Advances in DNA- and RNA-based technologies have made gene therapy suitable for many lung diseases, especially those that are hereditary. The main objective of gene therapy is to deliver an adequate amount of gene construct to the intended target cell, achieve stable transduction in target cells, and to produce a clinically therapeutic effect. This review focuses on the cellular organization in the normal lung and how gene therapy targets the specific cell types that are affected by pulmonary disorders caused by genetic mutations. Furthermore, it examines the pulmonary barriers that can compromise the absorption and transduction of viral vectors and genetic agents by the lung. Finally, it discusses the advantages and limitations of direct intra-tracheal gene delivery with different viral vectors in small and large animal models and in clinical trials. There are no current conventional pharmacological treatments that can act effectively on intracellular targets. Instead, intracellular targets must be regulated by genes. Effective transcription and translation of DNA into a protein product can change cell behavior to achieve a successful therapeutic outcome. Therefore, gene-based therapy holds promise as a new, effective treatment. The main objective of gene therapy is to deliver an adequate amount of gene construct to the intended target cell, achieve a stable transduction of a target cells, and to produce a clinically therapeutic effect.Several pulmonary diseases have dysregulated factors that can be therapeutic targets. Thus, gene therapy approaches that can be well suited for hereditary lung diseases with monogenetic mutations are especially attractive given that the correction of autosomal recessive or dominant disorders could be resolved through the exogenous delivery of the wild-type gene. Furthermore, it is a viable approach to modulate disease-causing factors or to help upregulate the factors downregulated in the disease state.Over the last few decades, much progress has been made in the gene delivery approach to design vectors that achieve specific cell transfer, minimize innate and adaptive immune responses, create new candidates for therapeutic genes, and determine the best route of administration to introduce these genes. To achieve significant pulmonary gene transfection, various vectors have been used so far. These vectors can be classified into three main categories: non-viral, recombinant viral, and engineered hybrid. Compared to other vectors, viruses have an evolutionary advantage in their interactions with the cellular receptors and intracellular trafficking to the nucleus. In this review, we will focus on using only viral vectors for intratracheal gene transfer. The ideal viral vector should be trophic to the lung tissue, result in long-term expression, minimize the risk of innate and adaptive immune response, and possess an enough coding capacity to incorporate the gene and enhancing promoters . Today, A substantial limitation of AAV vectors is their small packaging capacity that is generally considered to be 4100–4900 bp. This limitation makes vector design for some of the lung diseases challenging. For example, cystic fibrosis trans-membrane conductance regulator (CFTR) gene has a cDNA of 4450 bp, therefore, the application of AAV viral vector in CFTR is limited. To address this problem, strategies based on trans-splicing and homologous recombination were developed . Some stThe development of gene-based therapeutics for pulmonary application requires a detailed knowledge of the lung’s cell structure. The lungs are paired organs of the respiratory system and can be divided into two major components: the conducting airways, consisting of the trachea, bronchi and bronchioles to transport air from upper respiratory tract to the peripheral lung, and the parenchyma, including alveoli, where gas exchange takes place. The upper respiratory system includes the nasal cavity, paranasal sinuses, and pharynx, while the larynx, trachea, bronchi and bronchioles comprise the lower respiratory tract. The nose and paranasal sinuses act as a first line of defense against inhaled particles through the filtration function and irregular structure of the nasal bones and mucosa. The trachea divides into the right and left main stem bronchi. Each of these give rise to lobes and segmental bronchi. The bronchi branch progressively branches into the bronchioles. The bronchioles terminate in the acinus, which is with capillary net involved in gas exchange and contains alveolar ducts, alveolar sacs, and the alveoli. There are about 40 types of cells in the respiratory tract . The epiThe other main cell functional compartment of the lung is comprised of pulmonary vascular endothelial cells, which form micro capillary bed that surround the alveoli where gas-exchange occurs. The lung also contains supporting cells such as: (i) smooth muscle cells lining both airways and vascular structures; (ii) supporting fibroblasts in the alveoli units; (iii) hematopoietic and immune cells; and (iv) neuronal cells of the pulmonary nerves. Many of these supporting cells make up part of the interstitial space, which is located between the alveolar epithelium and capillary endothelium membrane. The interstitium is formed by an extracellular matrix and contains the cells listed previously as well as alveolar macrophages, capillary pericytes, myofibroblasts, mast cells, lymphocytes and granulocytes.Currently, genetic etiologies have been identified for several lung diseases. With advances in nucleic acids research and engineering, gene transfer targeting specific cells is an emerging technology.2 range with extremely thin absorptive mucosal membranes and receive multiple arterial blood supplies from the bronchial and pulmonary arterial systems [Lungs are highly permeable and have large, absorptive surface areas in the 70–140 m systems . The res systems . With th systems ,17. MoreDepending on the underlying disease, the target cells in the lung can vary from epithelial cells, alveolar cells, macrophages, smooth muscle or endothelial cells. Below we will review several genetic lung diseases and the cell types involved in their pathogenesis.bone morphogenetic protein receptor type-II (BMPR-II) gene, a member of the transforming growth factor superfamily of receptors was documented in ~70% of patients with hereditary PAH and ~20% of patients with idiopathic PAH. The possibility of treating PAH via modulating dysregulated BMPR-II signaling is thus a rational consideration. Targeted delivery of adenoviral vector containing the BMPR II gene to pulmonary vascular endothelium in rodent PAH models resulted in substantially reduced severity of PAH, providing evidence for the rationale for gene therapy in the setting [Pulmonary arterial hypertension (PAH) is a devastating disease characterized by abnormal proliferation of pulmonary vascular endothelial and smooth muscle cells. The disease causes progressive dyspnea and right heart failure. A heterozygous mutation in the  setting . The celcystic fibrosis trans-membrane conductance regulator (CFTR) gene. The CFTR gene regulates chloride and sodium ions and the balance of electrolytes in the respiratory system across epithelial membranes. Thus, this gene has an important role in a number of cell functions. With a broad spectrum of effects, it is unlikely that one pharmacologic drug would be capable of restoring the multiple regulatory functions of CFTR. Mutations of this gene result in different effects as a function of pulmonary cell type. CFTR is located in the apical membrane of the airway epithelial cells. There is extensive evidence that the pulmonary abnormalities in CF are initiated by a deficiency of CFTR function in the epithelium. Therefore, the primary target for gene therapy of cystic fibrosis is the epithelium of the lower respiratory tract, which is mostly contained in the well-differentiated ciliated columnar airway epithelial cell. In addition, it was reported that mesenchymal stem cells (MSC) are an attractive therapeutic in murine models of CF. MSCs were shown to improve lung morphology, decrease neutrophil recruitment into the lung, and promote the resolution of infection in CF lungs [Cystic fibrosis (CF) is an autosomal recessive hereditary disorder resulting from mutations in the CF lungs . ProbablCFTR by delivering a normal CFTR gene to the respiratory epithelium is very attractive for a number of reasons. Cystic fibrosis is a recessive genetic disease, so the cellular defect can be corrected by a single normal copy of the gene. Furthermore, the respiratory epithelium, may be accessible by application of vector/gene construct within the airway, allowing for directed introduction of reparative genes to the primary target organ [CFTR cDNA could be transferred and expressed in the airway epithelium in vivo, it is logical to hypothesize that this could be a prospective approach to managing this disease [The strategy to repair the mutated et organ . With th disease .Alpha-1 antitrypsin (A1AT) is an enzyme produced by the liver that inhibits the action of proteases and protects the lungs from the damaging effects caused by the activation of neutrophil elastases and other proteinases such as proteinase-3, cathepsins, and metalloproteinases. A1AT deficiency is a genetic disorder and results from an inherited mutation in the gene that controls the production and release of the enzyme. The disorder results in a protease/antiprotease imbalance, causing interstitial lung damage and destruction of alveolar wall. Destruction of the alveolar wall severely inhibits gas exchange in the alveolar capillary membrane and causes excessive cell death of structural cells comprising the alveolus, leading to lung inflammation and emphysema ,22. It wSurfactant Protein B (SP-B) deficiency is a congenital lung disorder characterized by severe respiratory insufficiency and is unresponsive to any treatment except lung transplantation. SP-B deficiency is an example of inherited disease which is the result of a mutation in a single gene . ReplaceIn general, there are several biological barriers in the lungs that make absorption of foreign materials difficult. These barriers can be divided into: the conducting airway barriers, air–blood membrane  barriers, cellular barriers, and the immune system  Figure . The conThe mucus layer lays on top of the cilia in the nasal airway, pulmonary airway, and trachea. It consists of glycosylated macromolecules. Respiratory mucin containing secretions by Goblet cells and submucosal glands contribute to the formation of a mucus layer. The inhaled vectors are captured in the mucus via three mechanisms: electrostatic interactions, hydrophobic forces, and hydrogen bonding. They are cleared from the lung via mucociliary defense clearance by moving gene particles to the pharynx. An additional mechanism to clear mucus is cough clearance. Interestingly, lung infection and inflammation enhance this barrier ,28. The Pulmonary gene absorption is largely determined by the epithelial cells and their basement membrane, and the lung’s interstitial space containing a variety of cells, collagen, elastic fibers, interstitial fluid and lymphatic vessels. Absorption across the alveolar epithelium includes several mechanisms like passive and active movement through the cells and intracellular pores, vesicular transport, and drainage through the lymphatic’s vessels. Drugs are absorbed from the alveolo-capillary membrane into the blood must via the surfactant layer, the lining fluid, the epithelium, its basement membrane, the interstitium and the endothelium. The last layer, the alveolar–capillary endothelium, is extremely thin and has a relatively large number of endocytic vesicles and is not considered the major barrier to vector transport. The alveolar epithelium lines the alveoli with their cytoplasm, basal lamina, and capillary endothelium. On reaching the alveoli, many vectors can be degraded by proteases or removed by alveolar macrophages. The alveolar epithelium is also covered with a layer of pulmonary surfactant, which comprises phospholipids and specific surfactant-associated proteins. It is an active defense complex, which is synthesized and secreted primarily by type II alveolar pneumocytes. The major function of surfactant is to reduce surface tension at the air–liquid interface of the terminal airways, thereby reducing the tendency of alveoli to collapse . HoweverPre-existing antibodies and T- cell response against viral vectorsNeutralizing antibodies generated against capsid proteins of viral vectorT-cell-mediated response against viral vector capsidNeutralizing antibodies and T cell-mediated response against transgenesAlveolar macrophages are the most numerous cell type in the alveolar space and are capable of suppressing the alveolar inflammation and inducing an adaptive immune response through antigen presentation to T-cells. The pulmonary macrophages secrete peroxidases, and immunomodulatory mediators like interleukins, leukotrienes and proteases as part of the host defense mechanism. They are long-lived cells with a relatively slow turnover rate of ∼40% per year. It has been shown that they participate in the resolution of inflammation by clearing cellular debris, apoptotic neutrophils, and recruiting anti-inflammatory monocyte-derived macrophages. Displaying a unique phenotype, these cells maintain the lung’s homeostasis and help remodel the lung parenchyma after tissue injury and infection . AlveolaApart from pre-existing antibodies, cellular and humoral immune responses can develop after administration of the viral vector, which may render subsequent virus injection much less effective. The epithelial portion of the lung is another barrier for gene transfer into the lung due to the low efficiency of endocytosis across the apical membrane and tight junctions between cells that disturb the uptake of gene vectors into the baso-lateral surface ,36. ThisPulmonary gene transfer has become an attractive therapeutic approach, as there is a clinical need for long-term pharmaceutical interventions to address both inherited and acquired pulmonary diseases. Compared to other organs, the lung has a unique surface area for deposition and uptake of proteins including different vectors with gene constructs either for local or systemic aims. Through the pulmonary airway routes, genes can be administered intranasally or orally using inhalation devices, intratracheal instillation, and intratracheal inhalation with and without a bronchoscope. Intravascular pulmonary artery injection is an additional but invasive option .Direct airway delivery to the lungs is a very desirable and noninvasive route of gene administration. Compared to intravascular delivery, airway delivery has much lower endonuclease activities that can destroy DNA and RNA molecules . Direct Flexible bronchoscopy (FB) is a minimally invasive procedure that is widely used in humans and large animals, like sheep and pigs, for intratracheal delivery. FB allows the investigator to perform endoscopic examination of the tracheobronchial tree and choose the anatomical structure where the gene will be administered. In humans, the trachea is a continuation of the larynx that extends from the lower border of the cricoid cartilage to the carina. At the carina, the trachea bifurcates into the right and left main stem bronchi. The right main stem bronchus branches off at an angle of about 25 degrees with respect to the trachea. The right upper lobe bronchus trifurcates into the apical, posterior, and anterior divisions. The right main stem bronchus continues as the bronchus intermedius, which gives rise to the middle lobe bronchus and lower lobe bronchus. The middle lobe bronchus bifurcates into the medial and lateral branches. Segmental bronchi from the right lower lobe bronchus include the superior, anterior basal, medial basal, lateral basal and posterior basal divisions. The left main stem bronchus divides into the upper lobe bronchus and lower lobe bronchus. The left upper lobe bronchus has a superior division and an inferior division  that supply the upper lobe and lingula, respectively. The superior division bifurcates into the apicoposterior segmental bronchus and anterior segmental bronchus. The lingular bronchus bifurcates into superior and inferior lingular segmental bronchi. The lower lobe bronchus consists of superior, lateral basal, anteromedial basal, and posterior basal segmental bronchi.In rodents, unlike in humans, the right lung consists of four lobes: cranial, middle, caudal and accessory lobes, and the left lung has only one lobe. The rodent’s trachea divides into the left and right primary bronchi. The right primary bronchus divides into bronchi, which conform to the lobes of the right lung, namely the anterior , middle (cardiac), median (azygous or accessory) and posterior  lobes. The left primary bronchus supplies the left lobe. Each primary bronchus gives rise to smaller secondary bronchi from which the bronchioles originate. The rodents lack respiratory bronchioles, characteristic of primate lungs including those of humans. Several techniques are used for the tracheal access in rodents including oro-tracheal intubation, tracheal instillation and tracheotomy.Clinically relevant airway administration has some advantages and limitations. Advantages include: (i) Rapid onset of action, (ii) High local gene concentration, (iii) Delivery directly to the target lung tissue, (iv) The gene stays in the lung instead of being metabolized and excreted, and (v) Needle and pain-free administration . DisadvaThe deposition of genes by administration in the pulmonary airway mainly takes place by three mechanisms: gravitational sedimentation, inertial impaction, and diffusion. If the particle size is comparatively bigger than a nanoparticle, the deposition takes place by first two mechanisms. When the particle size is smaller, the diffusion mechanism turns on .The most common and successful method used in laboratory animals is the intratracheal instillation. In this method, a gene solution or dispersion is delivered into the lungs by a special syringe with a microsprayer tip. This provides a fast and quantifiable method of gene transfer. Thus, the instillation process is simple and has uniform drug distribution. In preclinical animal studies, intratracheal instillation has frequently been used to assess pulmonary uptake and systemic bio-distribution, especially with regard to the precise dosing and effectiveness associated with this method. However, intratracheal instillation for application in humans requires endotracheal intubation and anesthesia thus making it difficult to use in clinic. LacZβ-galactosidase gene stimulated a localized and systemic antibody response to the vector [CFTR to patients with CF demonstrated that Ad vector can deliver sufficient levels of CFTR cDNA to the airway epithelium and can protect the lungs from the respiratory manifestations of CF. However, expression was transient, lasting less than 30 days and was achieved with a second administration [CFTR showed that infected cells with the vector included mononuclear inflammatory cells, cuboidal and columnar epithelial cells [TIMP-1 gene was administered. Rats treated with the gene had less severe pulmonary vascular remodeling evidenced by a decreased right ventricular hypertrophy, decreased masculinization of peripheral pulmonary arteries and increased lung-cell apoptosis compared to controls [Adenovirus vectors are the most frequently used viral vectors due to the high levels of transgene expression that can be obtained in a many host cells and can be effectively delivered intratracheally. Current versions of adenovirus exhibit reduced toxicity and decreased immune response. The vectors are predominantly nonintegrating, episomal, nonenveloped, double-stranded DNA viruses. In models of bacterial sepsis, a cDNA encoding CTP:phosphocholine cytidylyltransferase (CCTα) enzyme was delivered intratracheally in mice using a replication-deficient adenovirus 5. Gene transfer produced high-level gene expression, increased alveolar surfactant (DPPC) levels and improved lung surface tension and pressure–volume relationships relative to control mice . Repetite vector . Adenovie vector . Endobrostration . Intratral cells . To inhicontrols . BMPR-II gene in all major lung lobes after intratracheal nebulized gene therapy with adenoviral BMPR-II delivery [vascular endothelial growth factor (VEGF) gene resulted in reduced endothelial apoptosis, increased vascularization, and improved PAP due to reduced remodeling but worsened PF [Kv1.5 gene is an important O2-sensitive, voltage gated K+ channel and potential therapeutic target in PH. Administration of Kv1.5 to the pulmonary circulation via an intratracheal aerosol is feasible and effective in eliciting transgene expression in PA smooth muscle cells (SMCs). This treatment reduces pulmonary vascular resistance (PVR) and restores hypoxic pulmonary vasoconstriction (HPV) in rats with established CH-PHT [Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) demonstrated robust and similar expression of delivery . Treatmesened PF . The Kv1d CH-PHT . Adeno-associated viruses are becoming increasingly investigated as gene delivery vectors due to a lack of pathogenicity, low immunogenicity in humans, lack of integration into the host genome, ability to infect dividing and non-dividing cells of many tissues, and prolonged transgene expression. However, studies have also revealed challenges of AAV’s as gene delivery vectors, including species specific variable transfection rates, low organ targeting efficiencies, and neutralization by pre-existing circulating antibodies. A1AT gene expression in serum was ≈60-fold better than that of AAV2/5. The AAV2/9-mediated LacZβ-galactosidase gene transfer in lung airways was stable for 9 months, suggesting that a progenitor airway cell population was transduced [Intratracheal delivery of rAAV5 and rAAV1 vectors resulted in transduction of lung Clara cells and alveolar type II cells that proliferated following lung injury. Both of these AAV vectors appeared to preferentially transduce conducting airway epithelial progenitors with the capacity to clonally expand, both in vivo following lung injury and in vitro . AAV2/9 ansduced . Studiesansduced . Viral vansduced . Anotheransduced . Large animal models offer testing of gene therapy approaches in more clinically relevant conditions in terms of lung size, vector dose, and deliver method. A huge advance in CF gene therapy was the recent generation of a pig CF model . This moAdenoviral gene therapy for CF advanced rapidly into clinical trial after demonstration of effective expression in rats ,62. ArouAs the limitation of adenovirus for long-term expression became aware in clinical trial, CFTR gene therapy was tested using AAV2 in rabbits and then macaques ,65. ThesCFTR gene therapy in clinically relevant condition, their chimeric AAV failed to transduce human airway epithelia in vitro. Another in vitro study also showed that airway epithelia from human and Indian Rhesus monkeys exhibited significant differences in gene transduction efficiency [In a recent study, bronchoscopy delivery of different AAV serotypes encoding GFP in 1 week-old sheep resulted in transgene expression of small and medium sized airways with AAV1, AAV6, AAV8, AAV9, and AAVrh10, but not AAV5 . In contficiency . These rSERCA2a gene delivery in a rodent model, this program was advanced to a large animal experiments [SERCA2a gene delivery with use a bronchoscopy sprayer and nebulization approach prevents progression of vascular remodeling and development of pulmonary hypertension [One emerging avenue of airway gene therapy is the targeting of vascular cells for treating pulmonary hypertension using vascular tropic AAV serotype. Following the demonstration of pulmonary vascular remodeling inhibition with airway AAV1.eriments . Using artension . No safeFor the majority of clinical applications that aim to deliver therapeutic genes to the lung with AAV vectors, long-term expression of the therapeutic proteins will be essential. Due to the turnover of lung epithelial and other target cells in the lung, the over-expression of delivered genes will decline with time. Therefore, infrequent vector re-administration will be necessary for pulmonary epithelial targets. The reason for this is that AAV genomes largely persists episomally, and cell division of the pulmonary cells will eventually result in a progressive reduction in vector genomes per cell and a concomitant decrease in expression of the therapeutic protein. Systemic re-administration of recombinant AAVs is recognized as challenge and remains an unmet need for systemic re-administration. The potential for repeated administration to the lung, however, has not been established yet.LacZ β-galactosidase essentially blocks subsequent transduction by an AAV2 vector expressing alkaline phosphatase [LacZ β-galactosidase followed by the delivery of AAV6 encoding alkaline phosphatase only modestly reduced transduction by the latter vector. These data suggest that administration of a different serotype might be a promising approach for re-administration of viral vectors [Data obtained from preclinical animal experiments is complex. For instance, Miller and colleagues reported that, in mice, administration of recombinant AAV serotype 2 expressing sphatase . In cont vectors . Deliver vectors . Taken t vectors .The same group reported earlier that even if the transgene is delivered with a AAV serotype 2 vector, re-administration is possible if the immune system is transiently suppressed by the co-administration of an antibody against CD40 and a fusion protein of an IgG constant region with the extracellular domain of CTLA4. The former inhibits a B-cell antibody response, whereas the latter reduces T-cell activation by binding to the CTLA4 receptor CD86 ,76,77. Wilson et al. group demonstrated that when an AAV5 vector encoding erythropoietin is delivered by intranasal instillation expression of erythropoietin progressively decreased over a time span of 5 months. Strikingly, however, intranasal administration of an AAV5 vector encoding β-galactosidase to the mice that were previously injected with AAV5. erythropoietin resulted in near identical expression levels of β-galactosidase when compared to naïve mice . Given tTo date, very little data is available for the efficiency of repeated administration of aerosolized AAV in humans. To our knowledge, the repeated administration of an AAV2 vector encoding the CF transmembrane regulator to treat CF is the only clinical trial that tested the hypothesis that repeat application of aerosolized AAV can be used to treat lung disorders. Unfortunately, this trial failed to meet its primary endpoint .Clearly, repeated application of aerosolized AAV as a feasible therapeutic modality will need extensive further investigation for further translation to the clinic. Regrettably, the immune system of mice and humans are quite different, and only future clinical trials will ultimately yield definitive answers whether repeat administration of aerosolized AAV is a viable treatment strategy.Taken together, these studies demonstrate the therapeutic potential of AAV vectors for pulmonary gene transfer in a preclinical setting, suggesting further development of an AAV-based gene therapy for human airway diseases.For many lung diseases, effective gene therapy will require stable genetic transfection of terminally differentiated cells or of progenitor cells that divide infrequently. Lentiviral vectors can integrate reliably into the chromosomes of non-dividing cells. Lentiviruses are a family of complex retroviruses typically associated with infection of lymphocytes and macrophages. Retroviral vectors, unlike other vectors are highly efficient at integrating into the genome of the target cell and do not transfer any viral gene, thus alleviating the risk of vector-mediated toxicity and immune response against the transduced cells. The results support the hypothesis that after intratracheal delivery, lentiviral vectors can overcome physical and biochemical barriers present on normal airway epithelium and efficiently transduced the proximal and distal surface epithelia, and to a lower extent, alveolar epithelium . It was Intratracheal gene delivery is a promising approach to treat both acquired and inherited lung diseases. Compared to systemic and local gene delivery routes including intra-oral and intra-nasal routes, intratracheal instillation/spray with bronchoscope has many benefits including rapid onset of action, achievement of high local gene concentration in targeted cells with minimizing systemic effects and homogeneous distribution. Though there are hurdles to gene therapy that still exist and testing that still needs to be done before treatment options are available in clinic, much progress has been made in the gene therapy approach to design vectors to achieve specific cell transfer, minimize innate and adaptive immune responses, create new genes, and determine the best route of administration. Producing a successful gene therapy treatment for genetic lung diseases may prove useful for treating other genetic diseases in other organs and cells in the body.


Skeletal muscle has remarkable regenerative capacity, relying on precise coordination between resident muscle stem cells (satellite cells) and the immune system. The age-related decline in skeletal muscle regenerative capacity contributes to the onset of sarcopenia, prolonged hospitalization, and loss of autonomy. Although several age-sensitive pathways have been identified, further investigation is needed to define targets of cellular dysfunction. Autophagy, a process of cellular catabolism, is emerging as a key regulator of muscle regeneration affecting stem cell, immune cell, and myofiber function. Muscle stem cell senescence is associated with a suppression of autophagy during key phases of the regenerative program. Macrophages, a key immune cell involved in muscle repair, also rely on autophagy to aid in tissue repair. This review will focus on the role of autophagy in various aspects of the regenerative program, including adult skeletal muscle stem cells, monocytes/macrophages, and corresponding age-associated dysfunction. Furthermore, we will highlight rejuvenation strategies that alter autophagy to improve muscle regenerative function. The physiological process of aging consists of diverse cellular changes including proliferation, metabolism, inflammation, and apoptosis. To identify and provide new therapeutic targets and approaches to counteract the physiological decline of advanced age, a thorough understanding of cellular processes involved is paramount. Autophagy has emerged over the years as an important regulator of age-related changes in various tissues including skeletal muscle. Aging is directly associated with adverse changes to skeletal muscle including a decline in functional capacity which contributes to lower quality of life, reduced independence, and greater mortality in the elderly. The capacity of skeletal muscle to regenerate following injury is, likewise, negatively affected by aging and appears to be associated with alterations in cellular autophagy ,2. The rAtgs). Atgs facilitate specific stages in the autophagic process such as initiation, formation, elongation, and fusion [Macroautophagy (hereafter referred to as autophagy and distinct from micro- and chaperone-mediated autophagy as reviewed in ) was orid fusion .A number of molecular signals are involved in the coordination and control of the process (readers are referred to a previous review ). TranscSaccharomyces cerevisiae, screens for short-lived mutants include various autophagy loss-of-function Atg mutants [Ceanorhabditis elegans [Drosophila melanogaster [Aging is a complex process associated with diminished ability for tissues to maintain biological homeostasis. This is especially relevant in tissues that exhibit age-related changes in autophagic function. In numerous cell types tested, autophagy upregulation is capable of mitigating aging-induced apoptosis and necrosis . Prolife mutants . Similar elegans  and Beclnogaster . In humanogaster , circulanogaster , connectnogaster , and carnogaster . Wound rnogaster . Taken tSkeletal muscle is a dynamic tissue that is constantly adapting and changing to physical and metabolic demands. As such, autophagy seems to be a key step in healthy muscle homeostasis and physiology . PathophIn addition to maintaining muscle homeostasis, the activation of autophagy becomes an important factor for skeletal muscle remodeling after stimuli like contractile activity/exercise ,42. Not mdx mice [Skeletal muscle has extensive regenerative capabilities due to resident stem cell functions and a highly coordinated interaction with hematopoietic/immune cells during the repair process. This complex, yet efficient process results in complete return of morphology and function in healthy muscle. Blocking autophagy by genetic  or pharmacological (chloroquine) methods causes accumulation of sarcomeric and nuclear debris following muscle damage in zebrafish showing the key role autophagy plays in the repair process . In the mdx mice . Geneticmdx mice . While tmdx mice . These dmdx mice . TogetheSuccessful muscle regeneration requires a coordinated effort from various other cell types including mast cells, neutrophils, T regulatory cells, eosinophils, and CD8+ T cells . While e+/Myod− cells that are capable of asymmetric cell division and forming mature myonuclei when transplanted into injured muscle. Successful regeneration requires two stages of satellite cell function: proliferation and fusion/myogenesis. Both phases of repair show evidence of dysfunction with advanced age. The function of satellite cells is measured by their ability to activate from quiescence and undergo asymmetric proliferation into (1) daughter cells that are capable of further proliferation and differentiation into mature muscle fibers and (2) daughter cells that are capable of reentering quiescence. Therefore, we refer to satellite cell function as their ability to proliferate and/or undergo myogenesis. A summary of the general mechanism of myogenesis and myofibrillar regeneration is shown in Adult skeletal muscle maintains a relatively remarkable ability to regenerate and recover following mild damage or insult throughout much of the lifespan . The funPax7-expressing satellite cells suggests a minimal role in the development of sarcopenia (under sedentary conditions) as measured by specific force and muscle morphology comparing young (~5 months) and aged (~22 months) mice [Pax7-dependent contribution of BrdU to myofiber nuclei was also shown to be minimal in unchallenged muscle [Pax7 positive satellite cells in regeneration, several groups have reported age-associated decrements in the number of satellite cells [Aging has long been known to be inversely related to the proliferative potential of satellite cells ,57,58. Dhs) mice . Similard muscle . While tte cells ,65,66. Nte cells . The mecte cells ,69,70. Ink4a [Satellite cells isolated from aged mice can be transplanted into injured muscles of young recipients and contribute to regeneration but fail to repair myofibers and replenish the quiescent resident stem cell pool to a similar extent of young donor cells ,71. ThisInk4a ,67,70,71Ink4a . AlteratInk4a  which, iInk4a . Age-relKip1 pathway [Kip1 signaling to promote autophagy and subsequent proliferative/myogenic function. Aged MuSCs had suppressed AMPK/p27Kip1 signaling associated with reductions in autophagy and proliferation with increased apoptosis and senescence. Genetic or pharmacological activation of AMPK or p27Kip1 was effective in inducing autophagy and returning myogenic potential of aged MuSCs in addition to blocking the induction of senescence markers [The importance of skeletal muscle autophagy is evident as impairments in autophagy in muscle tissue cause myopathy , altered pathway . MuSC ac1/2 = 3 days) and highly mobile cell residing in the blood and spleen which respond to inflammation by differentiation into macrophages and dendritic cells as well as tissue specific cells such as the Kupffer cells of the liver. The process of differentiating from a monocyte to a macrophage dictates the extent and duration of both immune responses and tissue remodeling. Without appropriate stimulation to differentiate, monocytes undergo apoptotic cell death and are cleared from the system. Here we will focus on the role autophagy plays on monocyte differentiation with age and the impact this has on macrophage function during regeneration. In order to fully appreciate the regenerative function of these immune cells, one must appreciate the phenotypic spectrum and heterogeneity of the macrophage population recruited during the regenerative process.Alongside satellite cells, the innate immune system, specifically myeloid cells, plays a principal role in development, homeostasis, and regeneration of skeletal muscle ,80,81,82Monocytes are derived from quiescent HSCs found in the hypoxic bone marrow environment. This environment limits oxidative metabolism and nutrient availability to HSCs which are typical activators of the autophagic process ,87,88. IMonocytes are short-lived in circulation and undergo apoptosis unless stimulated by inflammatory factors such as TNF to upregulate survival pathways. Upon activation by inflammatory signals, monocytes use autophagy as a means to differentiate into macrophages or dendritic cells . The speDuring the early stages of the recovery process (roughly 0–48 h post injury), neutrophils help to condition the tissue environment at the site of injury helping to direct subsequent regenerative immune events. Monocyte extravasation in to the muscle is controlled by chemotaxis towards pro-inflammatory cytokines such as interferon-γ (IFNγ) and tumor necrosis factor (TNF). Following classical activation and recruitment to the damaged muscle, monocyte differentiation will lean towards a pro-inflammatory macrophage (M1) designed to ingest cellular debris and necrotic tissue to combat the initial insult and direct the subsequent immune response. Following the initial M1 response, monocytes will begin to differentiate towards alternatively activated, anti-inflammatory macrophages (M2), which reduces local inflammation and tampers the immune response to promote tissue remodeling see . The chaAtg-deficient mouse models to show that there is a need for effective autophagy to promote an anti-inflammatory environment through LC3-associated phagocytosis and appropriate cytokine signal release [It is clear that autophagy is a critical component to the efficient function of immune cells to clear pathogens or cellular debris. This is highlighted by macrophage function during skeletal muscle repair ,111. We  release ,114. fl/flAtg7 mice, macrophage-specific autophagy was shown to play a role in M1/M2 polarization potential. When differentiated macrophages are unable to use autophagy, the macrophage population shifts towards a proinflammatory M1 phenotype along with greater glycolytic metabolism [In addition to regulating macrophage function, autophagy appears to play a role in phenotype as well. By using a LysM-Cre recombinase system with tabolism . Converstabolism . The inttabolism . When mTtabolism ,119. Immune cells are subjected to an age-associated decline in effective response, termed immunesenescence . AlthougEvidence supports age-associated changes in macrophage function across the lifespan. Early studies showed altered response and expression of toll-like receptors in aged mice compared to young . MonocytAfter having established the importance of autophagy in various aspects of skeletal muscle repair, autophagy, and related pathways present an obvious therapeutic target to enhance muscle function and regenerative capacity. Exercise and caloric restriction (CR) are two established methods shown to induce autophagy in several tissues ,134,135.Exercise is an established method to prevent the onset of sarcopenia. In addition, exercise may also aid to enhance regenerative capacity in aged muscle. Moderate intensity treadmill running is capable of increasing the number of MuSCs isolated from hindlimb muscles in young and old mice while increasing aged MuSC proliferative function ,141. In Kip1 in activating aged MuSCs [An additional intervention shown to induce global autophagy and related extension in health/lifespan is caloric restriction . The bened MuSCs . These dLittle has been shown on how exercise training might improve immune function in the elderly or mitigate the immunosenescent phenotype. Early studies have shown a connection between exercise and macrophage phagocytic function in athletes . A 10-weSimilar to the MuSC, macrophage polarization and function seems to be sensitive to caloric restriction . GreaterAs has been alluded to, the pharmacological induction of autophagy represents a promising strategy to improve stress resistance and regeneration of skeletal muscle. Spermidine and rapamycin are two examples of drugs that have been studied for their autophagy-inducing effects and lifespan extension in rodent models ,155. WhiThe ability to repair damaged tissue and continuously respond to stressful stimuli is essential to preserve whole body function throughout life. Muscle stem cells and macrophages are an integral part of this process and their loss of function with age contributes to degenerative diseases. This review highlights how muscle stem cells and monocytes/macrophages are essential for skeletal muscle homeostasis and regeneration. A common theme among these cell populations is the idea that autophagy is a key process that is altered in aged cells leading to functional decline. Autophagy is no longer an emerging regulator of cellular function but has consistently been shown to play a central and important role, especially in the context of aging. Stem cells, in particular, show dysfunctional autophagy during initial stages of activation while caloric restriction and physical activity allow a sensitization to autophagy with beneficial outcomes in cellular activation and function. The exact role for autophagy in muscle regeneration will be complex considering the temporal nature and diverse cell types contributing to the regenerative program. However, global induction of autophagy appears beneficial to the regenerative capacity in the aged muscle. Continuing to uncover the molecular events responsible for age-related perturbations in these pathways is critical for exposing pharmaceutical targets to combat the aging process and improve tissue regeneration in aged individuals.


Challenges in diagnosing primary immunodeficiency are numerous and diverse, with current whole-exome and whole-genome sequencing approaches only able to reach a molecular diagnosis in 25–60% of cases. We assess these problems and discuss how RNA-focused analysis has expanded and improved in recent years and may now be utilized to gain an unparalleled insight into cellular immunology. We review how investigation into RNA biology can give information regarding the differential expression, monoallelic expression, and alternative splicing—which have important roles in immune regulation and function. We show how this information can inform bioinformatic analysis pipelines and aid in the variant filtering process, expediting the identification of causal variants—especially those affecting splicing—and enhance overall diagnostic ability. We also demonstrate the challenges, which remain in the design of this type of investigation, regarding technological limitation and biological considerations and suggest potential directions for the clinical applications. Primary immunodeficiency disorders (PID) result from altered, poor, or absent function in one or more components of the immune system, rendering the affected individuals with increased susceptibility to immune-related ailments including increased frequency and severity of infection, autoimmunity, aberrant inflammation, and malignancy . The undThe most common form of PID is selective immunoglobulin A deficiency, which is usually typically asymptomatic but can manifest with a variety of clinical presentations including coeliac disease, type 1 diabetes mellitus, and increased infections. With an estimated prevalence of 1 in 300–500 persons , and whiThe importance of early diagnosis In PID cases is high, with relation to both the patient’s qualitative experience and the economic cost to healthcare services. Sources vary in cost analysis of undiagnosed PID. Some say that while a diagnosed US patient costs healthcare services over US$250,000 per annum, largely due to treatment costs, an early diagnosis of the disorder can save as much as US$6,500 per patient, per annum . An alteChallenges in diagnosing primary immunodeficiency are numerous and diverse. Studies which correlate the phenotype and genotype have been useful in diagnostics, developing an understanding of various PID disorders . AdditioDNA sequencing-based genetic testing is used where possible, as it provides the best diagnostic capability of existing clinically adapted methods . Whole-eDue to the improvement that WES and whole-genome sequencing (WGS) bring to diagnostics, researchers are calling for universal molecular gene testing for the diagnosis of primary immune deficiencies . Evidenccis-acting regulatory elements of transcription  elicit a powerful, sometimes synergistic effect on the expression of a large number of genes. Single-nucleotide polymorphisms (SNPs) on eQTLs affect the transcriptional level of other RNAs, modifying protein expression and causing phenotypic changes to the abilities and behaviors of cells in some immunological cases . These e effects . Large-s effects . This re effects . The resAlternative splicing is the method through which the cell can produce an array of transcript isoforms derived from a single gene or multiple genes spliced together . IntronsDeep surveying on alternative splicing has shown that 95% of genes which contain multiple exons undergo alternative splicing, and even when only considering moderate to high abundance events, there are reportedly 100,000 individual splicing events in major tissues .Alternative splicing occurs both co-transcriptionally and post-transcriptionally, and the action of transcription factors as well as splicing factors regulates and influences splicing events in some of the most crucial mechanisms of the adaptive immune system . ImportaCARMA1, BCL10, and MALT1  to complement genomic sequencing, Cummings et al. evidenced this in the n muscle . Part ofn muscle . Indeed,n muscle . Analysin muscle . These sn muscle .RNU4ATAC that produces a small nuclear RNA (snRNA) termed U4atac was discovered to cause Roifman syndrome  and microRNA (miRNA), the significance of which is continually being elucidated in the molecular pathology of disease . Indeed,c manner . Subsequc manner . It is ac manner .Microfluidic technology adaptations have allowed the development of robust, single-cell transcriptomic profiling . In combex vivo tests, which are difficult to standardize and mostly available in research laboratories. RNA profiling to identify alternative splicing, gene expression-level variation monoallelic expression may contribute a further insight into candidate variants derived from proband or family-based WES/WGS sequencing results. We propose the introduction of RNASeq-based analysis for patients who have a clinical presentation of PID, but who, despite normal baseline immune testing, cellular analysis, and having undergone WES/WGS remain undiagnosed  will require further dissection.Utilizing candidate gene lists and large control datasets for comparison enhances the power of the transcriptional profiling through RNASeq and improves resolution for differential gene expression. Existing projects have developed these datasets for whole blood and immune cells, which provide a starting point for the interrogation of clinical samples for diagnostic research.+ T cells, for example, show a high degree of heterogeneity in the context of temporal changes through the life course of the individual, and CD4+ T cells and monocytes are heavily influenced by sex  professorship to DB.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.


Recent findings regarding early lophotrochozoan development have altered the conventional model of neurogenesis and revealed that peripheral sensory elements play a key role in the initial organization of the larval nervous system. Here, we describe the main neurogenetic events in bivalve mollusks in comparison with other Lophotrochozoa, emphasizing a novel role for early neurons in establishing larval nervous systems and speculating about the morphogenetic function of the apical organ. We demonstrate that during bivalve development, peripheral sensory neurons utilizing various transmitters differentiate before the apical organ emerges. The first neurons and their neurites serve as a scaffold for the development of the nervous system. During veliger stage, cerebral, pleural, and visceral ganglia form along the lateral  nerve cords in anterior-to-posterior axis. The pedal ganglia and corresponding ventral  nerve cords develop much later, after larval settlement and metamorphosis. Pharmacological abolishment of the serotonin gradient within the larval body disrupts the navigation of “pioneer” axons resulting in malformation of the whole nervous system architecture. Comparative morphological data on neurogenetic events in bivalve mollusks shed new light on the origin of the nervous system, mechanisms of early axon navigation, and sequence of the tetraneurous nervous system formation. Furthermore, this information improves our understanding of the basic nervous system architecture in larval Bivalvia and Mollusca. Lophotrochozoa, a morphologically varied group of animals, include worm-like forms, shelled animals, and soft-bodied slugs, which makes searching for common anatomical and morphological features in adults daunting and unsuccessful to date. However, during lophotrochozoan early larval development, ancestral morphological and molecular patterns are present with formation of the nervous system being among the most well-conserved and prominent features of this development. Therefore, development of the nervous system has long attracted the attention of comparative biologists for testing evolutionary and phylogenetic hypotheses.Although for more than 100 years, researchers have attempted to understand the details and general patterns of this development utilizing available methodology , some maBivalves are seldom used in studies of lophotrochozoan neurodevelopment. These animals, with sedentary or free-living lifestyles as adults, develop from free-swimming larvae that hatch as trochophores, develop through the veliger stage, undergo metamorphosis, and then settle –4, 6.Previous investigations on bivalves have concluded that the central nervous system (CNS) of adults consists of paired cerebropleural and visceral ganglia, the latter typically fused and connected to the cerebropleural ganglia by longitudinal lateral cords –12  and lateral  nerve cords together build a tetra-cord structure [Accordingly, although morphology varies, the nervous system of bivalve veligers consists of three paired ganglia  connected by the lateral cord Fig. b. Howevetructure , 6, 20. Here, we first review the findings on later developmental stages (after the settlement and metamorphosis of pediveligers), when pedal ganglia with their cerebropleuro-pedal connections  arise Fig. c. ImmunoIt is generally accepted that the first neurons to appear in larvae are sensory cells, responsible for converting external stimuli from the environment and appropriately regulating larval behavior and development in Lophotrochozoa –30, EchiHowever, some evidence indicates that simultaneously with or even before the neurotransmitter expression in AO cells, solitary sensory cells appear on the periphery of the body of several trochozoan larvae. The function of these pioneer sensory neurons remains unknown. One hypothesis is that they help to provide scaffolding for the definitive nervous system , 37–39.Crassostrea gigas confirm the early appearance of peripheral sensory neurons [Mytilus trossulus, larvae presented here indicate the morphogenetic significance of these cells in connection with formation of the nervous system.Recent studies on the bivalve mollusk  neurons  and, togC. gigas prior to the emergence of the AO. The neurites of the FMRFamide-immunoreactive (FMRF-ir) neurons in the dorsal sensory group  extend towards the neurons of the ventral sensory group   Fig. a. As neuSC) Fig. b. HoweveC. gigas, the anlagen of the lateral nerve cords have already been formed  and FMRFamide-related peptides (FMRF-RP) are the primary transmitters expressed by the first sensory neurons that appear during bivalve development. FMRFamide and related peptides, in addition to 5-HT, influence multiple physiological processes, including heart rate, blood pressure, motility of the gut, feeding, and locomotory behavior in both adult and larval invertebrates –47. The + and associated water, rendering larvae resistant to osmotic pressure [Neurons with different immunoreactivity (FMRF-RP and 5-HT) innervating several larval organs may perform common functions, e.g., affecting the beating of cilia or innervating muscles , 54–58. pressure , 59–62. pressure , 21, 48,Mytilus trossulus, they appear in the episphere and their processes organize into two nerve bundles that run in parallel along the ventral side of the larvae [Ischnochiton hakodadensis, the bodies of these early cells are located in the episphere and their long axons extend underneath the neuropil of the AO and thereafter caudally in two bundles on the ventral side [Neurogenesis in several groups of Lophotrochozoa has been found to begin with the emergence of peripheral neurons. Importantly, the locations of these early larval neurons vary in different species. In the bivalve mollusk e larvae  , where specific neurons secrete transmitter via their varicose neuropil . Apical al death .Mytilus trossulus as described for Platynereis [−6 M to early trochophores, up to the veliger stage)  Fig. b–b2. NumImmunolabeling is widely utilized to visualize differentiation in specific nerve elements of various taxa, but, unfortunately, only a limited number of commercial antibodies are applicable for invertebrates. Among these, antibodies against 5-HT and FMRF-RP are most commonly used. In addition, antibodies directed towards acetylated alpha-tubulin provide a marker for neuronal processes and some neuronal cell bodies in annelids and mollusks .C. gigas, we showed that an antibody against the vesicular acetylcholine transporter (VAChT) is a reliable marker of ACh-containing elements [Histochemical reactions and immunochemical labeling against enzymes involved in biochemical degradation have been employed in attempts to visualize acetylcholine-containing (ACh-containing) regions as well as individual cells and nerve bundles; but these approaches in many cases have produced inconsistent results which were hard to interpret. In our recent study on neurogenesis in elements . With thelements . In addielements , 77–81.FIn summary, neurogenesis in bivalve mollusks begins with the emergence of peripheral FMRF-ir cells with their growing processes and apical 5-HT-ir sensory cells. Together, these early elements delineate the main structure of the nervous system, including the apical/cerebral ganglia and paired nerve cords. VAChT-ir cells later appear in the periphery and their processes follow the pathway already established by early “pioneer” neurons Fig. . Both eaThe second neuronal cord, i.e., the pedal  cord with associated pedal ganglia, appears late in bivalve development, after pediveliger settling. Further research on bivalve neurogenesis, with a particular focus on VAChT-ir structures, is required to determine the cellular compositions of the AO and the central and peripheral nervous systems of veligers in detail.Are some of the early neurons a prerequisite for the main ganglia of the definitive nervous system in mollusks?When do ganglia fuse and what is the underlying mechanism and in response to what stimulus it starts?Why does the pedal nervous system appear later in the development of bivalve mollusks and how is its development connected to that of the foot? What happens to the pedal ganglia in mollusks where the foot is resorbed (cementing species)?Do mollusks have a peripheral (autonomous) nervous system?How and where  do neural precursors form and neuroblasts migrate?What characters of the early development are common for various taxa and thus may reflect the neuroarchitecture of the last common ancestor?At present, many major questions and gaps remain regarding the cellular and molecular mechanisms of neurogenesis in bivalve mollusks:


Periodontitis is a chronic inflammatory disorder that causes destruction of the periodontal attachment apparatus including alveolar bone, the periodontal ligament, and cementum. Dental implants have been routinely installed after extraction of periodontitis-affected teeth; however, recent studies have indicated that many dental implants are affected by peri-implantitis, which progresses rapidly because of the failure of the immune system. Therefore, there is a renewed focus on periodontal regeneration aroundnatural teeth. To regenerate periodontal tissue, many researchers and clinicians have attempted to perform periodontal regenerative therapy using materials such as bioresorbable scaffolds, growth factors, and cells. The concept of guided tissue regeneration, by which endogenous periodontal ligament- and alveolar bone-derived cells are preferentially proliferated by barrier membranes, has proved effective, and various kinds of membranes are now commercially available. Clinical studies have shown the significance of barrier membranes for periodontal regeneration; however, the technique is indicated only for relatively small infrabony defects. Cytokine therapies have also been introduced to promote periodontal regeneration, but the indications are also for small size defects. To overcome this limitation, ex vivo expanded multipotent mesenchymal stromal cells (MSCs) have been studied. In particular, periodontal ligament-derived multipotent mesenchymal stromal cells are thought to be a responsible cell source, based on both translational and clinical studies. In this review, responsible cell sources for periodontal regeneration and their clinical applications are summarized. In addition, recent transplantation strategies and perspectives about the cytotherapeutic use of stem cells for periodontal regeneration are discussed. Periodontal disease is mainly caused by oral bacteria. Without dental treatment, bacteria-induced inflammation can spread and destroy the periodontal ligament, alveolar bone, cementum, and gingiva. When the destruction of alveolar bone is evident radiographically, it is diagnosed as periodontitis, which is generally considered an irreversible condition. Once periodontitis occurs, it does not heal spontaneously. Therefore, gingival recession usually occurs followed by functional and esthetic problems, such as root caries and black triangles . MoreoveBecause the regenerative therapies mentioned above have limited indications and do not provide good results for a wide range of defects, such as one-wall infrabony defects, class III furcation defects, and horizontal defects, cytotherapeutic approaches were introduced in the 2000s. Based on recent developments in stem cell biology and tissue engineering, stem cells from patients or healthy volunteer donors can be harvested and amplified in vitro. Stem cells can then be manufactured with or without scaffolds and transplanted for periodontal regeneration.It is thought that there are two main modes of action of cytotherapy in periodontal regeneration. One is the supply of favorable cells for periodontal regeneration, such as periodontal ligament cells and/or osteoblastic cells. The other is the support of endogenous favorable cells through the paracrine effects of transplanted cells. Sometimes these two modes of actions are combined.This section reviews the current literature about cytotherapy, mainly using multipotent mesenchymal stromal cells, for periodontal regeneration.Multipotent mesenchymal stromal cells (MSCs) can be isolated from several tissues including bone marrow, fat, periodontal ligament, dental pulp, and periosteum. Because the MSCs used in each laboratory were different, the MSC Committee of the International Society of Cell Therapy (ISCT) published a statement paper in 2006 that defined the minimal criteria of human MSC characteristics, including the following: (1) adherence to plastic under standard culture conditions; (2) specific surface antigen expression ; and (3) in vitro differentiation capacity into osteoblasts, adipocytes, and chondroblasts [Periodontal ligament (PDL) tissue is the soft connective tissue between the tooth and the alveolar bone. PDL tissue mainly consists of fibroblastic ligament cells that function as a cushion at the time of chewing. PDL tissue also contains abundant nerves and vessels and, thus, transmits information about food texture to the brain and retains an immune response against oral bacteria. Clinically, PDL tissue is important in autotransplantation because the PDL tissue of the donor’s teeth has the potential to induce the formation of new bone, gingiva, and PDL at the recipient site . In factN-isopropylacrylamide (PIPAAm) for the surface of cell culture dishes. At temperatures lower than 32 °C, PIPAAm is fully hydrated with an extended chain conformation; however, at temperatures higher than 32 °C, PIPAAm is extensively dehydrated and compact. Cells generally adhere to hydrophobic surfaces but not to highly hydrated hydrophilic surfaces. Our laboratory grafted PIPAAm to develop temperature-responsive culture dishes by irradiation with an electron beam. The advantage of a temperature-responsive dish is the possibility of harvesting intact cells and proteins with only low-temperature treatment. By reducing the temperature, cells spontaneously detach from the surface of dishes. Compared with the process of retrieving cells with enzymes such as trypsin or dispase, loss of cell viability and degradation of surface proteins are minimized. Recent reports have clearly demonstrated that various kinds of single cells harvested from temperature-responsive dishes have highly preserved functions as well as intact proteins. Additionally, the preserved subcellular matrix proteins of harvested cell sheets provide adhesive properties for stacking. Therefore, cell sheets can be stabilized at the recipient sites without any glue or sutures in a corneal reconstruction model [It is important that ex vivo expanded MSCs are transplanted in an appropriate position. The injection of a single cell suspension is known to be inefficient because it is unstable and will diffuse. Our research group introduced cell sheet engineering technology, using temperature-responsive cell culture dishes, in which cells can be harvested in a sheet form . We deveon model . It is aPDL cell sheets were manufactured and transplanted, then periodontal regeneration was observed in two animal models ,18. FollThe results of this clinical study validated the safety and efficacy of autologous PDL-MSC sheets in severe periodontal defects. All the findings, including reduced periodontal probing depth , clinical attachment gain (2.5 ± 2.6 mm), and increased radiographic bone height (2.3 ± 1.8 mm), indicated improved periodontal conditions in all 10 cases at 6 months after the transplantation. These therapeutic effects were sustained during a mean follow-up period of 55 ± 19 months, and there were no serious adverse events .5 cells/well with 30 μg/mL of l-ascorbic acid for approximately 10 days. PDLSC sheets were mechanically detached, rolled up to pack the xenogeneic bone substrates (Bio-Oss®), and placed 1 mm over the crest height of the defects. Collagen membrane (Bio-Guide®) was placed on the PDLSC sheet/bone substrate complex to cover. The authors observed the safety of the autologous PDLSCs, although they did not find any significant therapeutic efficacy of the PDLSCs compared with that of the control group (collagen membrane and bone substrate without cells). It was possible that the bone substrates used in this clinical trial may have masked the potential of the PDLSC sheet because they were considered to be slow-resorbing materials. Further research should be undertaken with mechanically detached PDLSC sheets to clarify this point.A randomized clinical trial of autologous PDLSCs with GTR membranes and bovine-derived bone substrates was reported in 2016  based onAs mentioned above, PDL-derived cells may have the potential to induce true periodontal regeneration around the natural teeth. However, there is no standard protocol for isolation and cultivation of PDL-derived cells. It may be important to define the optimal culture conditions and an evaluating system for PDL-derived cells. Additional research should be undertaken to determine the characteristics of transplanted cells for further development of cytotherapy with PDL-derived cells.Iliac bone marrow MSCs (BMMSCs) are a well-characterized cell source for bone regeneration. BMMSCs combined with biomaterials have been studied in relation to periodontal regeneration. BMMSCs mixed with platelet-rich plasma (PRP) were transplanted in 17 patients, and reduced probing depth, clinical attachment gain, and bone gain were observed, although the defect shape was not mentioned .Periosteal cells possess the capacity for bone regeneration, and they are easily harvested. Membranous cultured periosteum was transplanted in the same way as the GTR membrane, and its safety and efficacy were confirmed ,34.Recently, a clinical study with a single-cell suspension of adipose stem cells with fibrin glue was undertaken in Osaka, Japan. Abdominal fat tissue was harvested from each patient and cultured under specific culture conditions. The results have not yet been disclosed.Mechanically dissociated dental pulp tissue has been used in clinical settings ,36. AlthThis section reviews the recent progress of tissue engineering for periodontal regeneration.Recent polymer engineering has enabled the development of scaffolds with functions. Cell orientation and cell type can be controlled by cell culture surface modification  or polymIn addition to the cell sheet engineering technology mentioned above, various cell manufacturing technologies have been established. A spheroid culture technique was introduced for PDL-MSCs with enhanced osteogenic potential ; therefo4OH/Triton X-100 and DNase solutions, resulting in an ideal scaffold, with intact extracellular matrices and resident growth factors, and that can support repopulation by allogeneic cells [Recently, decellularized materials have been introduced for periodontal regeneration. hPDL cell sheets were chemically decellularized by NHic cells . The samic cells . The decOur autologous transplantation study of PDL-MSC sheets proved their safety and feasibility for clinical use . However5)–(4 × 105) cells/dish and cultured in an osteoinductive medium  for 9–10 day. Culturing PDL-MSCs with osteoinductive supplements enhances bone regeneration in vivo [Because wisdom teeth are routinely extracted from young patients, there is a limitless source of cells to establish a cell bank of allogeneic PDL-MSCs. Extracted teeth were collected, and the periodontal ligament tissue of each tooth was dissected out. MSCs were isolated, cultured, and stocked at passage three as master cell banks. The master cell banks were thawed and cultured to construct working cell banks at passage five. Working cell banks were further cultured to make PDL-MSC sheets on temperature-responsive culture dishes. PDL-MSCs were seeded on temperature-responsive culture dishes at a cell density of  cells because they are considered to proliferate permanently. Human ES cells derived from the inner cell mass of blastocysts are pluripotent stem cells capable of differentiating into multiple cell lineages. iPS cells possess characteristics similar to those of ES cells and can be created from somatic cells by overexpression of four key transcription factors: Sox2, Klf4, Oct4, and c-Myc. Although the pathway and protocol for inducing PDL cells are not well understood, a method for inducing MSCs via neural crest cells from ES/iPS cells has been established . The devMSCs produce and secrete proteins and extracellular vesicles into the culture medium, and a recent study suggested that the conditioned medium may have regenerative potential. Nagata et al. collected and concentrated the conditioned medium of PDL-MSCs and used it in rat periodontal defect models . ProteomTo date, four clinical studies in which ex vivo expanded stem/progenitor cells were transplanted with biomaterials have been published, and all studies reported the safety and efficacy of their products for periodontal regeneration. Taking into account the concept of GTR, it is clear that the stem cells within the PDL tissue are the key cells for periodontal regeneration and homeostasis; therefore, the cytotherapeutic approach for periodontal regeneration has started with PDL-derived cells. Although many researchers have used MSCs derived from a variety of tissues when using a cytotherapeutic approach for periodontal regeneration, it may be more effective to use MSCs derived from periodontal tissue for cell therapy, considering that they may inherit the characteristics of the tissue of origin. Indeed, PDL-MSCs have shown the greatest potential to regenerate periodontal ligament tissue when compared with other sources of MSCs . In otheThe field of regenerative medicine is rapidly evolving through the fusion of stem cell biology and tissue engineering. Transplantation of MSCs is under trial not only for regenerative medicine but also in clinical applications for intractable diseases such as graft versus host rejection (GVHD) in expectation of its immune-regulatory function, and it has been approved in some countries. It is clear that an allogeneic approach is essential for the widespread use of regenerative therapy. We also investigated the allogeneic use of PDL-MSCs, and a clinical trial began in 2018. It is important to consider not only the efficacy, but also the safety, of cellular products, and industry-academia collaboration is necessary to expand this treatment. Although verification by clinical trials has not yet occurred, it is hoped that hMSC can assist in addressing unmet medical needs.


Male and female infants respond differentially to environmental stimuli, with different growth and neurodevelopmental trajectories. Male infants are more likely to be disadvantaged when subjected to adversity and show a higher risk of perinatal complications. However, the underlying causes of this sex-bias are not well defined and optimising the early life nutritional care may be necessary to minimise the “male disadvantage” that may be experienced early in life. Experimental models have demonstrated that animal milk composition differs according to offspring sex, suggesting that the tailoring of early life nutrition may be one mechanism to maximise health protection and development to infants of both sexes. However, evidence for a sex-specificity in human milk composition is limited and conflicting, with studies documenting higher milk energy content for either male or female infants. These data show sex differences, however, there has been limited compositional analysis of the current data nor strategies proposed for how sex-specific compositional differences in early life nutrition may be used to improve infant health. The present narrative review highlights that an improved understanding of sex-specific human milk composition is essential for promoting optimal infant growth and development. Human milk (HM) is the gold standard for infant nutrition, providing the necessary building blocks required for postnatal growth and development ,2. The bDespite international recognition of the importance of breastfeeding, not all infants can be exclusively breastfed. Some mothers indeed encounter challenges in establishing and/or maintaining lactation over the first six months of life ,27,28,29The present work constitutes a narrative review of the existing evidence regarding sex-specificity in HM composition in relation to sex-specific health outcomes such as the higher perinatal risk of mortality and morbidity observed in male neonates.Differences in perinatal outcomes between males and females have been recognised since the 1970’s as the “male disadvantage” . The conAt present, sex-specific differences in perinatal health outcomes, including neurological, metabolic and respiratory complications, with males having higher risk for poorer health ,38, remaMacaca mulatta) mothers of male offspring produced a lower volume of milk but with a higher energy content, whereas mothers of female offspring had greater volume of milk which was less energy-dense, resulting in similar total energy content of the milk produced for both sexes [Macropus giganteus) [Macropus eugenii) [Cervus elaphus hispanicus) mothers were observed to produce greater yields of milk with higher energy content for male calves, reflected by higher protein, fat and lactose content [taxa, the range of milk-borne compounds analysed in each study is limited and no information is available around hormonal concentration in milk produced for male and female offspring. Furthermore, the mechanisms driving sex-specific milk synthesis are currently unclear and need to be investigated in order to enable a better understanding of the phenomenon.Evidence from animal models  suggeststh sexes . Yet, mith sexes . In a both sexes . Evidencganteus)  and Tammeugenii)  were fou content . While tDespite data derived from experimental models, there remains a paucity of data around the potential for sex-specificity in HM composition in regards to nutrients and bioactive compounds. The potential for a sex-specificity has however been suggested by clinical research on twins . In thisNonetheless to date only six human studies have reported on the relationship between HM composition and infant sex ,52,53,54Importantly, there is emerging research demonstrating a sex-specific differential effect of animal milk bioactives on offspring outcomes. As an example, elevated cortisol in the milk of lactating macaques has been found to correlate with a more nervousness and less confident temperament, impacting male offspring to a greater extent than female . However.Evidence to date suggests that infants of different sex may have different nutritional and hormonal requirements. This underpins a need for sex-specific nutritional strategies for male and female infants, in order to optimise their growth and development. Experimental and limited clinical observations that maternal HM composition changes dependent on male or female sex, support this hypothesis. However, these reported sex-specific differences in HM composition require validation and clarification in additional human cohorts. These studies should aim to describe sex-specific differences in HM composition across the course of lactation and correlate differences with sex-specificity of infant outcomes, including sex-specific growth trajectories and sex-specific morbidity risk.


Chemical and biological sensing are crucial tools in science and technology. Plasmonic nanoparticles offer a virtually limitless number of photons for sensing applications, which can be available for visual detection over long periods. Moreover, cellulosic materials, such as paper, represent a versatile building block for implementation of simple, yet valuable, microfluidic analytical devices. This mini review outlines the basic theory of nanoplasmonics and the usability of paper as a nanoplasmonic substrate exploiting its features as a (bio)sensing platform based on different mechanisms depending on localized surface plasmon resonance response. Progress, current trends, challenges and opportunities are also underscored. It is intended for general researchers and technologists who are new to the topic as well as specialist/experts in the field. The quantitative description of plasmons in gold nanoparticles Mie, , has porThe most common configuration employed in nanoplasmonics is via the colloidal suspensions of noble metal nanoparticles. Several researchers have deeply studied and optimized the synthesis of nanoparticles in suspension with the aim of tuning the LPSR response by means of their size, shape and separation distance  has been employed for analyte separation and identification, a technique named as “Paper Chromatography” that takes advantage of the hydrophilicity of paper to run samples passively sensor devices from design, fabrication and potential applications. Although other reports have introduced the importance of paper-based devices and nanoplasmonics  can unfold a nanoplasmonic behavior depending on its size, composition, shape, geometry, and dielectric environment. At nanoscale dimensions, an incident electromagnetic wave behaves like a planar front wave and therefore, focusing of light is not required see . What haTypically, plasmonic nanoparticles are made of noble metals such as Au and Ag. On the one hand, gold nanoparticles are generally reported to be biocompatible  are single-use innovative analytical platforms capable of handling fluids and analyzing biochemical samples. The incorporation of microfluidics into paper-based devices results advantageous, in comparison to soft polymers  format where the user can employ a set of prefabricated microfluidic tools or manufacture the device on his/her own. For example, Lu et al.  developeThe flow control approaches discussed above are often crucial to engineer and implement different sensing mechanisms resulting in advantageous (bio)sensing devices whose results are visually observable. It is worth mentioning that keeping in mind the human vision system and visible spectrum features, the cyan to green transition zone at ~500 nm has been recently reported to be the optimal starting wavelength aiming at visualizing changes in plasmonic color . In this context, inter-particle distance can be controlled using specific interactions between the target molecule and MNPs. For example, methimazol, 2-mercaptobenzothiazole, and D-cysteine have been reported to agglomerate silver nanoparticles (AgNPs) embedded in nanopaper see , thus trScattering and UV-Vis absorbance of MNPs can also be strongly modulated by modifying their size. Consequently, etching of MNPs can be judiciously employed as a visually observable sensing mechanism, as well. For example, food spoilage monitoring can be carried out via paper-based devices incorporating MNPs; since food spoilage involves the release of ammonia accompanied by other volatile organic compounds that are able to etch AgNPs 2]+, forming AgNPs and leading to a uric acid sensing platform that can be implemented using paper-based analytical devices  can be controlled using catalase  can be considered a gold standard for protein detection. This immunoassay involves a series of immunoreactions between biomolecules  reported by chromogenic agents activated by enzymes labeling such immunoreactions . Importantly, Raman spectroscopy facilitates the determination of (bio)molecules via their highly specific Raman scattering sensing mechanisms which can be visually observable. Hence, the synergy between nanoplasmonics and paper-based substrates leads to simple, single-use and cost-efficient analytical devices. In fact, these devices are likely to meet the ASSURED criteria , which is crucial to further point-of-care and care-at-home devices with potential pre/clinical applications.EM-N conceived the overall manuscript. SM and EM-N contributed to writing, editing, and literature review.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.