreover, no immunological response was observed [rAAV9 has proven to be particularly efficient in transfecting the heart . Mini-dyfunction ,133. Desfunction ,186. Morobserved .Overall, viral vectors are promising candidates for the delivery of the dystrophin gene. However, special care should be taken regarding immunogenicity as the human population carries antibodies towards many prevalent viruses, such as AAV ,187, thoin vitro [With cell-based therapy, healthy dystrophin-expressing cells are transplanted into DMD patient tissue which not only integrate into healthy adult muscle cells, but ideally also co-populate the satellite cells. These cells can be derived either from healthy precursor cells or from patient cells that are genetically modified in vitro . Severalin vitro ,190,191.mdx mouse models [A more recent shift to the use of induced pluripotent stem (iPS) cells has been observed. Indeed, human iPS cells have been differentiated into muscle precursor cells and injected into TA muscles of immunocompromised e models . Followie models ,144. Thein vitro with the use of nucleases [in vitro to induce exon skipping, shift the reading frame or insert the missing exon with successful results. Subsequently, these iPS cells were differentiated into skeletal muscle cells and dystrophin expression was observed. The knock-in of the missing exon was most effective [Several groups have aimed at utilizing genetically modified patient precursor cells as treatment. This would circumvent immunological suppression needed when introducing foreign material into the body. The genome of the cells are modified ucleases ,194,195,ffective . Given tOverall, utilizing genetically modified cells from patients would be a more valuable treatment when considering immunological responses. However, the ability to induce and sustain adequate dystrophin levels remains difficult. Currently, delivery of progenitor cells is most successful if done intramuscularly , yet thiAntisense oligonucleotides (AONs) are another viable approach for treating DMD patients, and are specifically utilized in the skipping of exons with mutations or deletions in the dystrophin gene in order to correct the aberrant reading frame . AONs arThe predominant AON chemistries which are currently undergoing clinical evaluation are phosphorodiamidate morpholino oligomers (PMOs)  and 2′O In an effort to improve the delivery capacity of AONs and reduce the dose required, short peptide sequences, ‘cell penetrating peptides’ (CPPs), have been chemically conjugated to neutral AONs such as PMO to facilitate their delivery across the plasma membrane. A multitude of studies have investigated peptide conjugated AONs in the context of DMD and can broadly be classified into 3 distinct groups namely arginine rich, Pip , and phage and chimeric peptides.Most importantly the arginine rich and Pip peptides have demonstrated cardiac dystrophin expression. Arginine rich CPPS conjugated to PMO were the first to show activity in the heart  and indeAnother method to improve delivery of charged AONs is to encapsulate them in liposomes  or nanopTo summarize, with the exception of tricyclo-DNA, naked AONs have demonstrated limited systemic delivery and an inability to restore dystrophin in the heart, whereas conjugation to CPPs or the use of encapsulation technologies have significantly improved upon this. From a CPP perspective, both poly-arginine and Pip CPPs have displayed widespread dystrophin restoration in multiple skeletal muscle tissues and the heart. Nanoparticles are another promising delivery strategy, particularly for charged AONs such as 2′OMePS. This field is rapidly evolving and it is hoped that one or more of these technologies is advanced to clinical trial in the near future.Whilst these experimental therapies have inspired new hope for patients and families affected by DMD, the feasibility of clinically translating some of these therapies has been brought into question. Not only should species differences be considered in terms of cellular processes and pathways, but also concerning immunological responses. Mouse models of DMD are somewhat resilient, and studies in dog models have highlighted inevitable obstacles particularly due to our complex immune system. This is impossible to avoid given the requirement of a systemic approach to treat a disease which is devastating to the entire body. Not only is preventing an immunological response particularly relevant for cell-based therapies and viral vectors, but also for peptide-conjugated AONs which may cause complications. Certainly, long term administration of ‘naked’ PMO did not elicit an immunological response. Other therapies such as aminoglycosides may induce off-target or other toxicological affects. Ultimately, in order to substantially ameliorate or abrogate this disease, targeting the underlying defect, specifically the lack of dystrophin protein, is required. Current pharmacological interventions can only delay or partially ameliorate the symptoms of this disease. Therefore it is imperative that one or more, or indeed a new therapy altogether, is able to overcome these obstacles and by-pass our innate immune system.2+, NO, mitochondria and other second messengers and organelles in the cell. However, given the gaps that still remain in our knowledge, it seems likely that other unknown proteins or pathways also affect disease pathology. Furthermore, as dystrophin is expressed body-wide, it is important that individual systems such as the heart are considered, as specific mechanistic findings from skeletal muscle pathology may not necessarily be applied to the heart. Moreover, the comparison of molecular aspects of dystrophin function between different cell types could provide valuable information.Our current understanding of Duchenne muscular dystrophy has increased significantly over the last few decades. The absence of the dystrophin protein gives rise to a complex pathology with the involvement of many different processes in the cell all playing an intricate part in further disease progression. Much research has already been performed which indicate the importance of CaThe knowledge gained from improved understanding of molecular pathological pathways provides more ways of developing and assessing the effectiveness of treatment. With the availability of steroids and other drugs, improvements have been made to ameliorate the skeletal muscle phenotype. Whilst pharmacological treatments to stabilize cardiac function, such as ACE inhibitors and beta-blockers, are in use, mortality from cardiorespiratory complications in DMD patients remains high. Moreover, whilst some experimental therapies are under development, these may not benefit cardiac aspects of the disease and most clinical trials to date have focused exclusively on the beneficial effects of drug treatment on skeletal muscle function, without considering the impact on heart function. Therefore, an increased focus on the heart is essential, both in terms of existing treatments and the effects on heart function, but also more critically on novel treatments that could be developed to specifically restore dystrophin or upregulate utrophin in cardiac muscle, resulting in clinical benefit for patients.


Genetic and phenotypic tumour heterogeneity is an important cause of therapy resistance. Moreover, non-uniform spatial drug distribution in cancer treatment may cause pseudo-resistance, meaning that a treatment is ineffective because the drug does not reach its target at sufficient concentrations. Together with tumour heterogeneity, non-uniform drug distribution causes “therapy heterogeneity”: a spatially heterogeneous treatment effect. Spatial heterogeneity in drug distribution occurs on all scales ranging from interpatient differences to intratumour differences on tissue or cellular scale. Nanomedicine aims to improve the balance between efficacy and safety of drugs by targeting drug-loaded nanoparticles specifically to tumours. Spatial heterogeneity in nanoparticle and payload distribution could be an important factor that limits their efficacy in patients. Therefore, imaging spatial nanoparticle distribution and imaging the tumour environment giving rise to this distribution could help understand (lack of) clinical success of nanomedicine. Imaging the nanoparticle, drug and tumour environment can lead to improvements of new nanotherapies, increase understanding of underlying mechanisms of heterogeneous distribution, facilitate patient selection for nanotherapies and help assess the effect of treatments that aim to reduce heterogeneity in nanoparticle distribution.In this review, we discuss three groups of imaging modalities applied in nanomedicine research: non-invasive clinical imaging methods , optical imaging and mass spectrometry imaging. Because each imaging modality provides information at a different scale and has its own strengths and weaknesses, choosing wisely and combining modalities will lead to a wealth of information that will help bring nanomedicine forward. In 2015, 17.5 million people were diagnosed with cancer globally and its incidence is increasing. Although the prognosis of most cancer types has improved, still 8.7 million people died of cancer in that year Therapy heterogeneity, a spatially heterogeneous treatment effect, is another important source of variability between patients and between tumours within an individual. Spatial heterogeneity in drug distribution contributes to therapy heterogeneity and can lead to pseudoresistance: the treatment does not have the desired effect, not because of cellular or genetic mechanisms of resistance, but because the drug simply does not reach all tumour cells at a high enough concentration Heterogeneous drug distribution occurs for drugs of all sizes. In this review we will focus on its impact in the field of nanomedicine. Nanomedicines are sub-micron size drug delivery systems, which are designed to improve the drug delivery to tumours while reducing systemic side effects As reviewed by Garattini et al., heterogeneous drug distribution leading to therapy heterogeneity can occur on many scales On patient scale, the inter-patient variability of nanomedicine pharmacokinetics (PK) is influenced by many factors such as age, gender, body composition, prior treatments, and drug-drug interactions Also on the organ and tumour scale, many factors can contribute to heterogeneity in nanoparticle and drug distribution between different tumours in an individual patient. Accumulation of nanoparticles is tumour type and organ dependent Finally, on tissue and cellular scale, there are numerous causes for heterogeneity in drug and nanoparticle distribution within a single tumour. These intratumour differences relate among other things to endothelial cell gaps across the vessel wall, perfusion, extracellular matrix composition and immune cell presence  Some nanomedicine formulations are currently used in the clinic Many imaging methods are available to visualize at a variety of scales the three main factors  that influence spatial therapy heterogeneity and thus efficacy. These methods can be used to:1. Evaluate the effect of the therapy in a preclinical setting, to facilitate the development of new treatments.2. Improve understanding of the underlying mechanisms that lead to heterogeneous distribution of nanoparticles and drugs.3. Select patients and predict their treatment response for a personalized treatment plan: which patient likely benefits from a certain therapy and in which patient is adaptation of therapy necessary?4. Evaluate the effect of methods that aim to address therapy heterogeneity, such as modulating the tumour microenvironment, hyperthermia or sonopermeation Over the last years, several excellent review papers on imaging in nanomedicine were published focusing on imaging the biodistribution of nanoparticles Nuclear imaging techniques, namely scintigraphy, Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) provide highly sensitive quantitative information about the distribution of an administered radiopharmaceutical. They are often combined with CT (SPECT/CT or PET/CT) to add anatomical information and perform attenuation correction. Since the spatial resolution is lower than that of MRI, CT and US imaging, clinical PET and SPECT are mainly informative on patient and organ scale. In the preclinical setting, high-resolution PET and SPECT techniques demonstrate expansion to the tissue scale 18F-fluorodeoxyglucose (FDG-) PET/CT is widely used in the clinic for diagnosis and monitoring therapy response. Traditionally several parameters are analysed for diagnosis and prognosis, including maximum standardized uptake values (SUVmax), peak standardized uptake values (SUVpeak), metabolic tumour volume (MTV), and total lesion glycolysis (TLG). Recently, intratumoural heterogeneity of baseline 18F-FDG uptake measured by PET texture analysis has been introduced as new predictive and prognostic factor for neoadjuvant chemotherapy Currently, the metabolic activity measured with 64Cu-labelled tracer dose of the same liposomes and their distribution was imaged by PET/CT. This study was able to demonstrate and quantify the EPR effect in patients and found that tumour liposome concentrations were similar to those found preclinically. Heterogeneity in nanoparticle distribution on the patient and tumour scale was observed and an association was found between the amount of 64Cu-labelled liposome uptake in the tumour and the overall tumour response and progression free survival 89Zr-labelled docetaxel nanoparticles in five patients with various tumour types and observed uptake of the nanoparticles in 35% of the tumours, which they attributed to the EPR effect. Heterogeneous patterns of accumulation were seen on patient and organ scale Nuclear imaging has already gained widespread acceptance in the management of cancer using standard chemotherapeutic agents, but may also play an important role in the advancement of nanomedicine towards clinical practice. Historically, nuclear imaging is used to image the distribution of radiolabelled nanoparticles on the patient and organ/tumour scale 18F-fluorothymidine (FLT-PET) 18F-fluoromisonidazole (F-MISO)-PET Furthermore, clinical studies with different tracers are being conducted to extend the use of nuclear imaging to gaining insight into the different aspects of tumour physiology, such as proliferation (e.g. 18F-fluoroestradiol (FES-) PET Other tracers visualize expression of receptors that can be targeted by drugs (e.g. 89Zr-trastuzumab and FDG-PET/CT after just one treatment cycle. These two types of PET imaging where used to characterize distribution of the nanoparticle (trastuzumab in T-DM1) and the tumour environment (FDG uptake). In metastatic breast cancer patients, who all had HER2 positive disease on biopsy, the combination of early metabolic response on FDG-PET (significant reduction in FDG uptake in >50% of the tumour load) and positive 89Zr-trastuzumab uptake (>50% of the tumour load) on the HER2-PET could predict treatment response to the antibody-drug conjugate (ADC) trastuzumab-emtansine (T-DM1)  is widely used in daily clinical practice for tumour diagnosis, characterisation and response evaluation. MRI is increasingly being investigated in image-guided therapy such as MR-guided radiotherapy (MR-LINAC) Because of the lack of radiation, its high spatial resolution and excellent soft tissue contrastTumour vascular development and density, as well as perfusion and hypoxia, are key regulators of nanoparticle distribution and nanotherapy effect. Dynamic Contrast Enhanced (DCE-) MRI has been used in clinical trials to evaluate the effect of antivascular treatment on perfusion and vascular permeability Iron-oxide nanoparticles have been approved as MRI contrast agent for clinical use. However, they can also function as companion diagnostic or as (imageable) drug delivery systems co-glycolide) nano/microspheres, encapsulated with gadolinium-DTPA, SPIONs and the chemotherapeutic drugs 5-fluorouracil. In vivo, release of gadolinium-DTPA was seen from 30 minutes after intravenous injection, in the same tumour regions where most of the nanospheres had accumulated in vivo drug release and spatial distribution at tumour and tissue scale. However, these MR contrast agents may influence the stability of nanoparticle Moreover, MRI contrast agents have been encapsulated in nanoparticles as model drugs, to visualize and quantify drug release triggered by temperature, pH or ultrasound sensitive nanoparticles using MRI Computed tomography (CT) imaging is very commonly used in the clinic for diagnostic purposes and response evaluation after treatment. More recently, it was shown that CT could derive tumour transport properties in patients with pancreatic cancer that correlated with gemcitabine incorporation, pathological response, and oncologic outcome The added value of CT imaging for nanomedicine through identifying tumour transport properties was already shown in the preclinical setting. Dynamic Contrast Enhanced (DCE) CT has been used in several studies to measure the intratumoural perfusion, permeability and the accumulation of CT contrast agent-containing nanoparticles in mice In addition to imaging a contrast agent encapsulated in a nanoparticle, CT can also be utilized to image metallic nanoparticles with a high attenuation of x-rays Ultrasound (US) is a low-cost, radiation-free and patient-friendly clinical imaging method that is mostly used for tumour diagnosis and image-guided biopsies. The introduction of contrast-enhanced ultrasound (CEUS), i.e. using microbubbles as ultrasonographic contrast agents, has extended the application of ultrasound in many fields due to improved image quality and new information that cannot be obtained with standard US CEUS can provide anatomical as well as functional information about the vasculature of the tumour (micro-) environment Tumour environment modelling treatments to improve nanoparticle delivery have been investigated using CEUS. Changes in tumour physiology (i.e. vessel fraction and blood flow) measured by US imaging after collagenase treatment corresponded with changes in IFP, therefore US imaging can be used as an earlier marker of tumour response Recent advances in US imaging such as ultrafast ultrasound and super-resolution techniques provide also information on microvascular properties In addition to imaging the tumour environment, the distribution of nanoparticles can be imaged by US, using echogenic nanoparticles Although drugs cannot be imaged directly with ultrasound, drug distribution can be visualized. To achieve this, Ektate et al. developed low-temperature sensitive echogenic liposomes, loaded with doxorubicin and perfluoropentane. Tumour hyperthermia led to increased US contrast in mice, which was correlated with increased doxorubicin delivery in vivo optical imaging modalities are steadily substituting “old-school” ex vivo methodology - “dead mice tell too few tales” ex vivo / in vitro optical imaging techniques can provide supportive structural and functional information (immunohistochemistry (IHC) on tissue slices, electron microscopy (EM), flow cytometry imaging). Optical imaging is widely used in the development and evaluation of nanotherapies: molecular imaging helps unravel nanoparticles' complex in vivo fate Optical imaging modalities are used to collect a variety of information on various spatial and temporal scales in vivo release profile Traditionally, macroscopic optical imaging modalities are used in preclinical small animal experimental procedures as alternatives to conventional non-terminal / non-invasive whole-body imaging modalities . BLI, one of the most commonly used optical imaging techniques, allows real-time detection of protein-derived native light emission Fig. A. Even tin vivo is highly complex. Therefore, a deeper understanding of in vivo behaviour, targeting mechanisms and nanoparticles' specific engagement with cell populations  is essential. Extensive use of real-time imaging techniques like IVM, could potentiate our efforts to characterize the tumour microenvironment on tissue and cellular scale  PEG surface density proved to contribute to a higher targeting specificity of arginylglycylaspartic acid (RGD) nanoparticles, than high (50 mol %) surface PEG density Interestingly, IVM has been used to identify differences in nanoparticle diffusion to tumour sites or nanoparticle tumour targeting, and to correlate them to different physicochemical properties, providing an excellent tool for head-to-head nanoparticle comparisons. The size-dependent diffusion of nanoparticles was studied after administration of a library of small fluorescent quantum dot nanoparticles with diameters of 12, 60 and 125 nm, revealing that 12 nm nanoparticles diffused twice as far in comparison to the largest size particles In addition to addressing nanoparticle tumour targeting and accumulation profiles, IVM has been used to unravel more intricate interactions between nanoparticles and immune cells; an interesting option given the increasing appeal of cancer immunotherapy. One of the first studies providing real-time insight in the behaviour of TAM, developed magneto-fluorescent nanoparticles enabling the visualization of nanoparticle-labelled TAM on tumour/organ scale  and on tissue and cellular scale (IVM). Among other findings, TAM phagocytosed more nanoparticles than other myeloid cells, they were situated in close proximity to tumour cells and displayed low motility in vivo behaviour in a similar manner regardless of the selected nanomaterial, i.e., nanoemulsions versus quantum dots. The multimodal nature of nanoparticles allows for visualization of angiogenic endothelium and neovasculature on tumour/organ scale , tissue scale (IHC and IVM), and cellular scale (IVM) Given that angiogenesis is a hallmark of cancer, the attention of intravital microscopy users has been directed particularly to nanoparticle-mediated vessel wall visualization and targeting. Already in the mid '00s, a successful attempt using vascular cell adhesion molecule 1 (VCAM-1) decorated nanoparticles paved the way for future success The new mechanistic and molecular insights that were obtained through IVM procedures inspired researchers to develop IVM-specialized imaging agents. Biocompatible organic dots for MPM in vivo imaging of inherently fluorescent drugs is hampered by their relatively low fluorescence quantum yield, which limits their detectability at therapeutic concentrations. Another approach could be the conjugation of fluorescent molecules to the drugs that are carried by nanoparticles, despite the fact that this could result into alternation of their properties. The conjugation of fluorescent dyes to macromolecular drugs has been successfully applied before Visualization of fluorescent-labelled nanoparticles and related aspects of the tumour microenvironment provide valuable information on therapy heterogeneity in nanomedicine. Imaging the administered drug itself would complete the picture. However, direct imaging of drugs remains elusive. In this respect, inherently fluorescent drugs are convenient ex vivo techniques like the tissue optical clearing strategies Utilization of fluorescent molecules is often restricted by factors such limited imaging depth. Therefore, more sophisticated in vivo optical imaging is mainly constricted to preclinical applications. Clinically, optical imaging is of course widely used on ex vivo biopsy or surgical samples. Although this consists mostly of immune histochemistry, some work has been done to complement this with fluorescent imaging Due to the limitations in tissue penetration and size of the imageable subject, Optoacoustic (photoacoustic) imaging is an emerging hybrid technique that combines the benefits of US and optical techniques, i.e. deep imaging depth, high spatial resolution and high contrast in vivo spatiotemporal changes of macrophage activity In the oncology domain the endogenous contrast is typically used to study tumour vasculature in vivoNanocarriers not only serve as optoacoustic contrast agent, but can also act as vehicles for drugs. Several nanoparticles have been loaded with drugs and combined with optoacoustic imaging for non-invasive and real-time monitoring of biodistribution and pharmacokinetics In order to monitor the drugs themselves using optoacoustic imaging they should exhibit NIR-absorbing properties. Unfortunately, few if any clinically prescribed drugs have strong intrinsic absorption in the NIR. As an alternative, small molecule NIR dyes are co-loaded with drugs of interest to monitor drug release and distribution 284Optical and optoacoustic imaging have frequently been combined with photothermal therapy PTT , 285 andMass Spectrometry Imaging (MSI) is a label free, multiplex technique that is used to visualize the molecular distribution of endogenous compounds such as metabolitesIn MSI spatially defined desorption/ionization methods are used to collect sequentially mass spectra from a small region (pixel) of a tissue sample. Among the multitude of surface sampling techniques, matrix-assisted laser desorption/ionisation (MALDI) uses a laser beam for desorption/ionization of tissue-representative molecules co-crystalized in a solidified matrix; while desorption electrospray ionization (DESI) makes use of an electrically charged solvent spray and in secondary ion mass spectrometry (SIMS), a beam of high energy primary ions  is used to release secondary ions from the sample surface. In contrast to MALDI and SIMS where the sample is analysed under vacuum, DESI is non-destructive and performed at atmospheric pressure, which renders the technique more user friendly and through appropriate solvent selection, more tuneable to increase selectivity and/or sensitivity. Depending on the ionization method used spatial resolution, sensitivity, and the molecules that can be analysed change. Mostly, MALDI is used at 10 to 20 µm spatial resolution, whilst DESI resolution spans 50 µm till 200 µm and SIMS allows for sub-µm resolution. Sensitivity wise DESI outperforms MALDI and SIMS, partly due to increased pixel size  and partly due to improved ionization efficiency. Although sensitivity is determined by e.g. physiochemical properties of the compound, typically one requires low µg/g concentrations in the case of DESI and approx. 20 µg/g tissue for detection by MALDI and SIMS.The number of drugs that have been detected using MSI is extensive, ranging from anti-cancer drugs , was introduced in the life sciences by Giessen et al. in 2014 in vivo fate after administration. However, this requires additional chemical development and the introduction of the probe may influence the biodistribution of the nanoparticle. Recently, Zandanel et al. showed that MALDI-MSI allows for the simultaneous visualization of the polymeric nanoparticle, the encapsulated drug (doxorubicin) and its metabolite (doxorubicinol) in treated mouse liver MSI also provides the opportunity to image nanocarriers, such as lipid- and metal-based nanoparticles. Typically nanoparticles are labelled or loaded with a radioactive or fluorescent probe in order to follow the in situ drug (doxorubicin) release from molybdenum disulphide (MoS2) nanosheets Xue et al. developed an MSI method that enabled not only the visualization, but also the quantification of the As indicated earlier, mass spectrometry offers unique capabilities for untargeted exploration of biological samples and provides simultaneous information of the distribution of the drug, the nanoparticle and endogenous compounds such as metabolites, lipids and proteins. Therefore, MSI can be used to detect biomarkers associated with disease, molecular changes due to drug treatment and tumour components limiting nanomedicine distribution and effect or augmenting off-target effects.Several studies report that the heterogeneous distribution of lipids and proteins could reflect the effect of therapy and/or could be used as prognostic/predictive marker for outcome. A very nice example of identifying and using endogenous proteomic profiles for distinguishing between responders and non-responders to chemotherapy for oesophageal adenocarcinoma is given by Aichler et al. 18F-fluoromisonidazole (FMISO), a widely used PET hypoxia imaging probe Also tumour hypoxia is investigated by many groups since it is associated with tumour aggressiveness and resistance to cancer treatment. Manscini et al. used MALDI-MSI to simultaneously detect pimonidazole, a clinically used hypoxia marker, its metabolites and associated biomolecules in a single experiment Interestingly, most MSI studies that investigate drug distributions do not yet exploit the multiplex capabilities of MSI. Instead of using the wealth of information on endogenous molecule distributions  already available in the acquired MSI image they superimpose the drug MSI images with standard H&E stained tissue images and/or various immunohistochemistry (IHC) images. As most of these IHC images are specific to a certain protein, this can be a laborious and time consuming effort. Moreover, correlating drug distribution with the distribution of these endogenous molecules would allow for non-supervised investigations to discover new factors that impair drug transport in tumour tissue and could be used as biomarker for prognosis and therapy response prediction.Spatial heterogeneity in nanoparticle distribution occurs at all scales and can reduce nanotherapy efficacy. A wide range of imaging modalities help visualize nanoparticle distribution or factors contributing to heterogeneous distribution, by imaging the drug, the nanoparticle and the tumour environment.in vivo applications and intravital techniques offer the possibility to visualize dynamic processes. Clinical use of optical imaging is still hampered by the limited penetration depth and therefore requires tissue sampling or intraoperative use. MSI provides information on tissue and even cellular scale. As it requires a tissue specimen it is inherently an invasive method, regardless of preclinical or clinical use. Due to this limitation, the technique is most suitable and currently most used in the preclinical setting, where an entire tumour or even the whole animal can be analysed at once. In contrast, in the clinic MSI approaches will most likely follow a workflow similar to standard histopathology. Emerging technologies allow for more accurate tissue sampling using intra-procedural multimodality imaging during biopsies When selecting imaging modalities, researchers need to take into account the desired imaging scale. Non-invasive clinical imaging methods provide three-dimensional information of the intact body on patient, organ and tumour scale, and recent developments, such as super-resolution ultrasound, have broadened their application to provide even tissue scale information. Optical imaging modalities have a superior spatial resolution, procuring images on the tissue and cellular scale, and in the preclinical setting they can provide (sometimes non-invasive) organism and organ scale information as well. Besides that, Nuclear imaging methods and most optical imaging methods require labelling of nanoparticles, drugs or aspects of the tumour environment, which has some disadvantages. First, the stability of the link between a label and a nanoparticle determines its usefulness in tracking the nanoparticle, for one could be imaging the label on itself after disconnection from the nanoparticle. Second, attaching a label could change the pharmacokinetic and drug release properties of the nanoparticle leading to erroneous prediction of the distribution of an unlabelled equivalent. Third, administering a labelled version of a previously approved therapeutic nanoparticle or drug could cause additional toxicity and obtaining clinical approval is cumbersome and costly. Fourth, administration of radiolabelled theranostic nanoparticles may decrease the effect of subsequent therapeutic administration through the ABC phenomenon The integration of imaging data collected from multimodality techniques offers a unique opportunity to combine information related to drug and nanoparticle distribution and tumour environment on multiple scales and provide synergistic advantages over the use of a single modality.in situ hybridization (FISH), creates a new dimension to molecular pathology. Now, drug levels within tumour regions can be correlated to different degrees of vascularization of the tumour - highlighted with specific vasculature staining Clinical imaging modalities are generally combined to merge functional  with anatomical information  collected on the same scale. A range of nanoparticles for multimodality imaging have been developed To achieve multiscale information, optical imaging and MSI have been paired with clinical imaging ex vivo MSI or optical imaging with 3D / in vivo images, such as those generated with SPECT, PET, CT or MRI. This challenge is inherent to the fact that the images are acquired at different scales, can be subjected to different deformations, and lack common fiducial markers. A first step towards combining two-dimensional MSI with three-dimensional MRI is the ability to monitor MRI contrast agents with MSI, which was demonstrated by Tata et al. A challenge remains the integration of molecular information provided by 2D / in vivo behaviour of nanoparticles. Additionally, it has led to valuable information on how specific characteristics within the tumour environment can affect nanotherapy outcomes. Continued application of these imaging approaches, and especially the combination of different techniques, will further strengthen our understanding of therapy heterogeneity on organism, tissue, cellular and even subcellular scale. Meanwhile the role of imaging for monitoring PK and biodistribution is well established in research and development stage; however in the clinical setting it is not yet adopted. Clinically, spatial heterogeneity distribution of therapeutic nanoparticles, poses the threats of under- and overtreatment. Despite the fact that many studies have shown that drug concentration does not correspond with tissue drug levels, phase I and II clinical trials mainly rely on blood samples and spatially sparse biopsies to measure PK on patient scale and biodistribution on organ scale. Imaging drugs or nanoparticles in clinical studies is still rare and often no information is acquired about spatial heterogeneity of nanomedicine on organ, tumour or tissue scale. However imaging drug/nanoparticle distribution can help predict treatment effect and therefore select which patients will benefit most and in which patients a therapy adjustment or a combination therapy that reduces heterogeneity are warranted. Combining imaging of nanoparticle and drug distribution with imaging of tumour environment characteristics or early response indicators promises to help personalize treatment further. Currently, only optical imaging is commonly used to investigate, in the preclinical setting, the interplay between environment, nanoparticle and drug. It is our hope and recommendation that non-invasive clinical imaging and MSI will play a central role in future preclinical and clinical research on the interaction of drug, nanoparticle and environment. Choosing and combining imaging modalities wisely will lay the foundation for successful future nanotherapies.Integration of imaging techniques in preclinical lab practice has contributed to an improved understanding of the


Staphylococcus aureus to platelets and endothelial cells involves binding of bacterial cell surface protein A (SpA) to the large plasma glycoprotein von Willebrand factor (vWF). SpA-mediated bacterial adhesion to vWF is controlled by fluid shear stress, yet little is currently known about the underlying molecular mechanism. In a recent publication, we showed that the SpA-vWF interaction is tightly regulated by mechanical force. By means of single-molecule pulling experiments, we found that the SpA-vWF bond is extremely strong, being able to resist forces which largely outperform the strength of typical receptor-ligand bonds. In line with flow experiments, strong adhesion is activated by mechanical tension. These results suggest that force induces conformational changes in the vWF molecule, from a globular to an extended state, leading to the exposure of cryptic binding sites to which SpA strongly binds. This force-sensitive mechanism may largely contribute to help S. aureus bacteria to resist shear stress of flowing blood during infection.Attachment of  Staphylococcus aureus is a leading cause of endovascular diseases, such as infective endocarditis or heart valve prosthetic infection. During endovascular infection, the pathogen needs to attach to the endothelium and to resist shear stress of flowing blood. For that purpose, S. aureus expresses a repertoire of surface associated proteins (adhesins) that mediate bacterial attachment to extracellular matrix and endothelial cell surface components. A prototype of such interaction is the binding of protein A (SpA) to von Willebrand factor (vWF), a glycoprotein found in the vascular basement membrane and in the plasma. The mature vWF monomer consists of 2,050 residues and contains a number of domains, each involved in binding specific ligands, including FVIII, GPIbα and collagen. Endothelial cells and megakaryocytes are the only cells that synthesize vWF. vWF monomers are organized in large compacted multimers stored in organelles called Weibel-Palade bodies, which can be secreted upon extracellular stimuli.S. aureus adhesion to vWF is influenced by fluid shear, but the molecular details behind this interaction remain mysterious. This prompted us to investigate the strength and dynamics of the SpA-vWF bond using single-molecule atomic force microscopy. We discovered that SpA-dependent bacterial adhesion to vWF involves specific molecular bonds that are much stronger  than most receptor-ligand interactions studied to date (∼200 pN). Remarkably, we found that the SpA-vWF bond is tightly mechanoregulated, being weak at low tensile force, but extremely strong at high force, thereby explaining why in high shear flow conditions bacteria adhere in large amounts to vWF surfaces .SpA-dependent Extremely strong forces  were recently reported for the other staphylococcal adhesins SdrG, ClfA and ClfB, which bind to their ligands (e.g. fibrinogen) through the dock, lock, and latch (DLL) mechanism involving conformational changes in the adhesins that greatly stabilize the complex. Single-molecule experiments and steered molecular dynamics simulations have shown that the high mechanical stability of the SdrG-fibrinogen DLL complex results from a hydrogen bond network between the ligand peptide backbone and the adhesin. Our findings are novel and unexpected since SpA is structurally and functionally very different from other adhesins investigated so far, and does not involve DLL binding. We therefore believe that the force activation of the SpA-vWF bond primarily involves conformational changes in vWF rather than in the adhesin. Supporting this view, vWF is a mechanosensitive protein capable to respond to external forces, such as hydrodynamic shear in flowing blood. Under force, the vWF molecule transitions from a globular state to an extended chain conformation with exposure of intra-molecular globular domains. We speculate that force-induced extension of vWF may lead to the exposure of a cryptic high-affinity binding site to which SpA strongly binds. In addition, we cannot exclude that force-induced structural changes in the adhesin domains also contribute to the formation of such strong bonds. In the future, new structural biology data on the SpA-vWF complex should allow to clarify the molecular nature of this interaction.E. coli to epithelial cells, involving binding of FimH adhesin to mannose residues. This is achieved through so-called “catch bonds” that are strengthened by mechanical stress. Several studies have suggested that staphylococcal SdrG, ClfA and ClfB may use catch bond mechanisms to resist high shear stress during infections. These experiments suggest that in flow conditions the binding strength of bacterial proteins, measured under force at non-equilibrium, are more relevant than bulk affinity values obtained at equilibrium.Our study contributes to a growing body of literature showing that physical stress can profoundly impact bacterial behaviors. Specifically, there is increasing evidence that the adhesion of bacterial pathogens during infection can be strongly enhanced by fluid flow conditions. A prototypical example is the attachment of S. aureus to withstand high shear forces of flowing blood during endovascular infections . The force-activated bond might represent an interesting target for anti-staphylococcal therapy. Blocking of the SpA-vWF interaction is potentially important as entry of S. aureus into circulation can cause life-threatening vascular diseases. S. aureus adhesion to endothelial cells is a critical step in the destruction of cardiac valves with subsequent endocarditis and systemic dysfunction in the case of sepsis. Currently, treatment of blood stream infections involves aggressive antibiotic therapy for long periods of time. The greater the duration of exposure of an antibiotic to bacteria, the greater the risk is of developing antibiotic resistance. So, there is great interest in developing new strategies focusing on the prevention of bacterial attachment to the endothelium.In conclusion, the mechanoregulated SpA-vWF bond rationalizes at the molecular level the ability of


Extracellular matrices (ECMs) have emerged as promising off-the-shelf products to induce bone regeneration, with the capacity not only to activate osteoprogenitors, but also to influence the immune response. ECMs generated starting from living cells such as mesenchymal stromal cells (MSCs) have the potential to combine advantages of native tissue-derived ECMs  with those of synthetic ECMs . MSC-derived ECMs could be tailored by enrichment not only in osteogenic cytokines, but also in immunomodulatory factors, to skew the innate immune response toward regenerative processes. After reviewing the different immunoregulatory properties of ECM components, here we propose different approaches to engineer ECMs enriched in factors capable to regulate macrophage polarization, recruit host immune and mesenchymal cells, and stimulate the synthesis of other immunoinstructive cytokines. Finally, we offer a perspective on the possible evolution of the paradigm based on biological and chemico-physical design considerations, and the use of gene editing approaches. Bone disorders have a worldwide prevalence since they can be derived from multiple causes, including orthopedic trauma, cancer or congenital diseases. Since it emerged in the early 90s, bone tissue engineering has aimed to develop innovative biological materials to improve bone repair and regeneration , 2. AmonDespite many advances, the need for quality improvement of engineered ECM (either synthetic or MSC-generated) for bone healing is still quite large . Along tIn this review, we describe firstly key aspects of the interplay between innate immunity and bone healing. Then, we highlight how some ECM components are able to modulate the innate immune response. Finally, we summarize different strategies proposed for ECMs enriched with innate immunoinstructive factors to improve bone regeneration.All bone substitute materials, as any other foreign structure, trigger a host immune reaction after implantation, which recapitulates the first steps of the classical immune response after bone injury , 17. In Immediately after any bone injury, vascular disruption generates a hematoma and triggers a quick and potent inflammatory reaction. Multiple blood and interstitial fluid proteins  adsorb the injury site and activate the blood coagulation cascade as well as the complement system . In thisCirculating PMNs are quickly recruited by this chemoattractant protein matrix to the injury site. While they might contribute to fibrin clot formation , their mFollowing these principles, several studies have attempted to improve bone regeneration modulating either macrophage number or their polarization toward M1 or M2 phenotypes . On the in vivo, since the exposition to multiple signaling leads to activation of macrophages with mixed functions. This is especially prominent in pathological conditions, where abnormal signaling might prime macrophages toward a profibrotic phenotype , contained in basement membranes components such as entactin or presented in photopolymerizable poly(ethylene glycol) (PEG)- based hydrogels, has been shown to enhance myeloid cells adhesion to the ECM , 62, whiThe anti-inflammatory properties of glucocorticoids are well-known. In particular, dexamethasone delivery in polydimethylsiloxane-based 3D scaffolds has been used to promote macrophage polarization toward an anti-inflammatory (M2) phenotype and suppress inflammatory pathways during the first week post-implantation . Dexametin vivo. Nevertheless, the delivery of exogenous factors is subjected to several drawbacks including poor matrix penetration, diffusion, enzymatic degradation and thus uncontrolled doses. In addition, the delivery of few specific agents has been revealed inefficient in triggering a complete immune response in vivo. For this reason, different strategies have been developed to control the spatial and temporal delivery  and anti-inflammatory  signals in order to promote macrophages recruitment and enhance bone healing . In contInterestingly, inorganic compounds like magnesium-doped calcium phosphate cement are also able to elicit a favorable innate immune reaction modulating macrophage activity to improve osteogenesis and angiogenesis. This compound represses TNFα and IL6 expression while it upregulates TGFβ1 in macrophages . Beyond To sum up, these studies attempt to improve bone regeneration by targeting endogenous MSC/immune cells to produce themselves the cues critical for an orchestrated repair upon bone injury .In this work, we have reviewed some relevant aspects of the interplay between the innate immune system and osteogenesis in the context of bone healing. Then we have focused on the interactions between ECM components and innate immune cells to finally discuss some strategies followed to immune-instruct ECMs. However, many other critical aspects have not been discussed here.As previously mentioned, the innate immunity plays an essential role during the initial phases after bone injury, promoting cell immunorecruitment and modulating the inflammatory environment (M1-to-M2 paradigm). Importantly, the adaptive immune response takes slowly part in this regulation to instruct the bone formation phase. Multiples studies have attempted to engineer ECM-based materials to modulate the adaptive immune response, specially targeting T cells . Indeed,in vivo (We have discussed how different ECM components, even in the absence of immunomodulatory factors, could modulate the innate immune response. The works studying these interactions reveal that ECM composition is an important factor to consider prior to any further immunoregulatory engineering. However, beyond its chemical properties, ECM physical features can also decisively modulate the immune response in vivo . Therefoin vivo .Different approaches have been proposed to improve the spatiotemporal delivery of growth factors to engineer “smart” ECMs. However, in most cases they only focus on osteogenic and angiogenic factors. Immunomodulatory ECM-like microspheres have been recently used to improve IL4 delivery and accelerate bone regeneration modulating macrophage polarization . Future Genetic manipulation of MSCs has also emerged as an alternative to better control the dose and temporal delivery of osteogenic and angiogenic factors into engineered ECMs to improve bone regeneration process . GeneticIn summary, important advances have been achieved in the last years to improve the quality of immunoinstructive ECM-derived grafts and their immunogenicity after implantation. In the context of ECM engineering, immunoregulators can be exogenously delivered to enrich the biomaterial in specific cytokines and/or stimulate the endogenous synthesis of other factors by host cells. In this perspective, genetically modified MSCs represent a relevant alternative to control the spatiotemporal delivery of immunoregulators in order to engineer immunoinstructive ECMs promoting efficient bone repair.AG-G and IM prepared the figures and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many global infectious diseases are not well-controlled, underlining a critical need for new, more effective therapies. Pathogens and pathogen-infected host cells, like cancer cells, evade immune surveillance via immune evasion mechanisms. The present study indicates that pathogenic bacteria, endoparasites, and virus-infected host cells can have immune evasion mechanisms in common with cancers. These include entry into dormancy and metabolic reprogramming to aerobic glycolysis leading to excessive secretion of lactic acid and immobilization of local host immunity. The latter evasion tactic provides a therapeutic target for cancer, as shown by our recent finding that patient-derived cancer xenografts can be growth-arrested, without major host toxicity, by inhibiting their lactic acid secretion (as mediated by the MCT4 transporter)-with evidence of host immunity restoration. Accordingly, the multiplication of bacteria, endoparasites, and viruses that primarily depend on metabolic reprogramming to aerobic glycolysis for survival may be arrested using cancer treatment strategies that inhibit their lactic acid secretion. Immune evasion mechanisms shared by pathogens and cancer cells likely represent fundamental, evolutionarily-conserved mechanisms that may be particularly critical to their welfare. As such, their targeting may lead to novel therapies for infectious diseases. Infectious diseases constitute a major health problem worldwide and impose a substantial burden on societies in human suffering and economic loss. While diseases like smallpox have essentially been eliminated, many other pathogen-induced diseases are not well-controlled and new infectious diseases are continually emerging, thus underlining a critical need for novel therapeutic targets leading to more effective therapies Fonkwo, .innate immune system as “non-self” via cell surface pattern recognition receptors (PRRs) on innate immune cells which recognize pathogen-associated molecular patterns (PAMPs) on the foreign microbes; the latter are then eliminated by, for example, macrophage-mediated phagocytosis or secretion of cytotoxins by NK cells  and B-cell receptors (BCRs). Identification of pathogen-infected host cells is achieved by T cells via recognition of antigen fragments presented by major histocompatibility complex (MHC) molecules. Elimination of the intruders is accomplished via cytotoxic responses by CD8+ T cells and CD45+ lymphocytes, recruitment of neutrophils, monocytes, and mature B lymphocytes (mediated by CD4+ helper T cells), as well as antibody production by plasma cells.Recognition of foreign microbes by In addition to foreign microorganisms, host cancer cells can be recognized by the immune system as changes in their cell surface structure render them foreign Chaplin, .Despite vigorous immune surveillance by healthy, immunocompetent hosts, pathogens often manage to survive in the body. They accomplish this by employing a large variety of immune evasion mechanisms based on, for example, immobilizing immune responses or avoiding recognition by host immune cells  and human cancer cells in search of mechanisms common to both groups. Such shared tactics may represent fundamental, evolutionarily-conserved mechanisms of immune evasion, which could in turn be useful as targets for novel therapies of both cancer and infectious diseases.A literature survey led to the following information.A small percentage of bacterial species can cause globally significant diseases such as tuberculosis and pneumonia. A typical immune response to pathogenic bacteria consists of opsonisation of the microbes followed by phagocytosis by host macrophages and fusion of the phagosomes with lysosomes. Intra-lysosomal acid hydrolases, reactive oxygen species (ROS) and nitric oxide ultimately kill the pathogens  as distinct from the mitochondrial oxidative phosphorylation pathway used by normal cells Warburg, . The resAs highlighted by this study, pathogenic bacteria, endoparasites and virus-infected host cells employ, like cancer cells, a large variety of mechanisms to evade immune surveillance. These mechanisms are diverse and multifaceted and, even within the four classes, various species may have their own unique methods of evading immune surveillance. As such, it is beyond the scope of this perspective article to elaborate on them. Further information can be obtained from the following literature references for pathogenic bacteria  Metabolic reprogramming to aerobic glycolysis leading to excessive lactic acid secretion and consequent local host immunity suppression, a well-defined mechanism used by most human cancers  targeting MCT4-mediated lactic acid secretion led to a marked reduction in MCT4 expression and lactic acid secretion, increased intracellular lactic acid levels, and marked reductions in aerobic glycolysis, cell proliferation, cell migration, and tissue invasion  and the unique metabolic properties of dormant cells could eliminate a greater proportion of cancers and pathogenic microorganisms. While mechanisms underlying cellular entry into dormancy remain to be more fully elucidated, preliminary investigations in our laboratory have uncovered changes in amino acid metabolism as a potentially unique metabolic profile in dormant cancer cells post-therapy  metabolic reprogramming to aerobic glycolysis leading to excessive lactic acid secretion and host immunity suppression and (ii) entry into dormancy. Targeting fundamental, evolutionarily-conserved mechanisms of immune evasion could be particularly effective for treatments of both cancer and multiple infectious diseases.YW initiated the topic in July 2016. JW, SC, and PG organized the manuscript. RL and EX provided published evidence. JW, SC, JK, PG, and YW were involved in the further development of the ideas and the writing of the paper. PG finalized the write-up.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During the menopausal transition, which begins four to six years before cessation of menses, middle-aged women experience a progressive change in ovarian activity and a physiologic deterioration of hypothalamic-pituitary-ovarian axis function associated with fluctuating hormone levels. During this transition, women can suffer symptoms related to menopause . Neurological symptoms such as sleep disturbance, “brain fog” and mood changes are a major complaint of women transitioning menopause, with a significant impact on their quality of life, productivity and physical health. In this paper, we consider the associations between menopausal stage and/or hormone levels and sleep problems, mood and reduced cognitive performance. The role of estrogen and menopause hormone therapy (MHT) in cognitive function, sleep and mood are also discussed. During their lifetime women experience dramatic fluctuations in the levels of the sexual hormones estradiol, progesterone and also androgens when going through the different stages of life, from menarche to menopause . These fSex steroids play a role in the brain, on both cortical and sub cortical structures, through genomic and non-genomic paths . Even thSteroid hormones active in the CNS are called neurosteroids. They may be peripherally produced steroids able to cross the blood-brain barrier or synthesized in the central and peripheral nervous system by neurons and glial cells either via de novo synthesis from cholesterol or from local metabolism of intermediate steroids produced in the periphery . Even ifDifferent hormones can provide brain regulation in a sexually specific way: neuroprotective effects of estrogens are more evident in females than males, and androgens seem to be more active in males in the recovery from demyelinating events, while progestogens seem to be more effective in the male in the reduction of apoptosis and abnormal proliferation after a trauma or a stroke .In the CNS there is a wide distribution of estrogen receptors (ERs) localized in areas of the brain involved in memory and executive function. The ER isoform, ERβ, is expressed mostly in the cerebral cortex and hippocampus, whereas ERα signaling is largely represented in magnocellular cholinergic neurons of the basal forebrain . In the Estrogens elicit their function involving many neurotransmitter systems, such as acetylcholine, serotonin, noradrenaline and glutamate. In particular, the cholinergic neurotransmitter system is relevant in memory processes . The metSimilar to estrogen, progesterone is also a potent regulator of neurogenesis, cell survival and bioenergetic systems. They do not have synergistic action and their co-administration leads to a lower response than with the administration of a single compound ,16. ProgProgesterone, and especially allopregnanolone, is able to promote the GABAergic system inhibiting synaptic transmission, producing what is believed to be an anti-anxiety effect similar to that of benzodiazepines ,20,21. IThe brain is also able to locally produce the androgen dihydrotestosterone independently of the gonads . AndrogeDuring menopausal transition many women complain of memory problems such as difficulty with words, forgetfulness and “brain fog” , thus suHowever, in a recent cross-sectional study, women in their early midlife demonstrated better performance in detailed memory tasks when compared to age-matched men . These shttps://www.swanstudy.org/); in the next few years we believe that this study will provide a unique insight into the long-term effects of hormonal changes in middle-aged women. Longitudinal data considering the impact of menopausal transition on memory and cognitive function are scarce. Two longitudinal studies reported subtle decrements in cognitive function: the Kinmen Women-Health Investigation (KIWI) did not evidence significant cognitive decline, with the exception of verbal fluency, in an 18-month follow-up of pre-menopausal women . The StuIt should be noted that not only events occurring during menopausal transition but also lifelong hormonal processes seem to be significant for cognition. A recent observational study of 1315 women showed that a longer fertile period  was associated with better verbal memory . On the Despite medical literature which highlight the deep connection between estrogen and cognitive function, data regarding the relationship between menopause hormone therapy (MHT) and its neuroprotective outcomes remains conflicting. Although several observational studies have demonstrated a positive effect of hormone therapy on Alzheimer’s disease (AD) ,38,39,40WHIMS was a randomized, placebo-controlled clinical trial, and the first large, long-term study to address the cognitive effects of MHT (0.625 mg of conjugated equine estrogen (CEE) plus 2.5 mg of medroxyprogesterone acetate) in the prevention of AD, in 4532 post-menopausal women (over 65 years of age) enrolled in the larger WHI study. Results showed that after a mean follow-up of 4.2 years, MHT failed to reduce general cognitive decline and was associated with a substantially and clinically important increased decline in Modified Mini-Mental State Examination (MMSE) total scores when compared with placebo .Subsequent analysis of 2947 WHIMS estrogen-alone treated women (aged 65‒79) showed that mean MMSE scores were significantly lower in the estrogen group compared with placebo. The adverse effect of estrogens was more pronounced in women with lower cognitive function at baseline .In the analysis of data from the Nurses’ Health Study, a prospective cohort of over 13,807 women aged 70 to 81, little difference was found in cognitive decline between current hormone users and those that had never used them. Long-term users (at least 5‒10 years of use) of estrogen or estro-progestin showed an increased risk of substantial decline in most cognitive tests. The impairment was more evident in women starting hormones at an advanced age . SubsequIn 2008, a meta-analysis of clinical trials investigating MHT (estrogen or estrogen-progestin therapy) in post-menopausal women (over 60 years of age) concluded that MHT does not protect against cognitive function decline in normal women .It should be observed that studies involving older women (the WHIMS was over 65 years of age) did not consider the so called “timing hypothesis”. In fact, further studies, similar to the cardiovascular risk, have suggested that estrogen can be neuroprotective only if commenced shortly after the onset of menopause ,48. SustData from the Cache County Study revealed that women using MHT had a reduced risk of AD compared with non-MHT users only if treatment was started soon after menopause (within five years) ,39. SimiThere have also been other limited clinical trials regarding early MHT and cognitive performance later in life. The WHIMS‒Young (WHIMSY) study examined 326 post-menopausal women between 50 to 55 years of age randomized for MHT (CEE with or without 2.5 mg medroxyprogesterone acetate over a mean of 7.0 years) or placebo. CEE based therapies produced no overall sustained benefit or risk to cognitive function when administered to post-menopausal women aged 50 to 55 years. Cognitive evaluation performed up to 67 years of age did not show sustained benefits of MHT over placebo . Even thCognitive decline and dementia are a growing public health problem. The inability to recall information, defined as impaired episodic memory, is a potentially alarming symptom of the very early signs of AD or other forms of dementia. AD is the most common cause of dementia and emerges more frequently in women than in men, with some authors hinting at sex-specific differences in the incidence of AD . This coConsidering the significance of this disease, research in this field has attracted considerable scientific and public interest. The role of estrogen has been assessed not only in the maintenance of cognitive function in non-demented women but also in the prevention and/or treatment of AD. In vivo and in vitro preliminary data suggest that estrogen may play a role in the prevention of amyloid deposition. Estradiol has been shown to attenuate tau hyperphosphorylation  and depoIn the Cache County Study, a prospective study of incident dementia among 1357 men (mean age 73.2 years) and 1889 women (mean age 74.5 years), women using MHT had a reduced risk of AD when compared with non-MHT users (adjusted HR 0.59), whereas there were no apparent benefits with current MHT use unless they had been used for in excess of 10 years .Other large epidemiologic studies have addressed the role of exogenous estrogens and the risk of dementia. In 2001, the first meta-analysis of these observational studies reported a reduced risk of dementia in MHT users, even with some methodological limits of the studies , whereasRecently, the Kronos Early Estrogen Prevention Study (KEEPS) showed that transdermal 17-beta estradiol therapy was linked to a decrease in amyloid-beta deposition on neuroimaging studies, particularly in apolipoprotein E (APOE) E4 carriers .More recently, data provided by Savolainen-Peltonen et al. showed that systemic hormone therapy users have a higher risk of AD when compared to vaginal estradiol users and that long term use of systemic hormone therapy could be burdened by a globally increased risk of AD, independent from the age at initiation of systemic hormone therapy . The risSeveral small randomized clinical trials have considered the possibility of the treatment of women with Alzheimer’s with estrogen. In the largest study of 120 women, estrogen administration for one year (conjugated equine estrogens 0.625 mg or 1.25 mg vs placebo) did not slow progression of the disease nor did it improve global, cognitive or functional outcomes . SubsequWe believe that those studies should not modify our current clinical practice and decision making regarding MHT. In fact, even if some observational studies had inconsistent and mixed results, it should be remembered that several randomized clinical trials supported the safety profile of MHT on cognition in early menopause.Overall, young menopausal women without contraindication to MHT and with impaired quality of life because of night sweats, vasomotor symptoms or disrupted sleep can benefit from MHT with several studies providing reassurance that MHT does not adversely affect cognition in these women. On the counterpart, it should be noted that, consistently with guidelines, MHT should not be prescribed with the sole purpose of AD or memory loss prevention in women without these symptoms or in those not willing to start hormonal treatment.Despite the amount of data already available on this topic, future research should focus on long-term risks of MHT on cognition and AD and on the different effects on cognitive outcomes of various type of molecules.During menopausal transition, women are at higher risk of developing depression, stress, anxiety and emotional distress . SeveralLongitudinal studies show that there are many other factors that may have a significant influence on mood levels. In particular, these include demographic, psychosocial and health related characteristics such as unemployment, low level of education, being Black, Japanese or Hispanic, poor social support, smoking, chronic medical diseases, a history of anxiety, history of postpartum depressive symptoms, nulliparity, VMS, stressful life events, death of the partner, body mass index (BMI) and self-esteem. Body dissatisfaction may be common in midlife women . There iSome studies have examined the association between depression and hysterectomy with or without oophorectomy, obtaining mixed results ,71,72,73Considering the causes of mood alteration during menopausal transition, the role of estrogens should be considered, even if the link of estradiol (either declining levels or low levels) in the precipitation of peri-menopausal depression (PMD) is not yet fully understood . EstradiIn comparison with cross-sectional studies, longitudinal studies are a more suitable way for the assessment of the potential association between menopause transition stage and depression, as well as in the direction of association . The SWAThe existence of menopause-associated depression has been a controversial topic among clinicians, despite the undeniable evidence of a heightened increase of major depressive disorder (MDD) during menopausal transition. However, it is not possible to define an “estradiol withdrawal hypothesis”, since several hormonal and metabolic factors could confound the effects of estradiol withdrawal in peri-menopausal women. Furthermore, conflicting methodologies used to characterize reproductive staging or assess psychiatric conditions in various studies are confounding factors to the matter in question. Methodologic differences of note include menopausal state (peri-menopause vs. post-menopause), determination of state (earlier studies have used age as a proxy measure), baseline symptomatology (asymptomatic vs. depressive symptoms vs. syndromic depression), route of hormone administration , and symptom or syndrome measurement. For these reasons much of the literature on women’s mental health looks at the transition to menopause from a disease-risk point of view . AccordiIn peri-menopausal women, one randomized controlled trial assessed mood in 83 non-depressed peri-menopausal women during six months of hormone therapy with CEE for two weeks followed by two weeks of CEE plus medroxyprogesterone acetate (MPA). No significant effects on mood were observed . RecentlThe role of estrogen administration in the prevention of depressive symptoms in peri-menopausal and early post-menopausal transition has been assessed in one randomized trial. One hundred and seventy-two women were randomized for transdermal E2 (0.1 mg/d) plus intermittent oral micronized progesterone (200 mg/d for 12 days every three months) or placebo. After one year, women receiving estrogens plus progesterone were significantly less likely to present depressive symptoms compared with women receiving a placebo (32.3% vs. 17.3%), this effect being more evident in women in the early peri-menopausal stage .Considering post-menopausal non-depressed women, mood effects of hormone therapy have been assessed in several studies showing a lack of significant effects of estrogens on mood when used in non-depressed post-menopausal women. Haines et al. failed to recognize any effect on mood after one year of treatment with oral estradiol (1 or 2 mg) versus a placebo ; similarWhile the KEEPS study reported a positive impact of oral CEE versus transdermal E2, the Heart and Estrogen/Progestin Replacement Study (HERS), the Women’s Health Initiative Study of Cognitive Aging (WHISCA) and the Women’s International Study of long Duration Estrogen after the Menopause (WISDOM) did not report a significant impact of MHT on mood. For this reason, MHT should not be proposed to non-depressed, asymptomatic peri-menopausal women to prevent or alleviate mood symptoms. Estrogen seems to have a potential role among specific sub-populations at risk of depressive symptoms during menopausal transition. There is some evidence that hormonal therapy has potential anti-depressant effect similar to that of classic anti-depressant agents when administered to depressed peri-menopausal women . Even thPreviously reported data from Gordon et al. underline the “critical timing hypothesis,” which suggests that the beneficial effects of estradiol are observed only if it is administered proximate to the cessation of ovarian activity. Estrogen therapy is ineffective as a treatment for depressive disorders in post-menopausal women .Other findings from trials enrolling recently post-menopausal women with depression and several large observational studies, including women initiating MHT shortly after menopause, suggest that menopausal hormone therapy has either a neutral or beneficial effect on mood . Four smSchmidt and colleagues attempted to determine the existence of a diverse response to estradiol withdrawal in women with past pre-menopausal depression (PMD) compared with those without past depression . The stuIn conclusion, as suggested by the recent consensus recommendation by Maki et al., although estrogen may increase the response to anti-depressants in midlife and older women, their use should still be suggested when indicated for other concurrent conditions such as vasomotor symptoms . EstrogeAlthough only a small proportion of women experience depressed mood in relation to menopause, it is mandatory to accurately investigate the origin of depressive symptoms in order to detect those women who had history of depression before the menopause transition and those who had the onset during the menopausal transition. Antidepressants remain the first-line treatment of depression for patients with previous history of depression . In womeFuture research is needed to better understand the relationship between hormonal fluctuations and perimenopausal depression and the mechanisms of the windows of vulnerability across certain phases of women’s reproductive lifeWomen transitioning menopause typically complain of poor sleep quality, insufficient sleep, nocturnal awakenings and apnea. Sleep difficulties often begin during menopausal transition and their prevalence increases in post-menopausal life with rates of self-reported sleep problems ranging between 40% and 56%, compared to pre-menopausal women in their late reproductive stage, reporting rates of 31% . Sleep dKravitz et al. investigated the associations between E2, follicle stimulating hormone (FSH) and sleep disturbances. Their results indicated that only changes in hormone levels, but not baseline levels, are associated with sleep disturbances. In particular, serum E2 reduction correlated with both trouble falling asleep and staying asleep, whereas increments in serum FSH levels were associated with reports of difficulty staying asleep . Sowers Surgical menopause is associated with more severe sleep disturbances than with natural menopause . Indeed,Menopausal transition is accompanied by further typical risk factors for sleep disturbances other than by estrogen withdrawal itself. Vasomotor symptoms are one of the most characteristic manifestations of the climacteric, and many studies have reported a strong association between sleep disorders and vasomotor symptoms. Vasomotor symptoms arise with estrogen decrease through a complex mechanism of actions, more complex than just estrogen withdrawal, involving central noradrenergic activity, as well as serotonergic mechanisms , and, moMany other factors associated with the aging process can be involved in the onset of sleep problems during menopausal transition such as obesity, cardiovascular disease, gastrointestinal problems, urinary problems, endocrine problems, chronic pain syndromes, the use of neuroactive medications, smoking, caffeine, selective serotonin reuptake inhibitors, bronchodilators, antiepileptic medications, thyroid hormone preparations, among others . MoreoveOther factors related to sleep troubles are restless leg syndrome (RLS) and obstructive sleep apnea (OSA). RLS is a common condition of the nervous system that causes an overwhelming irresistible urge to move the legs, occurring during the evening or at bedtime. Its prevalence is higher among women; however, the precise cause is still unknown. A number of theories have been proposed that include iron deficiency secondary to pregnancy or persistently high levels of estrogen during pregnancy or the decrease of estrogen and melatonin at menopause . ConcernBetween 47% and 67% of post-menopausal women have been found to suffer from OSA ,115. PhyMHT composed of estrogen alone for women who have undergone a hysterectomy and combined with progestin for women with an intact uterus is the most effective treatment of menopausal symptoms  and their potential consequences  . SeveralIn most studies enhanced sleep quality resulted from an improvement in vasomotor symptoms ,119,120.The etiology of menopausal sleep disorder is not fully understood. However, current literature suggests multifactorial causes. Further investigations by randomized controlled studies are needed to investigate the efficacy of these treatments on preexisting insomnia, obstructive sleep apnea and restless leg syndrome, in peri-post menopausal women. Further research is also needed to determine if self-reported sleep quality in menopause is affected by different molecules, formulations and routes of administration of MHT.During menopausal transition women experience dramatic fluctuations in the levels of the sexual hormones estradiol, progesterone and also androgens, which are potentially responsible for modifications in behavior, cognition, mood and sleep. Cognitive function and cognitive disorders: despite the deep connection between estrogen and cognition, data regarding the relationship between hormone replacement therapy and the neuroprotective outcomes still remain conflicting. Several studies have excluded any cognitive benefits of estrogen or combined estrogen-progestin therapy in women over the age of 65 without underlying dementia. Young menopausal women without contraindication to MHT and with impaired quality of life because of night sweats, vasomotor symptoms or disrupted sleep can benefit from MHT, and in several studies, MHT does not adversely affect cognition in these women.Sleep disturbances: the etiology of menopausal sleep disorder is not fully understood, but MHT can play a role enhancing sleep quality. MHT can improve sleep, reducing night sweats, but it can also act through other mechanisms, as disturbed sleep during perimenopause can occur independently of hot flashes. Further research is needed to determine if self-reported sleep quality in menopause is affected by different molecules, formulations and routes of administration of MHT. For example, the GABAergic sedating effects of progesterone should be considered in women with sleep issues.Mood and depressive symptoms: even though some data support a potential beneficial effect of MHT on mood, it should not be proposed to non-depressed peri-menopausal women to prevent mood symptoms. Estrogens can be considered in menopausal women with other concurrent conditions such as vasomotor symptoms as they may increase the response to anti-depressants.The following short summary provides key concepts regarding MHT in menopause and perimenopause: