Cigarette smoking (CS) regulates both innate and adaptive immunity and causes numerous diseases, including cardiovascular, respiratory, and autoimmune diseases, allergies, cancers, and transplant rejection. Therefore, smoking poses a serious challenge to the healthcare system worldwide. Epidemiological studies have always shown that CS is one of the major risk factors for transplant rejection, even though smoking plays redundant roles in regulating immune responses. The complex roles for smoking in immunoregulation are likely due to molecular and functional diversities of cigarette smoke components, including carbon monoxide (CO) and nicotine. Especially, CO has been shown to induce immune tolerance. Although CS has been shown to impact transplantation by causing complications and subsequent rejection, it is overlooked whether CS interferes with transplant tolerance. We have previously demonstrated that cigarette smoke exposure reverses long-term allograft survival induced by costimulatory blockade. Given that CS impacts both adaptive and innate immunity and that it hinders long-term transplant survival, our perspective is that CS impacts transplant tolerance. Here, we review impacts of CS on major immune cells that are critical for transplant outcomes and propose the cellular and molecular mechanisms underlying its effects on alloimmunity and transplant survival. Further investigations are warranted to fully understand why CS exerts deleterious rather than beneficial effects on transplant survival even if some of its components are immunosuppressive. Cigarette smoking (CS) contributes to various diseases, including oral, respiratory, and cardiovascular diseases, infections, cancers, autoimmune diseases, and transplant rejection. It impacts both adaptive and innate immunity. On the other hand, CS is a well-known risk factor associated with the morbidity and mortality of organ transplantation. Numerous epidemiological analyses and retrospective reviews have shown that CS increases the risks of transplantation-related complications, allograft rejection, and recipient death in transplanted patients. Although the adverse impacts of smoking on outcomes of organ transplantation are well documented, the molecular and cellular mechanisms responsible for smoking-associated graft rejection are neither fully understood nor carefully proposed. Especially, it has been overlooked whether and how smoking directly impacts transplant tolerance, even with many of the original studies showing that CS has widely affected immune responses as well as transplantation outcomes. In the current review, we summarize the effects of smoking on the major immune cells that impact transplant survival or tolerance Figure  and propGrowing studies have demonstrated that smoking hinders long-term allograft survival by promoting various complications after transplantation. Pungpapong et al. reported that patients with a smoking history had higher risks of vascular complications, especially arterial ones than non-smokers after liver transplantation, while patients, who gave up smoking for 2 years prior to transplantation, had lower incidence of vascular complications . FurtherAlthough numerous epidemiological studies supported the finding that CS is an important risk factor for organ transplantation, there was a lack of the proof that smoking can directly cause allograft failure in a cause-effect manner. Recently, our studies have provided the first evidence that second-hand smoking (SHS) directly prevents long-term allograft survival, which would otherwise have been induced by the costimulatory blockade in animal models . We founIt is well known that allograft rejection can be suppressed or prevented by either global immunosuppression or targeted immunomodulation. The balanced activation of conventional dendritic cells (DCs), T cells and B cells, regulatory T cells (Treg) and regulatory B cells, and memory T and B cells is vital for the appropriate regulation of immunity, which determines transplantation outcomes. Since CS impacts both innate and adaptive immunity, it likely also affects immune-based transplant rejection or allograft tolerance Figure .Dendritic cells, a critical subset of innate immune cells that serve as antigen-presenting cells, play an important role in mediating T cell activation as well as regulation of T cell function. Oberhuber et al. demonstrated that DCs expressing CD11c in murine recipients facilitated rejection of cardiac allograft and promoted the proliferation of both CD4+ and CD8+ T cells . FurtherIt has been reported that CS interferes with DC function. Previous studies on airway inflammation have shown that CS increases the number of DCs , promoteIt is well known that Tregs play a key role in maintaining the immunologic self-tolerance and regulating immune responses to exogenous antigens in case of excessive immune reactions. Alterations in either functionality or number of Tregs may result in autoimmune diseases or loss of transplantation tolerance. Transfer of CD4+ CD25+ Tregs not only suppresses allograft rejection and graft-versus-host disease but also promotes transplant tolerance induction. While it is unclear whether CS alters Treg numbers and function in the context of transplantation, previous studies have revealed a significant downregulation of CD4+ CD25+ Treg cell numbers in bronchoalveolar lavage fluid and blood of smoking patients with COPD compared with healthy non-smokers , 24. SinMemory T cells can rapidly initiate alloimmune responses through the production of pro-inflammatory or effector cytokines . PreviouIt is well accepted that both memory T and memory B cells prevent the long-term survival of transplanted organs and hinder the induction of transplant tolerance. Thus, much attention has been paid to the actual effects of memory B cells and alloantibodies on transplant rejection. Using a murine model of cardiac transplantation, Burns et al. found that allograft survival induced by costimulatory blockade was prevented by memory B cells and alloantibodies of recipients , while aCigarette smoke contains large amounts of toxic components, including carbon monoxide (CO), nicotine, nitrogen oxides, and cadmium. These harmful toxins alter the immune homeostasis in oral or airway mucosa and solid organs, resulting in regional inflammation and abnormal immune responses to exogenous antigens. However, emerging evidence has shown that CO and nicotine, two of the major cigarette smoke components, are generally immunosuppressive. Therefore, they could suppress allograft rejection or promote transplant tolerance induction Figure .Carbon monoxide is produced when the heme is catalyzed by the enzyme heme oxygenase-1 (HO-1) expressed in immune cells , and it However, it is unclear why smoking, which also produces CO, has not been found to prolong allograft survival. Instead, it is well accepted that smoking generally promotes transplant rejection and even hinders the induction of long-term transplant survival or tolerance. Perhaps, CS itself does not provide transplanted smokers a significant amount of CO that is sufficient for suppressing vigorous alloimmunity. It is also possible that smoking generates a mixture of hazardous materials that cooperate to hinder long-term allograft survival. In other words, beneficial effects of CO on allograft survival may be well offset by other harmful substances contained in cigarette smoke. Therefore, how smoking-generated CO exerts its effects on alloimmunity and allograft survival in smokers remains unclear and deserves further investigations.in vitro and in vivo , reactive oxygen species (ROS), and free radicals. These biohazards contribute to inflammation, oxidative stress, and DNA damage. On the other hand, toll-like receptors (TLRs) expressed in innate immune cells are critical for recognizing exogenous pathogens. Previous studies presented the evidence that CS triggered the acute inflammation via TLR4/MYD88 signaling pathway . It was Taken together, the signaling pathways and molecular mechanisms underlying CS-mediated immunopathology, especially transplant rejection, are still not well understood due to the functional complexity and diversity of cigarette smoke components. Further investigations are warranted to understand the molecular mechanisms of their action, especially those involved in alloimmune responses.Smoking plays dual roles in immune responsiveness due to the molecular and functional diversities of cigarette smoke components, including RNS, ROS, CO, and nicotine. However, numerous experimental and clinical studies have shown that CS exerts a deleterious rather than beneficial effect on transplant survival even if CS downregulates immune responses under certain circumstances, for instance, smoking suppresses the immune system by reducing lymphocyte numbers in the blood of patients with chronic renal failure . TherefoFQ wrote the majority of the manuscript; PF and HL wrote a part of the manuscript; C-LL and WY prepared the literature; GN and ZD edited 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.


Prostanoids are products of cyclooxygenase biosynthetic pathways and constitute a family of lipidic mediators of widely diverse structures and biological actions. Besides their known proinflammatory role, numerous works have revealed the anti-inflammatory effects of various prostanoids and established their role in the resolution of inflammation. Among these, prostaglandins with cyclopentenone structure (cyPG) are electrophilic lipids that may act through various mechanisms, including the activation of nuclear and membrane receptors and, importantly, direct addition to protein cysteine residues and modification of protein function. Due to their ability to influence cysteine modification–mediated signaling, cyPG may play a critical role in the interplay between redox and inflammatory signaling pathways. Moreover, cellular redox status modulates cyPG addition to proteins; thus, a reciprocal regulation exists between these two factors. After initial controversy, it is becoming clear that endogenous cyPG are generated at concentrations sufficient to promote inflammatory resolution. As for other prostanoids, cyPG effects are highly dependent on context factors and they may exert pro- or anti-inflammatory actions in a cell type–dependent manner, or even biphasic or dual actions in a given cell type or tissue. In light of the growing number of cyPG protein targets identified, cyPG resemble other pleiotropic mediators acting through protein modification. However, their complex structure results in an inter- and intramolecular selectivity of the residues being modified, thus opening the way for structure-activity and drug discovery studies. Detailed characterization of cyPG interactions with cellular proteins will help us to understand their mechanism of action fully and establish their therapeutic potential in inflammation.


Over the past decades there have been exciting and rapid developments of highly specific molecules to bind cancer antigens that are overexpressed on the surfaces of malignant cells. Nanomedicine aims to exploit these ligands to generate nanoscale platforms for targeted cancer therapy, and to do so with negligible off-target effects. Aptamers are structured nucleic acids that bind to defined molecular targets ranging from small molecules and proteins to whole cells or viruses. They are selected through an iterative process of amplification and enrichment called SELEX , in which a combinatorial oligonucleotide library is exposed to the target of interest for several repetitive rounds. Nucleic acid ligands able to bind and internalize into malignant cells have been extensively used as tools for targeted delivery of therapeutic payloads both in vitro and in vivo. However, current cell targeting aptamer platforms suffer from limitations that have slowed their translation to the clinic. This is especially true for applications in which the cargo must reach the cytosol to exert its biological activity, as only a small percentage of the endocytosed cargo is typically able to translocate into the cytosol. Innovative technologies and selection strategies are required to enhance cytoplasmic delivery. In this review, we describe current selection methods used to generate aptamers that target cancer cells, and we highlight some of the factors that affect productive endosomal escape of cargoes. We also give an overview of the most promising strategies utilized to improve and monitor endosomal escape of therapeutic cargoes. The methods we highlight exploit tools and technologies that can potentially be incorporated in the SELEX process. Innovative selection protocols may identify aptamers with extended biological functionalities that allow effective cytosolic translocation of therapeutics. This in turn may facilitate successful translation of these platforms into clinical applications. Cancer remains one of the leading causes of death worldwide, with high morbidity and mortality. Though significant advances have been made in the fundamental understanding of cancer biology, the treatment of some cancer forms remains elusive, and many cancer treatments suffer from severe side effects ,2. Over Aptamers are short, single-chained DNA or RNA molecules that recognize molecular targets with high affinity (in nM to pM range) and specificity owing to their 3D conformation. Aptamers are emerging as a class of biocompatible ligands with huge potential in diagnostics and therapeutics ,16,17. CDespite the rapid development of diverse platforms that exploit aptamers to cell-surface receptors as vehicles to deliver peptides and siRNAs, none has made it into clinical trials yet . Some of4–105 copies/cell [4–105 of siRNAs would be delivered into endosomes during the course of one cycle of endocytosis, but only one to ten of those molecules (0.01%) would escape into the cytosol in that cycle. Given that plasma receptors typically recycle every 90 minutes for the major routes of internalization (caveolin and clathrin-mediated endocytosis), 500–5000 cycles of endocytosis (750–7500 h) would be required to deliver ~5000 siRNAs into the cytosol! In principle, the required time scale could be dramatically shortened by targeting receptors that have an expression >105 (but they are very rare) with fast uptake (<20 min), increasing the escape efficiency and delivering multiple cargo molecules for each receptor binding event . Using this simplified framework, an improved platform carrying 10 cargo molecules that binds 105 cell-surface receptors, internalizes rapidly and escapes from endosomes with an efficiency of 1% per cycle could quickly deliver 5000 siRNAs to the cytosol. In fact, many of the successful demonstrations of targeted delivery in vivo have exploited one or more of these strategies. For example, the asialoglycoprotein receptor (ASGPR) on liver cells and prostate-specific membrane antigen (PSMA) on prostate cancer cell lines are both expressed at very high levels (~106 copies/cell), and internalize rapidly (15min) [3 copies/cell). Even considering an efficiency of endosomal escape of 1% per cycle, the translocation of 5000 siRNAs in the cytosol of noncancerous cells would take a considerable amount of time (~ 1 week), thus reducing the risk of off target effects.The quantitative impacts of these factors can be illustrated by doing an admittedly over-simplified calculation. Recent studies suggest that significant siRNA-mediated gene knock-down requires approximately ~2000–5000 siRNA molecules in the cytosol . Currenties/cell . If targ (15min) ,23. HoweHere, in this review, we provide a comprehensive overview of the creative approaches being adopted to generate cell targeting aptamers. We describe recent strategies that lead to the selection of nucleic acid ligands with improved targeting properties and internalization abilities. In addition, we discuss the advantages and limitations of the most promising methods utilized to improve and monitor endosomal escape of therapeutic cargoes. An exciting frontier could be to incorporate these strategies into aptamer selection protocols to identify cell targeting aptamers that ensure improved cytoplasmic accessibility of their cargoes.14 species) [®) has been approved by the Food and Drug Administration in 2004 for the treatment of age-related macular degeneration (AMD) [165) [Aptamers are generated using a selection strategy called SELEX , an iterative process of exponential enrichment and amplification of desired sequences from a large combinatorial library (~10species) ,25. In especies) , to protspecies) , to compspecies) . To dateon (AMD) . PegaptaD) [165) . Other tD) [165) ,31.Many different SELEX protocols have been employed to generate aptamers with effective cell-targeting and internalizing properties, most of which reflect two main approaches: traditional protein-based SELEX for binding to purified membrane proteins and live cell-based SELEX ,33. SomeAs shown in Escherichia coli system, which lacks post-translational modifications. Selected aptamers were not able to bind the same protein when it was expressed in eukaryotic cells, which the authors concluded is due to the lack of one specific post-translation modification , significantly altering the structure of the target protein [Although, most cell targeting aptamers have been generated using protein-based selections, recombinant purified proteins may not fold into the correct 3D structure that is formed under physiologic conditions . In most protein . Live ce protein . TherebyAptamers from cell-SELEX can be used to identify novel tumor-associated biomarkers. For example, aptamers that recognize specific tumor cells can be used as affinity capture reagents to isolate their biomolecular targets ,54, follBcl2 gene in breast cancer cells [An ideal platform for targeted delivery should possess a high rate of endocytosis upon the binding of a highly-expressed receptor. Recent years have seen the emergence of improved and creative SELEX approaches to generate targeting aptamers that rapidly internalize into target cells. These strategies go beyond simply finding aptamers that bind a given cell by depleting sequences that lack internalization ability or that are endocytosed slowly while enriching sequences that rapidly internalize upon binding with a biomarker on the surface of target cells. To accomplish this, the Giangrande group pioneered a modified cell-SELEX methodology that they termed cell-internalization SELEX, in which they introduced a stringent, high-salt wash of target cells after incubation with the RNA library . As shower cells .Later reports highlighted that the stringent, high-salt wash alone was not sufficient to remove the totality of non-internalizing sequences bound to the cell surface. A second strategy, introduced by Levy group, exploits a cocktail of RNases to digest cell surface bound sequences. This group used aggressive nuclease treatment in several selections to generate 2′-fluoropyrimidine-containing RNA aptamers that readily internalize into various target cells ,56. In pA third method to remove cell-surface bound aptamers is to treat the surface of cells with proteinase K after incubation with aptamers. This treatment leads to the digestion of protein domains exposed on the cell surface, including those targeted by the aptamers. Thus, the RNA ligands that are no longer bound to their cognate epitopes are simply washed off. In a recent paper, the de Franciscis group exploited this approach to select cell-internalizing aptamers that bind insulin receptors to human glioma cells (U87MG cells) .+ Burkitt’s lymphoma cell sample. Single-chain DNA sequences that were internalized only into the viable cells were recovered and then amplified for more rounds of selection. The authors demonstrated that a DNA aptamer named C10 [An important consideration of cell-based selection protocols is that cells having a reduced membrane integrity, such as late apoptotic and necrotic cells, can indiscriminately take up nucleic acids, leading to enrichment of aptamers that non-specifically bind these cells. Partitioning methods that fail to eliminate these populations of cells drastically reduce the success rate of a selection process. To address this, the Mayer group developed what they termed fluorescent-activated cell sorting (FACS)-SELEX . In thisamed C10  and a tramed C10  were ablCertain receptors, such as GPCRs, are internalized only upon binding of their ligands in a process called ligand-induced uptake. Interestingly, targeting different epitopes on the same receptor can affect the route of endocytosis of a defined ligand. For example, Austin et al. demonstrated that an anti-EGFR monoclonal antibody and this receptor’s natural ligand, EGF, are taken up by two different mechanisms of endocytosis after binding to distinct epitopes on EGFR . AptamerThe therapeutic success of a given cell-internalizing platform can be strongly dependent on the ability of that platform to mediate efficient cytosolic translocation of cargoes. Thus, the efficiency of endosomal escape during a cycle of uptake needs to be improved significantly relative to that estimated for traditional delivery systems  . Over thA complex system of intracellular vesicles and trafficking is responsible for the fate of an internalized cargo. As shown in To increase the efficiency of endosomal escape, emerging strategies aim to delay endosome maturation of non-recycling vesicles, thus increasing retention of cargoes in these vesicles and avoiding fast degradation in lysosomes . For exaSimilarly, Wang et al. used an inhibitor of the Niemann-Pick type C1 (NPC1) protein, termed NP3.47, to delay maturation of non-recycling endosomes such as late endosomes and lysosomes . NPC1 isThe cellular endosomal trafficking machinery is complex. Understanding this complexity and elucidating the roles of its major components could rationally accelerate the development of more efficient strategies for targeted delivery. For instance, as detailed above, altering the function of ESCRT-I or using NPC1 inhibitors demonstrated that effective and careful manipulation of factors involved in the cellular endosomal trafficking machinery led to more efficient and apparently non-toxic endosomal escape of therapeutic RNA cargoes. The possible implications of these results are significant. In theory, delaying maturation of non-recycling vesicles is a strategy that could be adopted in a selection process. In the current cell-internalization SELEX, upon binding to a cell-surface marker, a fraction of the internalized nucleic acid ligands is recycled back to the plasma membrane, and then endocytosed again for a new cycle of internalization/recycling. On the other hand, other aptamer sequences are expected to be sorted first into late endosomes and finally to lysosomes, where they are efficiently degraded by lysosomal nucleases, showing an overall poor endosomal escape efficiency. However, the use of a modified selection in which one or more endosomal trafficking/sorting regulators (such as ESCRT-I or NPC1) are targeted to delay endosome maturation could enhance the retention of aptamers in non-recycling vesicles. Extending the window of time before their lysosomal degradation could offer the chance of a more favorable endosomal escape of a significant amount of nucleic acid sequences.An alternative to these strategies could be represented by the conjugation of aptamers to pH-dependent polymeric or peptide molecules that become protonated in acidic compartments. Interestingly, when located in mature endosomes (pH ≤ 6), these molecules can trigger an osmotic swelling of the vesicles , which enhances cargo release into the cytosol. An example of this approach was reported by Liu et al. . These aAn example of an aptamer that can, in some extent, gain cytosolic accessibility is the DNA aptamer (AS1411). This molecule is a G-quadruplex forming oligonucleotide that specifically binds nucleolin, a multifunctional nuclear ribonucleoprotein overexpressed in cancer cells where it shuttles among the nucleus, the cytoplasm, and the cell-surface. AS1411 was originally identified during a screen of antisense oligonucleotides for antiproliferative activity toward several human tumor cells . This DNGenerating efficient cytoplasmic delivery platforms requires highly sensitive and robust methods to measure cytosolic penetration and discriminate between internalization and cytosolic accessibility. Improvements upon current methodologies are needed to achieve this distinction and to evaluate aptamer uptake during the SELEX process. Fluorescence microscopy is one of the most used strategies to assess internalization of dye-labeled aptamers in target cells. Standard microscopy techniques enable qualitative analysis of the aptamer-mediated intracellular delivery of molecular payloads, especially when dual-labeled nanostructures are used, bearing one fluorophore on the targeting ligand and a different dye on the cargo ,76. UponIntriguing results were recently provided by the Giangrande group. To visualize internalized aptamers, Hernandez et al. developed a method called antibody amplification microscopy . In brieExploiting a novel and highly sensitive fluorescence microscopy technique, a recent study showed release of siRNA delivered via lipoplexes (i.e. lipofectamine-siRNA complexes) or LNPs from individual endosomes . FluoresAn attractive high-throughput approach used to assess both binding and internalization properties of fluorescently labeled aptamers is flow cytometry. In this case, to discriminate between surface bound aptamers and internalized species, a stringent, high-salt washing or an enzymatic treatment is required (such as using a cocktail of RNase or Proteinase K as described above). Therefore, the efficient removal of these surface-bound species plays a critical role in assessing aptamer endocytosis. Although flow cytometry is a high-throughput approach, it suffers from some of the same limitations as fluorescence microscopy, in that this method is not able to discern between cytoplasmic and endosomal localization of aptamers.The Dehua Pei group offered a potential solution that might overcome the difficulty in monitoring molecules that have escaped from endosomes. In a recent paper, Qian et al. exploited a pH-sensitive fluorophore called naphthofluorescein (NF) coupled to peptide sequences to assess their cytoplasmic translocation . InteresConjugating pH-sensitive fluorophores to known cell-internalizing aptamers could represent a promising and accessible strategy to compare both internalization and cytosolic translocation properties of sequences identified in previous selections. Moreover, a modified cell-SELEX that uses a nucleic acid library linked to a pH-sensitive dye could be used in each round of the protocol to monitor cytosolic entry via flow cytometry. This selection approach could be further modified by adopting subcellular fractionation as a partition method to separate aptamer sequences located in different subcellular compartments (such as cytosol and endocytic vesicles).Arguably, highly efficient methods to evaluate productive cytosolic translocation of aptamers and/or their associated cargoes should directly correlate the escape efficiency with a functional readout, such as a biological output that is only activated by internalized payloads that effectively access the cytosol. However, the biological activities for most therapeutic cargoes can only be monitored over time using a loss-of-function approach, such as target down-regulation by RNAi, and they often require a time scale  that is not compatible with a SELEX protocol due to accumulated digestion of internalized aptamers by lysosomal or cytosolic nucleases. Typically, validation of the biological activity of therapeutic cargoes delivered via aptamers can only be evaluated after some rounds or at the end of the selection. In contrast, a different functional readout that exploits rapid gain-of-function assays, such as monitoring increase of fluorescence or luminescence as a consequence of a biological effect, could be more easily incorporated in a SELEX protocol.Recently, Lonn et al. have developed a fluorescence-based quantitative cytosolic translocation assay to monitor peptide-mediated delivery of cargoes into the cytosol . They reAn intriguing alternative to this approach might exploit a split-aptamer system that generates a specific readout only upon formation of a fully-functional aptamer structure. One component of this split system could be directly connected to a combinatorial library, and the other component could be expressed in the target cells, such that signal is only observed upon successful delivery of the complementary strand. In this regard, our lab recently introduced a tool to monitor RNA-RNA interaction in bacteria. In brief, taking advantages of the light-up properties of fluorogenic RNA aptamers, we engineered a split version of the broccoli RNA aptamer . HybridiOver the last decade, aptamer-mediated targeted delivery of therapeutics in malignant cells has emerged as a promising cancer therapy approach with limited off-target effects. Current efforts are directed towards increasing the performance of these therapeutic aptamer nanostructures to facilitate their clinical translation. Particularly, several strategies aim to enhance the cytosolic accessibility of cargoes upon their aptamer-mediated internalization. Since its discovery, the SELEX process has undergone a continuous evolution. Existing methods have been modified and consequently evolved to meet new biological and technological requirements and ultimately to generate more versatile and potent nucleic acid ligands. In the case of cell targeting aptamers, early SELEX protocols aimed to identify nucleic acid ligands that bind recombinant purified cell-surface proteins. The evolution of this approach led to more complex selection strategies. In the early days, aptamers able to recognize whole tumor cells with high selectivity were generated through an innovative cell-SELEX method. Later, cell-internalization SELEX was introduced to select aptamers that not only bind cell-surface markers in their physiological environment but that also internalize into malignant cells. In each of these SELEX variations, additional properties, such as recognition of whole cells instead of recombinant proteins and rapid endocytosis in cancer cells, were included in the selected ligands. However, clinical translation is still limited by escape into the cytosol. Thereby, to overcome current limitations of the aptamer-mediated targeted delivery of therapeutics, a further evolution of SELEX is needed. Innovative selection protocols that incorporate technologies and tools developed for other biomolecules (such as peptides and proteins) could result in the effective identification of nucleic acid ligands with extended biological functionalities. New generations of aptamers will go beyond binding and internalization, and will be able to perform productive cytosolic translocation of therapeutics, thus accelerating clinical translation of cell targeting aptamer nanostructures.


Osseous dysplasia (OD) is the most common fibro-osseous lesion of the jaw affecting the periapical region. Early stages of OD can resemble periapical radiolucencies, thus mimicking the radiological aspects of an endodontic pathology. Such radiolucent lesions affecting previously decayed or treated teeth are even more complex to interpret.The aim of this paper is to report a case-series of representative clinical situations describing the radiological features and illustrating the diagnostic workup of patients with florid osseous dysplasia (FOD). Emphasis is given to the endodontic implications of such periapical bone disease and the complexity of accurate diagnosis in the context of endodontic retreatment. We then propose a practical radiological-based diagnostic algorithm to assist the clinician in the diagnostic of OD periapical lesions.Periapical lesions may be confused with bone diseases such as osseous dysplasia, especially in the radiolucent initial stage. Knowledge of clinical features associated with a careful reading of cone beam CT images, such as fine opacities within the hypodense periapical lesion, may help determine the right diagnostic. Osseous Dysplasia (OD) is the most common fibro-osseous lesion of the jaws affecting all parts of the maxilla and mandible, including the periapical region. OD lesions are classified as benign odontogenic neoplasms of the jaws with other fibro-osseous lesions in the 2005 World Health Organization classification, updated in 2017 .Different clinico-radiographic presentations are described, depending on whether the lesions are isolated or affect multiple anatomical areas and their specific location: focal, periapical, florid and a specific rare variant, the familial gigantiform cementoma , 5.Stage 1 – osteolytic stage: radiolucent lesions,Stage 2 – mixed stage: radiolucent and radiopaque lesions,Stage 3 – osteogenic stage: radiopaque lesions.Florid osseous dysplasia (FOD) is the most extensive form with bony lesions involving multiple quadrants of the jaws. The etiopathogenesis of FOD is unknown but could derive from the periodontal ligament, the medullar bone or both . Within As most FOD lesions are asymptomatic, diagnosis of such disease is based on incidental radiographic findings, usually during a routine dental exam, especially in middle-aged black female patients.Diagnosis of FOD is based on the typical radiographic appearance assessed with standard two-dimensional projection radiographs . Biopsy of such lesions is not recommended in typical presentations because of the risk of significant post-operative infection, probably secondary to the reduced vascular supply in these lesions .In atypical cases involving the periapical region, three-dimensional radiographic assessment via cone-beam CT (CBCT) may be mandatory, especially in the early stages of disease, to facilitate differential diagnosis with other radiolucent periapical pathoses. Indeed, in the early radiolucent stage, such FOD lesions can mimic apical periodontitis, often leading to unnecessary endodontic treatment of healthy teeth and secondary infection of the underlying hypovascular bone. Diagnosis becomes even more challenging when the periapical radiolucent FOD lesion is associated with an endodontically-treated tooth.The aim of this paper is to present a clinical case-series illustrating the radiological features of FOD involving the periapical region. Knowledge of such radiological features is essential for the differential diagnosis of apical periodontitis, especially on previously-treated teeth. Based on this illustrative case series and the authors’ experience of FOD, a practical radiological-based diagnostic algorithm is proposed to aid in the diagnosis of such perplexing radiographic presentations, a common clinical conundrum in routine endodontic practice.We report three representative clinical cases of incidental discovery of florid osseous dysplasia, illustrating the different radiographic stages of the disease, the associated pathological consequences and finally the complexity of accurate diagnosis of periapical FOD in previously endodontically-treated teeth.A 64 year-old African male patient, without any significant medical history, consulted the dental emergency department of the Bretonneau Hospital in Paris, with a mobile and painful upper left first molar resulting from a periodontal abscess. Aside from moderate to severe generalized alveolar bone loss, preoperative dental panoramic radiography revealed multiple periapical radiolucencies especially within the mandibular body Fig. . ConsideExtraction of the upper left first molar was performed under antibiotic prophylaxis (amoxicillin) and the patient was referred for a full periodontal treatment. At the one-week follow-up, the healing was uneventful.Upon further medical interview, the patient did not report any history of trauma or swelling and all the teeth were vital except the upper left first molar. Thus, endodontic treatment in such clinical context is certainly not justified.A 50 year-old African female patient, without any significant medical history, consulted the dental emergency department of the Bretonneau Hospital in Paris, with chronic pain and tenderness on the lower left second molar. Clinical and radiographic examinations  revealeHigh-resolution CBCT imaging of the lower left second molar also allowed to highlight the previous endodontic treatment failure of the lower left second molar, namely an untreated mesio-lingual canal. Treatment therefore consisted of an endodontic retreatment of the tooth, using mechanical Protaper® for root canal preparation followed by a vertical condensation obturation technique with heated gutta-percha. At the six-months follow-up, the patient was pain free and a ceramo-metallic crown was installed.Although the patient could not recall the history of the lower left second molar and more specifically the reasons for the initial endodontic treatment, it is plausible that the initial consultation was made when the radiographic aspects of the FOD (stage 1 - radiolucent) could mimic periapical pathology, thus potentially leading to an endodontic overtreatment, especially on a tooth that was probably previously decayed . This clinical case thus illustrates the difficulties of accurate diagnostic workup of periapical radiolucencies in previously treated and decayed teeth.A 72 year-old Cameroonian female patient, with a medical history of joint pain, gastric ulcer and hypertension, consulted the dental emergency department of the Bretonneau Hospital in Paris, with multiple tooth pain, more severe in the upper left maxillary region. After clinical and radiographic examinations, a diagnosis of symptomatic apical periodontitis on the upper left second molar was made and immediate root canal disinfection was undertaken. Orthopantomogram Fig.  and CBCTFollowing initial diagnostic workup, the endodontic treatment of the upper left second molar was performed using a conventional technique without need of any specific premedication. The healing was uneventful.In patients with advanced FOD lesions, diagnosis of such a condition is quite straightforward. Nevertheless, a doubt can still remain regarding teeth that present radiological aspects of OD but that also show significant tooth decay. Rigorous clinical exams including pulp sensitivity testing and periodontal probing as well as radiographic exams (2D and 3D imaging studies) are thus mandatory to allow precise diagnostic and suitable treatment of the periapical pathology. For instance, thickening of the Schneiderian membrane adjacent to periapical osteolytic image with micro-perforations of the sinus floor support the endodontic origin of the periapical radiolucency in the upper left second molar compared to the right side Fig. .Diagnostic workup of unexplained radiolucent lesions must follow a strict methodology to evoke or rule out a possible FOD lesion. This methodology is summarized in Fig. Lesions occurring in middle-aged woman, especially those of sub-Saharan origin,Unique or multiple radiolucent, mixed or radiopaque lesions in the tooth bearing area, especially if occurring on vital teeth,Absence of symptomatology (in most of the cases).First, clinical interview should focus on past history of dental pain, trauma, tooth decay or cracks. When faced with a previously endodontically-treated tooth, understanding the indication of such treatment is essential to determine if a possible misdiagnosis could have occurred. Indeed, spontaneous pulp necrosis in the absence of tooth decay, trauma or other local aggressive factors seems quite unlikely, especially in totally asymptomatic teeth. On the other hand, when faced with a periapical radiolucent lesion in an otherwise healthy tooth with a vital pulp, a non-endodontic origin of such lesion must be envisaged. Periapical osseous dysplasia should be considered in the following circumstances:If such arguments are present, a provisional diagnosis of osseous dysplasia can be provided. Management will consist in a close follow-up until these lesions develop the typical radiopaque appearance, confirming the diagnosis. To that extent, CBCT sections are of use in assessing the internal pattern of the lesion, with attention to minute microcalcifications, but also to rule out lesion expansion onto surrounding structures. In doubt, another origin must be evocated; typically odontogenic and non-odontogenic cysts or tumors of the jaws. A biopsy of such lesions is then required and treatment adapted to the underlying etiology.Osseous dysplasia (OD), a rare and benign fibro-osseous lesion of the jaws, can mimic a radiolucent endodontic periapical lesion in the early stage of its maturation. Whereas differentiating such conditions in a vital tooth can be relatively straightforward, the situation becomes more complex when faced with endodontically-treated teeth, especially in cases of presumed inadequate treatments. Correct differential diagnosis is thus of paramount importance for the clinician, in order to avoid unnecessary iatrogenic dental treatments.Florid osseous dysplasia (FOD) is the widespread form of osseous dysplasia; periapical OD is confined to the anterior mandible; focal OD occurs in a single sextant and familial gigantiform cementoma is an uncommon variant encountered preferentially in young patients that can lead to considerable bone expansion , 9.In FOD, the molar and premolar teeth are the most frequently involved. FOD is usually asymptomatic and thus an incidental finding during a routine dental radiographic examination. The rare clinical symptoms observed are pain, swelling and local drainage but these are only encountered in cases of secondary infection, when the calcified masses are exposed in the oral cavity.FOD follows an ethnic distribution, as shown in the systematic review of MacDonald-Jankowski in 2003: 59% of cases arise in African patients, 37% in Asians and 3% in Caucasians. In our patient series, collected within a French Parisian hospital, 92% of cases arose in African patients. Women are more affected than men and the disease occurring mostly around the fourth decade . SeveralBecause of its typical clinico-radiographic presentation, diagnosis of FOD is usually straightforward and does not require any additional investigations. For instance, the discovery of multiple periapical radiolucencies on vital teeth in different quadrants is a sufficient argument to evoke the diagnosis of Florid Osseous Dysplasia. There is no need for a biopsy in typical cases, especially considering the risk of secondary infection of these hypovascularized lesions. Nevertheless, when in doubt, a biopsy can be performed to rule out other fibro-osseous lesions or bone diseases .Differential diagnosis of early stage FOD Fig.  should iAs FOD is a benign usually self-limiting disease, treatment of such condition mostly consists of simple monitoring of the lesions, with an annual long-term clinical and radiographic follow-up , 14, 15.FOD is a condition of significant importance for the endodontist and general dental practitioner. Endodontic lesions and FOD may co-exist Figs.  and 3 anA classic clinical conundrum for the dental clinician is trying to accurately diagnose the radiolucent periapical lesion in an endodontically-treated tooth in a patient presenting with FOD. Several questions arise: Is the periapical lesion a FOD lesion, an apical periodontitis or a surinfection of a FOD lesion? Is the endodontic treatment a consequence of a misdiagnosed FOD lesion or an adequate treatment of a superinfected FOD lesion? Indeed, reports of lesions mimicking periapical endodontic lesions leading to inappropriate endodontic treatments are frequent in the literature .In this paper, we presented several clinical cases of patients presenting typical FOD lesions, in healthy teeth, endodontically-treated teeth or decayed teeth Figs. , 2 and 3Florid Osseous Dysplasia is a rare disease composed of slowly growing, quiescent fibro-osseous lesions presenting as radiolucent and/or radiopaque masses in the jaws and periapical region, which usually do not require any treatment except in case of complications.Whereas typical radiopaque lesions are highly suggestive of the condition, early stage radiolucent periapical lesions can easily be confused with apical periodontitis especially in endodontically-treated teeth. Diagnosis and management of such confusing presentations must thus follow a rigorous methodology to avoid unnecessary iatrogenic dental treatments and possible disease-related complications.


Yet, such strategies continue to fall short of delivering a sufficiently realistic physiological in vitro airway environment that cohesively integrates at true-scale three essential pillars: morphological constraints , physiological conditions , and biological functionality . With the advent of microfluidic lung-on-chips, there have been tremendous efforts towards designing biomimetic airway models of the epithelial barrier, including the ALI, and leveraging such in vitro scaffolds as a gateway for pulmonary disease modelling and drug screening assays. Here, we review in vitro platforms mimicking the pulmonary environment and identify ongoing challenges in reconstituting accurate biological airway barriers that still widely prevent microfluidic systems from delivering mainstream assays for the end-user, as compared to macroscale in vitro cell cultures. We further discuss existing hurdles in scaling up current lung-on-chip designs, from single airway models to more physiologically realistic airway environments that are anticipated to deliver increasingly meaningful whole-organ functions, with an outlook on translational and precision medicine.The entire luminal surface of the lungs is populated with a complex yet confluent, uninterrupted airway epithelium in conjunction with an extracellular liquid lining layer that creates the air-liquid interface (ALI), a critical feature of healthy lungs. Motivated by lung disease modelling, cytotoxicity studies, and drug delivery assessments amongst other,  I.in vitro models that mimic physiologically relevant organ functions, a field increasingly known as body- or organ-on-chips.in vitro microfluidic models of the pulmonary system that are robust and biologically faithful still remains a great technological challenge for advancing both fundamental and translational research in respiratory physiology. Notably, many of the hurdles faced in recreating biomimetic in vitro analogues of the pulmonary environment at the microscale are exemplified when addressing the gas-exchange region of the lungs.In recent years, microfluidics have gained significant momentum in lying the foundations for constructing 2 in an average adult), the lungs' principal function represents not only a cornerstone of human life, guaranteeing foremost oxygen and carbon dioxide exchange, but also a zone susceptible to insult and injuryin vitro microfluidic platforms of the respiratory airways are undergoing rapid development motivated amongst others by lung disease modeling,lung-on-chip models; a term we loosely define here as any microfluidic platform that mimics one or multiple features of the respiratory organ.With the body's largest surface area directly exposed to the external environmentin vitro models of the pulmonary environment, with a special emphasis on microfluidic platforms mimicking respiratory airways and the gas-exchange region. We first address three broadly identified areas  that may be regarded as the underlying pillars for devising biomimetic pulmonary models. We revisit traditional macroscale in vitro models replicating the air-liquid interface (ALI) and the persevering efforts undergone to miniaturize such platforms and integrate true-scale features of the airway barrier within microfluidic systems. Importantly, we emphasize some of the existing challenges in scaling-up current micro-technologies, from single airway models to more realistic airway networks that strive to better capture whole-organ functions. Altogether, existing and future endeavours are anticipated to advance such micro-platforms in becoming more mainstream assays at the hands of the end-user, compared to traditional macroscale in vitro cell cultures.In this article, we review and discuss ongoing developments in the field of II.μm). While the conductive region  does not participate in gas exchange, the pulmonary acinar region is designed for efficient oxygen and carbon-dioxide transfer into blood and accounts for >90% of total lung volume.3 with ∼10 000 alveoli arranged across ∼6 to 12 generations.−6μm3 with a characteristic length scale of ∼100–200 μm.The lungs' vast airspace is arranged within a bifurcating tree structure spanning ∼20 airway generations, where length scales cover several orders of magnitude, i.e., from the trachea ∼1 cm) down to the alveoli . While the shapes of alveoli are intrinsically heterogeneous, resembling spheroids, ellipsoids, and cylindroids,More recently, models of alveolated channelsin vitro airway microsystems, advances in microfabrication techniques have helped deliver 3D micro-engineered environments with the advent of 3D printing, additive manufacturing, and various stamping techniques.organ-on-chips.in vitro microcirculatory architectures have been motivated by tissue engineering applications as well as vasculo- and angiogenesis studies.in vivo requirements of gas exchange at the whole-organ level. Though some of the underlying 3D branching patterns and characteristic length scales  of the microvasculature are somewhat analogous to pulmonary airways, it is still unclear how closely the complexities of the lung morphology and the inherent three-dimensionality of the acinar airspace may be recreated [Fig. in vitro analogues of the airway environment, when replicating anatomy with high-fidelity is indeed critical.Beyond the aforementioned ted Fig. . NeverthIII.Following birth, the lungs rapidly adapt into an air-filled environment. This configuration distinguishes respiratory airflows from traditional microfluidic setups driven by liquid-based applications.The first specificity revolves around the organ's anatomy which departs from cylindrical airways due to the structure of the acinar ducts Sec.  and the μPIV), measurements in microfluidic alveolated tree modelsIn considering microfluidic designs, respiratory flows obey the laws of mass conservation such that flow magnitudes in the acinar airways rapidly decrease as air is distributed at each bifurcation into deeper generations. In turn, the coupling between oscillatory shear flows in acinar ducts and the alveolar cavity openings leads to distinct flow configurations that are sensitive to the location of an alveolus along the acinar tree.in vitro studies of aerosol deposition patterns under physiological breathing conditions.in vitro screening assays of inhaled airborne aerosols in true-scale acinar structures; an encouraging stepping-stone in eventually offering pre-clinical in vitro platforms that would circumvent, or at the very least reduce, animal studies.Despite limitations in the anatomical realism of recent microfluidic platforms see Sec. , experimin vitro environments to investigate the dynamics of plug propagation in single airwaysIn some instances, recreating airflows with microfluidics is less pertinent when addressing pulmonary diseasesMore recently, experiments have also demonstrated that high WSS can disorganize the F-actin cytoskeleton of surfactant-secreting alveolar epithelial cells and thereby limit fusion events of lamellar bodies with the cell membrane prior to exocytosis.ex utero, the developing pulmonary acinar environment during life in utero is fluid-filled and subject to spontaneous expansion and contraction motions known as fetal breathing movements.in vitro attempt to uncover respiratory flow physiology in utero following past efforts limited to numerical simulations.In contrast to life IV.μm), 75% of all lung cells (by volume and weight) are contained in the gas exchange regionThe continuous gaseous ventilation occurring throughout life facilitates the exchange of oxygen and carbon dioxide across a delicate architecture known as the alveolar-capillary barrier (ACB). The ACB separates air from blood and may be summarized as constructed of three minimal tissue layersThe lungs' epithelial cells form an uninterrupted, mosaic-like carpet that continuously lines the lumen of the entire respiratory tract including importantly the gas-exchange region, where the vast majority of lung surface area resides. Although the cellular makeup of the airway epithelium evolves along the respiratory tree from pseudostratified in the upper airways  to squamous cells in the deep airway regions, this mosaic of cells establishes consistently a tight monolayer at luminal interface under healthy conditions.ex utero, surface tension forces at the interface between liquid and gas become dominant and resist distention of the parenchymal walls during breathing. Pulmonary surfactant is thus necessary to mitigate the importance of surface tension along the ALI, such that insufficient concentrations of the surface-active material may render the simple act of breathing excruciatingly difficult. Instability within the parenchyma can then occur, causing alveoli to collapse throughout cycles of inhalation and exhalation. Breathing may be severely compromised, jeopardizing successful oxygen uptake; a condition that arises for example with acute (ARDS) or infant (IRDS) respiratory distress syndrome.An additional yet integral component of the defence barrier lies in pulmonary surfactant itself.μm or so in the trachea down to 2.5 μm in the bronchi, reaching finally 0.1–0.2 μm in the peripheral airwaysPulmonary surfactant is thus an essential component of the thin liquid lining layer. The latter constitutes an extracellular multiphase film that consists of a thin hypophase covered by the critical surface-active material.Alongside the physical barrier provided by the alveolar epithelium, components of the immune system are imperative to protect the fragile airway complex from external threats. To this end, distal airways accommodate populations of alveolar macrophages and dendritic cells above and beneath the epithelium,V.ex vivo human lungs are recognized to mimic most closely the appropriate physiological conditions of human airways in vivo.ex vivo organs are acknowledged to lack the fundamental features required for fast and low cost platforms when addressing, e.g., cytotoxicity and drug screening.ex vivo cultures are frequently regarded as offering insufficient advantages over conventional macroscopic cell culture approaches.The most prevalent approachesIn vitro cell cultures of the pulmonary environment may be distinguished according to the cell origin ; here, the reader is referred to extensive reviews on the topicin vivo environment. However, primary cells are rather heterogeneous with limited lifespan; each cell isolation from a subject is unique with high variability between donors or experiments, thus making reproducibility difficult to achieve. In contrast, cell lines are known to be more stable and homogeneous. Yet, their cancerous origin leads to phenotypic differences that render them less ideal for replicating in vivo conditions faithfully. We note that most recently, a new cell line, i.e., human Alveolar Epithelial Lentivirus immortalized2). While further detailed characterization is still needed, hAELVis are, however, the first cell line demonstrating barrier properties of the alveolar epithelium.Given the aforementioned drawbacks, cell culture models of the airway epithelium have hence been widely used: they enable as a first approximation close examination of the cellular, molecular, and biochemical features of pulmonary diseasesWhether operating with primary cells of alternatively cell lines, the most prevalent method for cell culturing consists of a macroscopic 2D sheet made of epithelial or “epithelial like” cell types and grown on specialized dishes Fig. . In genein vitro. Such setups mimic more faithfully the intrinsic epithelial structure where the topical side of the membrane is exposed to air, granting the cells its polarized nature and recreating the essentials of the ALI. Alternatively, other specialized culturing methods have strived to innovatively integrate dynamics of the liquid lining during breathing at the ALI. This includes, for example, a technique based on captive bubble surfactometry;in vivo environment, air-exposed culture conditions ultimately change the resulting cellular phenotype. While cell cultures grown in petri dishes have been traditionally conducted under immersed conditions, those at an ALI are known to increase monolayer integrityIn this context, cell cultures grown on permeable microporous membranes Fig.  represenin vivo. For example, immune-components have been integrated within the airway epithelium, including alveolar macrophages and dendritic cells.in vitro ACB composed of endothelial cells, a basement membrane, and epithelial cells.Similar to the classic cell cultures in dishes, integrating co-cultures of different cell types on permeable membranes Fig.  has incrVI.in vitro. In this context, the bulk of existing efforts still fall short in mimicking sufficiently adequately the airway milieu, both in a physiologically faithful manner and at true physiological scale. Notably, macroscale techniques do not capture the intricate 3D airway anatomy, as described earlier in Sec. in vitro cell cultures are still inherently confined to static culture conditions and largely omit respiratory airflows and the dynamic nature of the airway epithelium during breathing, as detailed earlier in Sec. The biological intricacies of the pulmonary environment represent an undeniable challenge to accurately recreate lung-on-chips, several limitations surrounding traditional macroscopic culture methods  have been tackled if not addressed with increasing success. As a brief remark, we comment here that lung-on-chip micro-devices have been instrumental in helping the general field of organ-on-chips evolve.lung-on-chip platforms first appeared during the development of in vitro models of liquid plug propagation in an effort to explore the effect of such plugs on the underlying epithelium (see Sec. in vitro models cultured directly in airway channels (rather than plates or inserts) and thereby replicate more faithfully physiological fluid shear forces  and solid mechanical forces  anticipated to act on the underlying lung epithelium. Together, this helped consolidate a departure from more traditional macroscopic approaches.With the advent of microfluidics and the birth of see Sec. . These met al.,organ-on-chips,Undoubtedly, the work of Huh airway-on-a-chip model for COPD studies.et al.airway-on-a-chip considers instead a bronchial epithelium representative of small airways susceptible to COPD. Culture of primary cells in such device produced ciliated epithelial cells as well as mucus-secreting goblet cells, club cells, and basal cells in proportions similar to those found in normal human lungs. Using COPD patient cells, important features of the disease were recapitulated including selective cytokine secretion, neutrophil recruitment, and exacerbation triggered by an infection.Other notable microfluidic designs under air-exposed ALI conditions have followed, with a keen eye on translational research and therapeutic applications. Such efforts have principally focused on airway disease models such as a micro-engineered ACB platform for pulmonary edema,et al.in vitro. Here, the airway platform  was designed to mimic mid- to small-bronchi . In order to identify the effects of smoking on healthy and COPD donors, the authors coupled the organ-on-chip device, seeded with a human bronchiole epithelium from either of the two groups, to a smoke generator and a micro-respirator to mimic human breathing behaviour. Briefly, the micro-respirator is programmed to breathe cyclically microliter volumes of air through the device's upper epithelium-lined channel and a smoke machine regulates smoking parameters . As the micro-respiratory and smoke generator work synchronously, whole cigarette smoke flows horizontally across the surface of the differentiated epithelium only during the inhalation phase of the respiration cycle and then flows back out during the exhalation phase. Upon inspection of the gene expression profiles of COPD samples exposed to smoke, the authors identified 276 genes differentially expressed compared to control samples without smoke, of which 147 genes were previously associated with COPD pathogenesis. Using real-time imaging, ciliary beating was monitored and changes as a result of smoke exposure were assessed. Overall, this work exemplifies important advantages of organ-on-chip platforms over more traditional alternatives devoid of real-time monitoring. Such efforts are also anticipated to help pave the way in discovering new therapeutic drug targets and more customized treatments.Most recently, Benam lung-on-chip models has inspired the design and development of other organ-specific microfluidic platforms such as the gut-on-chip;In parallel to these studies, innovations in microfabrication techniques themselves have led to reach out beyond the use of simple (rectangular) channels. For instance, a recent micro-device recreating the dynamic nature of an ALI has integrated a bio-inspired diaphragm-like actuation mechanism to mimic 3D cyclic strains that act upon epithelial layers.in vitro with the support of microfluidic platforms , whereas microporous membranes are typically on the order of 10 μm thick and thus cannot mimic the same translocation and diffusive properties. In turn, such artificial membranes are still a far reach from replicating the complex tissue barrier  and its associated in vivo transport characteristics . Nevertheless, the use of artificial porous membrane structures has been essential in advancing our general understanding of pulmonary physiology, notably in the areas of ALI barrier characteristics, drug translocation, and airway injury amongst other  models. Integrating the above is anticipated to open the door for advanced precision medicine: patients' cells can be collected from a biopsy, expanded, and grown inside devices allowing advanced diagnostic capabilities in addition to monitoring patient's cell response to different drugs for optimizing match.With an outlook on prospective directions, the schematic of Fig. lung-on-chips and other similar micro-devices will live up to the promise of becoming an accepted gold standard for various testing and screening assays, and thus fulfilling many if not most of the discussed aims herein. Even if such realizations are not necessarily met, the rapid progress witnessed in a single decade has already transformed our perception of in vitro macroscale approaches and underlined our criticism thereof, with an increasing urge to move beyond such traditional techniques.It remains still largely speculative whether


Preclinical studies suggest that fasting prior to chemotherapy may be an effective strategy to protect patients against the adverse effects of chemo-toxicity. Fasting may also sensitize cancer cells to chemotherapy. It is further suggested that fasting may similarly augment the efficacy of oncolytic viral therapy. The primary mechanism mediating these beneficial effects is thought to relate to the fact that fasting results in a decrease of circulating growth factors. In turn, such fasting cues would prompt normal cells to redirect energy toward cell maintenance and repair processes, rather than growth and proliferation. However, fasting is also known to upregulate autophagy, an evolutionarily conserved catabolic process that is upregulated in response to various cell stressors. Here, we review a number of mechanisms by which fasting-induced autophagy may have an impact on both chemo-tolerance and chemo-sensitization. First, fasting may exert a protective effect by mobilizing autophagic components prior to chemo-induction. In turn, the autophagic apparatus can be repurposed for removing cellular components damaged by chemotherapy. Autophagy also plays a key role in epitope expression as well as in modulating inflammation. Chemo-sensitization resulting from fasting may in fact be an effect of enhanced immune surveillance as a result of better autophagy-dependent epitope processing. Finally, autophagy is involved in host defense against viruses, and aspects of the autophagic process are also often targets for viral subversion. Consequently, altering autophagic flux by fasting may alter viral infectivity. These observations suggest that fasting-induced autophagy may have an impact on therapeutic efficacy in various oncological contexts. A clinical trial testing the feasibility of incorporating short-term fasting (72-h fasting around the time when the therapeutic agent is administered) into platinum-based chemotherapy  sources and MHC I from endogenous sources has been supplemented by findings indicating that phagocytosed particles could be expressed on MHC I , and conn MHC II . Short-tn MHC II . Similarn MHC II . SimilarIn fact, there is evidence that autophagy may play an important role in epitope presentation within an oncological context. As an example, autophagy, induced by either starvation or pharmacologically in antigen-donating cancer cells, enhances the cross-presentation of these antigens by dendritic cells . Also, rHRAS-transformed mouse kidney endothelium results in an altered proteome with a more pro-inflammatory profile, “priming” cells for an innate immune response  . In normHowever, fasting-induced autophagy may also have an impact on oncolytic viral efficacy, since autophagy is also implicated in the host’s immune response toward viral infections , 42. As Furthermore, viral subversion of autophagic processes is known to depend on the tissue type being infected . This woAutophagy as a catabolic process plays a key immunological role in enhancing cell-autonomous defenses, and, as such, pathogens such as viruses have evolved various strategies both to inhibit and to subvert this process , 48. Convia diverse mechanisms involving both an upregulation of autophagy and a decrease in circulating growth factors   . Even inor (FXR) . In addior (FXR) . As an eor (FXR) . It remaIn this article, we have highlighted the potential protective role of fasting-induced autophagy as a mechanism by which cells cope with the adverse effects of chemotherapy. In addition, we have pointed out that since autophagy is also involved in immune responses it is possible that an upregulation of autophagy may have an impact on immune function and, in particular, help or hinder the reinstatement of immune surveillance against transformed cells. Furthermore, fasting results in a range of physiological adaptations which may have an impact on the efficacy of chemotherapeutic agents. It is likely that the protective effects of fasting may be mediated by these processes operating in concert. A clear understanding of the mechanism invoked by fasting may lead to the development of pharmacological strategies that replicate these same processes. Such therapies may be of particular importance in patients who are already undernourished or where nutrient deprivation is contraindicated.GN, SH, and A-ME wrote the manuscript. All the authors read and approved of the final version of 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.


Age-related decline in immunity is characterized by stem cell exhaustion, telomere shortening, and disruption of cell-to-cell communication, leading to increased patient risk of disease. Recent data have demonstrated that chronic inflammation exerts a strong influence on immune aging and is closely correlated with telomere length in a range of major pathologies. The current review discusses the impact of inflammation on immune aging, the likely molecular mediators of this process, and the various disease states that have been linked with immunosenescence. Emerging findings implicate NF-κB, the major driver of inflammatory signaling, in several processes that regulate telomere maintenance and/or telomerase activity. While prolonged triggering of pattern recognition receptors is now known to promote immunosenescence, it remains unclear how this process is linked with the telomere complex or telomerase activity. Indeed, enzymatic control of telomere length has been studied for many decades, but alternative roles of telomerase and potential influences on inflammatory responses are only now beginning to emerge. Crosstalk between these pathways may prove to be a key molecular mechanism of immunosenescence. Understanding how components of immune aging interact and modify host protection against pathogens and tumors will be essential for the design of new vaccines and therapies for a wide range of clinical scenarios. Aging is a complex process that involves a gradual decline in critical cellular processes, signaling pathways, and regulatory mechanisms, leading to eventual disruption of tissue homeostasis . AccumulTo maintain telomere length and protect chromosomes against damage, cell types with high proliferative capacity such as hematopoietic progenitors , 4 and evia PRR signaling and have been identified as displaying significant impairment in various senescence-related disorders.Effective host immunity is essential for the maintenance of tissue homeostasis and health, but both innate and adaptive responses are subject to natural age-related functional decline termed “immunosenescence” . Key feaMyeloid cell-derived biomarkers of immunosenescence reportedly include increased production of the cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), which correlate with elevated serum levels of C-reactive protein to predict increased patient frailty and higher overall rates of mortality . IL-6 anvice versa, in particular, via interactions with the enzyme telomerase. Indeed, emerging data indicate that telomerase likely exerts a range of additional functions that could significantly impact on hematopoiesis and mitochondrial ROS production during age-related immune decline.Although immunosenescence occurs naturally as the human body ages, early activation of senescence pathways has been observed in a wide range of human disorders , 21. ImmImmunosenescence is strongly driven by persistent infections and/or tissue inflammation , 31, leaNatural age-associated changes in innate immune function have already been described in adults older than 40 years , whereasDirect roles for HSPCs in inflammation have only recently been described by Griseri et al. who identified progenitor cell infiltration of the gut mucosa in experimental colitis . Most stFormal demonstration of a direct influence of PRRs/inflammation on telomere length/telomerase activity in host leukocytes and stem cells is currently lacking, but experimental data consistent with this concept are continuing to accumulate. Indeed, age-related DNA damage and shortened telomeres have been observed in murine HSCs , and senvia a range of different mechanisms (Mitochondrial ROS production is a key antimicrobial function of specialized immune cells including macrophages, dendritic cells, and neutrophils. Accordingly, age-related impairment of mitochondrial function can significantly impair host immune responses . Increaschanisms . ROS accchanisms . While nchanisms , during chanisms . Furtherchanisms  and to dReactive oxygen species have also been strongly implicated in pathological changes in blood vessel structure and function that characterize age-related vascular diseases such as atherosclerosis . In thisTelomere shortening during cell division is a critical process in progression to senescence , and telEven in the absence of NF-κB signaling, prolonged low-grade inflammation is sufficient to induce telomere dysfunction, likely involving accumulation of mitochondrial ROS . TERT caThe central role of NF-κB in regulating chronic, low-grade inflammation has long been established, but only recently have experimental data begun to indicate a possible role for NF-κB in control of telomerase expression or activity in the context of senescence-associated disorders. For example, Gizard et al. showed that inflammation-induced NF-κB activation regulates TERT expression in macrophages and that human atherosclerotic lesions are characterized by high expression of TERT . Diseasevia PRRs should lead to significant progress in developing methods of combating immunosenescence in a range of human pathologies. Together, these data underscore the importance of inflammaging as a major driver of senescence progression and reinforce the concept that an array of different pathways likely interact to determine the rate of this process (graphically represented in Figure Immunosenescence is the culmination of a complex network of molecular processes. Despite intensive study over the last decade and improved understanding of the features of immunological aging, the molecular mediators of these events and the extent to which they interact remain poorly defined. Indeed, while the strong association of telomere length with cellular senescence has been known for decades, the direct/indirect relationship between telomerase activity and PRR signaling is only now coming to light. While the molecular basis of PRR interactions with telomerase activity has not yet been determined, better definition of this crosstalk will be essential to understanding the influence of PRRs and “inflammaging” on human hematopoiesis and tissue regeneration. The recently identified ability of stem cells to directly detect DAMPs and PAMPs SSJ prepared the figure and wrote the manuscript, KB wrote the manuscript and prepared the table, TK and ZK advised clinical research interpretations, and JF conceptualized, wrote, and critically reviewed 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.


To identify lipid markers of blastocyst implantation and ongoing pregnancy byday three culture medium mass spectrometry (MS) fingerprinting.For this study, 33 culture media samples were harvested on day three, from 22patients undergoing day five embryo transfers. All embryos achieved theblastocyst stage and were split into groups based on their implantation. Thepositive implantation cycles resulted in successful ongoing pregnancies. Thelipid extraction was performed by the Bligh-Dyer protocol and mass spectrawere obtained with a direct infusion into a Q-Tof mass spectrometer. Thedata obtained was analyzed by Principal Component Analysis (PCA) and PartialLeast Square Discrimination Analysis (PLS-DA). The statistical analysis wasperformed using the Metabo-Analyst 2.0.The variable importance in the projection (VIP) plot of the PLS-DA provided alist of four ions, in the positive mode, with an area under the curve (AUC)of 73.5%; and eight ions, in the negative mode, with and AUC of 72.0%. Forboth positive and negative modes, possible biomarkers for the negativeimplantation were identified by the lipidmaps: phosphoethanolamine,dicarboxylic acids, glycerophosphoglycerol, glycerophosphocholine,glicerophosphoinositol, phosphoethanolamine and unsaturated fat acids. Theother ions were not identified. These lipids are involved in the GPI anchorbiosynthesis and synthesis of lycerophospholipids and phosphateinositol.MS fingerprinting is useful to identify blastocysts that fail to implant, andtherefore this technique could be incorporated into the laboratory routine,adjunct to morphology evaluation to identify embryos that should not betransferred. In vitro fertilization (IVF) success rates have been remarkably improved since thefirst successful birth in 1978 . HoweverThis low efficiency contributes to the practice of multiple embryo transfer, whichfrequently leads to multiple pregnancies . In vitrIndeed, the need to decrease assisted reproduction-induced iatrogenic multiplepregnancies has become a health, economic, and legal issue in several countries. The novTherefore, the goal for the present study is to identify lipid markers of blastocystimplantation and ongoing pregnancy by day three culture medium MSfingerprinting.For this study, 33 culture media samples were harvested on day three from 22patients undergoing day five embryo transfers. All embryos achieved theblastocyst stage and were split into groups based on their implantation. Embryosecretomes were analyzed by MS.Patients in the positive implantation group presented 100% implantation andpositive implantation cycles resulted in successful ongoing pregnancies andseven successful live births, so far.Patients signed an informed consent form, in which they agreed to share theoutcomes of their cycles for research purposes. The local institutional reviewboard approved the study (CEP: 1095/2015).Controlled ovarian stimulation was achieved by using recombinant FSH , at a daily dose, starting on day three of thecycle. Pituitary blockage was performed by using a GnRH antagonist , starting when at least one follicle ≥14 mmwas visualized.Follicular growth was monitored using transvaginal ultrasound examinationstarting on day four of the gonadotropin administration. When adequatefollicular growth and serum 17β estradiol levels were seen, recombinanthCG  was administered to trigger the finalfollicular maturation. The oocytes were collected 35 hours after hCGadministration through transvaginal ultrasound ovum pick-up.Retrieved oocytes were maintained in culture medium for 5 hours. Surroundingcumulus cells were removed and oocytes were checked for oocyte maturation, andthose which had released the first polar body (metaphase II oocytes - MII) wereconsidered mature and used for Intracytoplasmic sperm injection (ICSI), whichwas performed using the technique described by Approximately 18h after ICSI, fertilization was confirmed by the presence of twopronuclei and the extrusion of the second polar body. Subsequently, embryos weretransferred to new drops of culture medium to be individually cultured for 48hours. On day three, the culture media was refreshed and spent culture media wascollected and stored at -20ºc. The embryos were transferred to another dish andcultured until day five when embryo transfer was performed.Subsequent to the confirmation of implantation, the culture medium samples weredivided according to their implantation outcomes.The lipids from culture medium were individually extracted using the Bligh andDyer method , driedaMass spectra were obtained with a direct infusion of both the negative andpositive ion modes into a Q-Tof mass spectrometer  equipped with an automated injector.The data obtained was analyzed by Principal Component Analysis (PCA) and PartialLeast Square Discrimination Analysis (PLS-DA), combined with variable influencein the projection (VIP) scores, to identify potential biomarkers of blastocystimplantation and ongoing pregnancy. Statistical analysis was performed using theMetabo-Analyst 3.0.The PCA analysis was performed to identify chemical differences between the Negativeand Positive Implantation groups. Moreover, we could note a better clustering in the negative implantation group forboth, positive and negative modes.The PLS-DA was applied to evaluate differences in the lipidomic profile between thegroups and to identify possible biomarkers of blastocyst implantation. Graphics onpositive  and negaThe variable importance in the projection (VIP) plot of the PLS-DA provided a list offour ions, in the positive mode ,with anA key step in assisted reproduction is to identify the embryo(s) that are most likelyto result in a pregnancy. In the present study, possible lipid biomarkers forblastocyst implantation fail were identified.Non-invasive determinants of embryo viability are limited, and therefore currentembryo assessment strategies involve embryo morphology, which is the easiest way topredict viability. However, the majority of studies suggest that the morphology ofembryos with high-quality morphological appearance is insufficient for predicting asuccessful implantation . FurtherWithin this context, many metabolic parameters for developing embryos have beenstudied using a variety of non-invasive methods . These sMore recently, studies have suggested that embryos with positive and negativeimplantation outcome alter their environment differently, which is reflected in thesurrounding metabolites (In a previous study of our group, MS associated with the PLS-DA mode has beensuccessfully employed for embryo viability prediction (For the present study, we specifically assessed the lipidomic profile. Althoughhaving been an intensive area of research already in the 1960s, lipid research hasrecently gained prominence with the emergence of lipidomics .The present study was able to identify possible lipid involvement in cellularmetabolism in culture media samples of non-implanted embryos. It could be arguedthat embryos who fail to implant may have used the cellular machinery to preventapoptosis to exhaustion.So far, the diagnostic power of such patterns is not completely conclusive sinceembryo implantation into the endometrium does not depend exclusively on properembryo development, it also depends on other critical events, which include theacquisition of a receptive endometrium, and the appropriate dialogue betweenmaternal and embryonic tissues (Nevertheless, this technology seems to provide a fast, reliable, and noninvasiveprediction tool to help the selection of the best embryo to be transferred, andshould be used as an adjunct to morphological evaluation, thus minimizing the risksof the undesirable outcome of multiple pregnancies.An important limitation of this study is that MS/MS to confirm the identification ofthe lipids was not performed, but this study is to be continued to confirm ourfindings.In conclusion, our evidence suggests that MS fingerprinting is a useful predictivetool for blastocysts that fail to implant, and therefore this technique could beincorporated in the laboratory routine, adjunct to morphology evaluation to identifyembryos that should not be transferred.


Background: Migraine is associated with increased cardiovascular risk and vascular dysfunction. Since aerobic exercise can reduce cardiovascular risk, the present randomized controlled trail aimed at investigating the effects of high-intensity interval training (HIT) vs. moderate continuous exercise training (MCT) on arterial stiffness in migraine patients.Methods: Forty-eight episodic migraineurs were initially enrolled in the study. 37 patients  completed the intervention. Central blood pressure, pulse wave reflection, and aortic pulse wave velocity (PWV) were obtained by an oscillometric monitor. Incremental treadmill exercise testing yielded maximal and submaximal fitness parameters. Participants were randomly assigned to either HIT, MCT, or a control group (CON). The intervention groups trained twice a week over a 12-week intervention period.Results: After adjustment for between-group baseline differences, a moderate meaningful overall reduction of the augmentation index at 75 min−1 heart rate (AIx@75) was observed . HIT induced a relevant reduction in central systolic blood pressure  with a 59% possibly beneficial effect compared to CON, while MCT showed larger effects in lowering central diastolic blood pressure , presenting 60% possibly beneficial effects compared to CON. Central aortic PWV showed no changes in any of the three groups. Migraine days were reduced more successfully by HIT than MCT .Conclusion: HIT but not MCT reduces AIx@75 as a measure of pulse wave reflection and indirect marker of systemic arterial stiffness. Both exercise modalities beneficially affect central blood pressure. HIT proved to be an effective complementary treatment option to reduce vascular dysfunction and blood pressure in migraineurs. Migraine is considered a debilitating neurological disease which recurs in the form of severe headache attacks accompanied with nausea, vomiting, phonophobia, and photophobia , moderate continuous aerobic training group (MCT), or control group (CON). Baseline parameters are depicted in Table The present study was designed as a three-armed randomized controlled trial with a primary endpoint set on the effect of exercise training on pulse wave reflection in patients with migraine. In order to determine a clinical baseline for disease severity, patients underwent a 4-week run-in period prior to the start of the intervention period. After the run-in period and pre-testing measurements, 48 patients were randomly assigned  and small effects for MCT . HIT showed a 91% likely beneficial effect while MCT revealed a 60% possibly beneficial effect over CON. Pairwise comparison of IAT revealed moderate effects in HIT .ANCOVA revealed a moderate but relevant between group effect for VOThe aim of the present randomized controlled trial was to investigate whether different aerobic exercise intensity modalities yield differential effects on central systolic and diastolic blood pressure, pulse wave reflection, and PWV in episodic migraineurs. To the best of our knowledge, no study to date has compared the effects of different exercise modalities and intensities on arterial stiffness and migraine. Our results show that HIT but not MCT was able to lower AIx@75 while both exercise programs were able to reduce migraine days and improve physical fitness. We could further elucidate that HIT is superior to MCT in improving AIx as an indirect marker of systemic arterial stiffness, which indicates cardiovascular risk reduction in migraineurs. The exercise-induced adaptations of arterial stiffness may be attributed in large part to an increased availability of nitric oxide (NO) and a consecutive improvement of endothelial function. Increases in shear stress during exercise induce NO production of the endothelium, which results in relaxation of vascular smooth muscle cells, arterial vasodilation, and a drop in vascular resistance (Alsop and Hauton, Interestingly, PWV showed no changes in any of the three groups even though HIT reduced central systolic blood pressure while MCT reduced central diastolic blood pressure. These results are in line with the few studies which have examined the effects of exercise on PWV (Heffernan et al., Migraine attacks are accompanied by repeated spells of vascular inflammation and repeated attacks of migraine have been suggested to induce inflammatory changes of cranial arteries (Bolay et al., p-values to estimate relevant between-group effects, as p-values do not sufficiently allow for continuous estimation of relevant interventional effect sizes (Pocock and Stone, The study comprises some limitations that need to be mentioned. The sample size of the pilot study might be considered low. In our group analysis, we refrained from interpreting our data on the basis of mere conventional Regular aerobic exercise has beneficial effects on blood pressure, vascular function, and cardiovascular risk. Since HIT was more effective in reducing systemic arterial stiffness and migraine days than MCT, we postulate that the exercise effects in migraine patients depend on intensity and modality of the exercise therapy. HIT should be considered as a complementary treatment strategy for migraine treatment and prevention. It is all the more important since drug treatment of migraine is oftentimes addressed with precautions in the presence of cardiovascular comorbidities. Exercise therapy seems to have the potential to play a crucial role in future preventive strategies to combat migraine complaints and development of associated CVD.Study design: HH, TS, and LD; data collection: AM, SM, AR, AP, and CK; statistical analysis: AM and LD; manuscript preparation: HH, AM, TS, and LD; manuscript revision: OF, AS-T, and LZ.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.