Limited drug delivery to the brain is one of the major reasons for high failure rates of central nervous system (CNS) drug candidates. The blood–brain barrier (BBB) with its tight junctions, membrane transporters, receptors and metabolizing enzymes is a main player in drug delivery to the brain, restricting the entrance of the drugs and other xenobiotics. Current knowledge about the uptake transporters expressed at the BBB and brain parenchymal cells has been used for delivery of CNS drugs to the brain via targeting transporters. Although many transporter-utilizing (pro)drugs and nanocarriers have been developed to improve the uptake of drugs to the brain, their success rate of translation from preclinical development to humans is negligible. In the present review, we provide a systematic summary of the current progress in development of transporter-utilizing (pro)drugs and nanocarriers for delivery of drugs to the brain. In addition, we applied CNS pharmacokinetic concepts for evaluation of the limitations and gaps in investigation of the developed transporter-utilizing (pro)drugs and nanocarriers. Finally, we give recommendations for a rational development of transporter-utilizing drug delivery systems targeting the brain based on CNS pharmacokinetic principles. Central nervous system (CNS) diseases including neurological disorders and brain cancers, remain to be one of the major causes of disability and death worldwide . DespiteKnowledge about transporters expressed at the BBB and structural requirements for substrate binding and translocation via the transporters have been used for development of transporter-mediated drug delivery systems targeting the brain via specific transporter proteins. The strategy includes 2 processes: (i) transporter-mediated permeation of a drug itself, or in form of a prodrug or nanocarrier through the BBB and in some cases the cellular barrier of brain parenchyma; and (ii) release of the active (parent) drug from a prodrug or nanocarrier at the site of action inside the brain.More than a hundred attempts to develop transporter-utilizing (pro)drugs and nanoparticles/liposomes (hereinafter referred as nanocarriers) have been made to improve the uptake of CNS drugs into the brain. However, the majority of the (pro)drugs and nanocarriers has not reached clinical trials. This fact raises the question about the effectiveness of the approach and its further potential to be used in humans. In the present review, we provide a systematic summary of the current progress in development of transporter-utilizing (pro)drugs and nanocarriers for delivery of drugs into the brain. We critically evaluate the effectiveness of the approach and give recommendations for evaluation of brain delivery efficacy by transporter-utilizing delivery systems based on CNS pharmacokinetic principles. In addition, the advantages and limitations of the approach as well as future perspectives and gaps in our knowledge about the approach are discussed.The main role of the CNS consisting of the brain and spinal cord is to regulate normal functioning of the body through integrating and coordinating input signals and providing a proper response. In this respect, the maintenance of the homeostasis of the CNS microenvironment is vital  and achi2 per gram of adult human brain , and it is a key player in drug delivery to the brain Fig. . The reaan brain , 6 availan brain . The resan brain , 9. TJs an brain . These pan brain , 12. TJsan brain . Moreovean brain .Fig. 1ThThe BBB is a dynamic barrier, and its protective function is achieved by the presence of metabolizing enzymes, multiple membrane transporters and receptors, as well as interaction with the brain parenchymal cells accounting for 80% of the brain tissue volume . The braDespite the restricted entry of compounds to the brain, there are several routes by which essential nutrients and other molecules, including drugs, can permeate the BBB Fig. . They inPassive diffusion Fig.  is an enLarge molecular weight compounds, such as peptides and proteins, required for normal brain functioning can pass through the BBB via transcytosis Fig. , which cReceptor-mediated transcytosis (RMT) occurs in 3 phases: endocytosis, intracellular vesicular trafficking, and finally exocytosis  is a non-selective process with a lack of targeting specificity. It is driven by nonspecific electrostatic interactions between cationic proteins, for instance histone and avidin, and anionic microdomains on endothelial cells   .transcytosis of transporter-binding nanocarriers has been investigated and utilized in order to deliver drugs into the brain. The transporter-mediated nanocarrier delivery of drugs into the brain is discussed in the present review.In addition to RMT and AMT, CMT of small molecules across the BBB occurs after the interaction between a substrate compound and the specific transport protein, which can be expressed in either the luminal or abluminal membrane or both sides of the brain capillary endothelial cells Table . The exp+, Na+, K+, Ca2+ and Cu2+ produce and maintain electrochemical ion gradients across the cellular membranes which are subsequently used by secondary active ion-coupled transporters to deliver molecules in antiport  or symport  of these ions  or active transport. While passive transporters, such as glucose transporter, enable delivery of solutes across the cellular membrane along their electrochemical gradient without energy consumption, active transporters generate solute gradients across the membranes and exploit various energy-coupling mechanisms. Active transporters are divided into primary or secondary carriers based on the source of cellular energy used to drive the process. Primary active transporters such as the adenosine triphosphate (ATP)-binding cassette (ABC) transporters and ion pumps (ATPases) efflux solutes from the cell or into the organelles against their concentration gradient utilizing the energy of the hydrolytic reaction of ATP , 44. Ionese ions , 46. In ese ions .Most transporters expressed at the BBB belong to 2 major superfamilies such as ABC transporters or solute carriers (SLCs) which possess differences in substrate translocation. Protein expression of several SLC and ABC transporters has been quantified at isolated human brain capillary endothelial cells by quantitative absolute proteomics. Information about these transporters and their substrates is given in Table The transporters mediate not only the BBB passage of essential nutrients such as glucose, amino acids, and vitamins, but also the influx and efflux of the CNS drugs to and out of the brain Table , therebyEfflux transporters expressed at the BBB limit the brain access of such drugs, which are substrates of these transporters. For instance, efflux transporters such as ABCB1, ABCG2, and ABCC4 are considered to be a major factor limiting drug delivery to the brain . In contGreat efforts have been taken to deliver drugs into the brain, which resulted in the development of several drug delivery strategies –70. One The transporter-mediated drug delivery approach includes 3 main strategies: (i) modification of the drug structure in a way that a specific BBB transporter will recognize it as a substrate, e.g. “pseudo nutrient”, and deliver the drug across the cellular membrane; (ii) conjugation of the parent drug with a promoiety representing nutrient-substrate of the particular BBB transporter known as a prodrug strategy; (iii) decoration of the drug-loaded nanocarriers with a moiety representing substrate of the particular BBB transporter. The knowledge about functional expression of transporters at the BBB and structural requirements for substrate binding and translocation via the transporters gives tempting opportunity for rational design and development of drugs, prodrugs and nanocarriers, which can utilize the specific BBB transporter for brain delivery of CNS drugs , 72.in vivo should occur in a predictable manner. In case of the transporter-mediated prodrug approach, the rational design of prodrugs should take into account the functional expression and structural requirements for substrate binding and translocation via the transporter, as well as capability to be bioconverted by an enzyme at the target site , LAT1-targeted nanoparticles reduced the plasma membrane expression of the transporter in HeLa cells when measured 1 h after exposure  reported that a GLUT1-targeted nanodisk underwent endocytosis by clathrin-mediated endocytosis and energy-dependent macro-pinocytosis pathway  studied the ability of carnitine-coated micelles to utilize OCTN2 for transcytosis across Caco-2 cell monolayers , carnitine conjugated nanoparticles were reported to undergo caveolin-mediated, clathrin-mediated endocytosis and micropinocytosis in Caco2 cells drugs and nanocarriers has to be done based on the CNS pharmacokinetic principles in order to provide accurate information about the effectiveness of the approach.The estimation of target‐site concentrations of the drug in the brain is vital for understanding of the pharmacodynamic response. Importantly, only unbound drugs can pass the cellular membrane, undergo biotransformation (in the case of prodrugs), and interact with a target. Therefore, the evaluation of the efficacy of the delivery via transporter-mediated approach should be performed with the main focus on unbound concentration of the active (parent) drug at the specific site in the brain where the target is located.In general, a (pro)drug or nanocarrier administered by a specific route follows numerous pharmacokinetic processes such as absorption , distribution, metabolism, and excretion Fig. . These pThe drug delivery to the brain is characterized by three main neuropharmacokinetic determinants, such as rate of BBB permeation, extent of brain delivery and intra-brain distribution of drug, allowing to assess the brain delivery efficacy of developed transporter-utilizing (pro)drugs or nanocarriers. The detailed explanation of the CNS pharmacokinetic concepts and evaluation of these parameters can be found in previously published review .in vivo brain uptake index (BUI) method (in situ brain perfusion (in vitro models of the BBB (Clout), which represents the net passage out of the brain and includes passive and active BBB transport as well as metabolism in the brain and elimination to the CSF by bulk flow  and permeability surface area product (PS product). Both parameters describe measurements of clearances, rather than rates per se  method , in situerfusion , cerebraerfusion , 91, and the BBB . The rats per se . In chrop,brain) or the ratio of unbound concentrations in the brain ISF and plasma   is a cerebral microdialysis technique, which is used for continuous monitoring of unbound concentrations of the drug in the brain ISF and blood over the time via inserted probes into the brain parenchyma  are used for calculation of Kp,brain or can be combined with unbound fraction determined by equilibrium dialysis to calculate Kp,uu,brain using a combinatory mapping approach  . The Kp,rocesses . If to ahe brain –96. The renchyma . In addiapproach , 97. In , which can be estimated using an in in vitro brain slice method drugs and nanocarriers are distributed in and out of the brain parenchymal cells via transporters expressed at cellular membranes, the ISF concentration of the unbound (pro)drug or nanocarriers is not necessarily equal to that in the brain parenchymal cells  inside the brain parenchymal cells cannot be achieved in practice, indirect methods combining the brain slice assay and brain homogenate method have been established to estimate Cu,cell  estimated using the brain homogenate method and cerebral microdialysis can be combined with Vu,brain to estimate unbound (pro)drug concentration ratio in intra- and extracellular compartments of the brain  drugs, while Kp,uu,cell lower than unity describes preferable extracellular distribution within the brain.After crossing the BBB via specific transporter, a drug, prodrug or nanocarrier can distribute from the brain ISF into the brain parenchymal cells via the same transporter expressed at the cellular membrane followed by the release of parent drug (in case of prodrugs and nanocarriers) or binding to the cell constituents, e.g. phospholipids or cellular proteins Fig. . The inte method , 99–101.rain ISF . In resprain,ISF , while Vhe brain . In contal cells . Moreove Cu,cell , 104. Thuu,cell) . The parOne of the factors limiting development of drug delivery to the brain via a transporter-mediated approach is the complexity of evaluation of brain delivery efficacy of drugs. Here, we critically evaluate and discuss advances in development of currently reported transporter-utilizing (pro)drugs and nanocarriers in light of the main CNS pharmacokinetic principles.The need for the improvement of drug delivery to the brain arises from an insufficient efficacy of drug treatments. The development of transporter-utilizing (pro)drugs and nanocarriers for improved brain delivery begins from the understanding whether the poor efficacy of the drug is caused by low extent of the brain permeation and inadequate concentrations of the drug at target site or due to other reasons. Second, information about the localization of the drug target in the brain, which can be extra- or intracellular, as well as functional expression of the selected transporter in the brain is essential for the selection of relevant transporter to be targeted at the BBB and brain parenchymal cells. Thus, targeting of the transporters expressed only at the BBB would be useful in terms of delivery of drugs with extracellular targets in the brain, while utilization of the transporters expressed at the BBB and cellular membrane of the brain parenchymal cells is essential for delivery of the drugs with intracellular targets. Among reviewed studies, the following SLC transporters have been targeted for delivery of various drugs to the brain: organic cation/carnitine transporter 2 (OCTN2), glucose transporters (GLUTs), LAT1, sodium-dependent vitamin C transporter 2 (SVCT2) and glutathione transporters Tables  and III.+- and pH-independent exchanger of large branched-chain and aromatic neutral amino acids coupled with a heavy chain subunit   –169. In  SLC3A2) , 170. In SLC3A2) , 172 as  SLC3A2) , 173–175 SLC3A2) , 177. LA SLC3A2) –182. How SLC3A2)  to impro+-independent facilitated transport of glucose and other hexoses across the cellular membranes. The expression of GLUTs is cell type-specific and can be affected by disease conditions . GLUTs are responsible for the Nanditions . High afnditions –186 and nditions . The hignditions . Neuronanditions .+-dependent transport of L-carnitine and Na+-independent uptake of organic cations  inhibitors ketoprofen, ibuprofen, indomethacin, and naproxen; neuronal gamma-aminobutyric acid (GABA) transaminase inhibitor valproic acid, neuronal β-secretase modulator ferulic acid, antiviral efavirenz and several anti-cancer agents Table , III. HoImportantly, the selection of the transporter for brain delivery of drugs can be affected not only by expression profiles at the BBB and brain parenchyma with regards to drug target localization, but also by the structural features of the substrates. The small size of substrates, which nutrient transporters can efficiently transport, limits the number of parent drug molecules for which the transporter-mediated prodrug delivery approach can be applied. In addition, in terms of the prodrug approach, not all drugs can be conjugated with the promoiety to be recognized as a substrate of the transporter due to the difficulties in synthesis implementation or limits of the chemical structures of the compounds. Furthermore, the availability of cost-efficient (pro)drug synthesis or nanocarrier preparation allowing high-scale production should be taken into consideration.in vivo cerebral microdialysis technique, in situ brain perfusion technique and PET imaging in transporter-knockout animals or using selective transporter inhibitors and substrates drugs and nanocarriers. Different in vitro BBB cell culture models including primary and immortalized brain endothelial cell models, have been developed to facilitate the investigation of drug permeation across the BBB drugs, or nanocarriers for the BBB uptake should be confirmed. In general, the transporter-mediated passage of compounds across the cellular membrane can be investigated using several methods including bstrates , 196–198 be used . Althoug the BBB . Howeverin situ brain perfusion in rodents with the corresponding transporter substrates, i.e., amino acids, glucose, etc. However, it is important to remember that binding to the transporter does not guarantee the utilization of the transporter for cellular passage. Therefore, the uptake of the compounds in the presence and absence of the selective transporter inhibitor provides important information about the translocation of the drugs via the specific transporter across the cellular membrane. However, confirmation of the utilization of the targeted transporter for cellular uptake was performed for a limited number of (pro)drugs (Table m) and transport capacity (Vmax), are essential for understanding the capability of the (pro)drugs to utilize the transporter and planning pharmacokinetics studies. However, in some studies the kinetic parameters (Km and Vmax) were calculated from linear or non-saturable cellular uptake data, which does not produce reliable results (In terms of the developed transporter-utilizing (pro)drugs, the transport mechanism has not been investigated for more than third part of (pro)drugs Table . Among tgs Table . In addigs Table . The tra results , 123.The majority of the published studies on nanocarriers Table  lack thoin vitro does not provide direct evidence of the utilization of the transporter at the BBB or brain parenchyma in vivo. In addition, it is important to remember, that the ability to utilize the transporter does not guarantee a high extent of brain delivery, which should be evaluated in pharmacokinetic studies drugs and nanocarriers  studies .The quantitative evaluation of brain delivery efficacy via transporter-mediated approach based on the CNS pharmacokinetic principles is critical for understanding the usability of the strategy and decision-making on further development of the transporter-utilizing (pro)drug and nanocarriers. The following criteria should be investigated during the development of the transporter-utilizing (pro)drug and nanocarriers: whether (pro)drugs or nanocarriers crosses the BBB in intact form; whether (pro)drugs or nanocarriers reaches the target site in the brain (extra- or intracellular) and releases the parent drug at the target site at the adequate concentration to produce pharmacological effect. Eventually, the main determinant of brain delivery efficacy is the concentration of the active (parent) drug at site of action in the brain or an increase of the relative distribution of parent drug between the brain and other tissues after administration of prodrug or nanocarriers compared to parent drug dosing.in situ perfusion techniques in rodents with a reported rate of permeation constant (Kin). It is important to remember that the rate of BBB permeation should be differentiated from the extent of BBB permeation, which is described by Kp,uu,brain. The rate of permeation mainly indicates whether the compound crosses the BBB and how fast this process is. Therefore, although some studies claimed improvement of drug delivery to the brain via developed prodrugs based on the higher rate of BBB permeation of prodrugs compared to parent drugs, in fact, the rate of BBB permeation cannot be used for prediction of brain delivery efficacy.The brain uptake of one third of the reported (pro)drugs and nanocarriers was not investigated Table ,III. Forp,brain, AUCu,brain/AUCu,plasma and Kp,uu,brain values for released parent drug after prodrug dosing and drug itself  in the study in mice with brain metastases of breast cancer  in brain metastases of triple negative and HER2/neu-positive breast cancers drug or nanocarrier administration ed Table . Among ted Table , 134, gled Table  and ibuped Table . In conted Table , 128, gled Table , modifieed Table , dopamined Table  as well ed Table  demonstrnic acid , 134 andnic acid  when comnic acid . Howevernic acid . Althougin vivo efficacy of the nanocarrier-delivered drug was compared to the drug itself in disease models in five out of 16 studies listed in Table in vivo delivery into the target tissue, brain or brain tumor, was evaluated in any capacity. Shao et al. (2014) showed in U87 xenograft glioma mouse model that the loading of paclitaxel into dehydroascorbic acid-decorated micelles increased the drug efficacy  reported dehydroascorbic acid modified micelles loaded with itraconazole, which enhanced the antifungal efficacy of itraconazole in the CNS infectious mouse model drugs and nanocarriers should possess high affinity to the transporter, which can affect the passage of physiologically important transporter substrates . The posechnique , althougmembrane . This iset al. (2011) revealed significant interspecies differences in transporter protein expression of the BBB transporters drugs or nanopartilce/liposomes has proceeded to clinical trials. However, differences in transporter and enzyme expression and function in preclinical models and humans may subsequently lead to interspecies variations in brain delivery of (pro)drugs and nanocarriers and the release of parent drug. Uchida sporters . In ordeThe present review provides a systematic up-to-date summary of progress in development of transporter-utilizing (pro)drugs and nanocarriers for brain delivery of drugs. We revealed, that despite many attempts, which have been made to develop transporter-utilizing (pro)drugs and nanocarriers for brain delivery of different drugs, there are several factors limiting the rational development and successful application of the approach to humans. First, the mechanism of transporter-mediated prodrug transport across the BBB is not investigated thoroughly in the majority of studies. Especially, prodrug binding to intracellular binding site of the abluminal BBB transporter and prodrugs’ efflux mechanism to brain parenchyma remains unstudied. In the case of transporter-mediated nanocarrier delivery, binding to the targeted transporter should be more thoroughly investigated. In addition, the transport processes across the BBB into target cells within the brain parenchyma are complex, and the optimal utilization of the drug delivery strategy will require more research. Especially, the detailed knowledge of the nanocarrier-transporter complex internalization and following sorting processes and exocytosis would enable rational design of nanocarriers capable of efficient transcytosis across the BBB by minimizing the lysosomal degradation of the nanocarrier in the endothelial cells. This, in turn, can enable delivery of drug cargo in the target cell within the brain parenchyma. Second, information about enzymatic conversion of prodrugs at the BBB and within the brain is not known. Third, the main limiting factor is that the main CNS pharmacokinetic principles are not followed during the evaluation of drug delivery efficacy of nanocarriers and (pro)drugs, which leads to inaccurate interpretation of the results or inadequate investigation of the brain delivery of drugs. Thus, in order to achieve the desirable therapeutic effect and adequate safety of the drugs delivered to the brain via transporter-mediated approach, the focus should be on the thorough investigation of utilization of transporter for cellular uptake and detailed evaluation of the efficacy of drug delivery into the brain and within the brain. All in all, although the transporter-mediated drug delivery approach can provide tempting opportunities for drug delivery to the brain, it is currently difficult to assess the potential of the approach due to the lack of pharmacokinetic evaluation of the prodrugs and nanocarriers. Therefore, the strategy requires more thorough mechanistic and pharmacokinetic investigation in order to be used for brain delivery of therapeutics in humans.The authors declare no competing financial interest.


Aim: The aim of this study was to show the efficacy of intravitreal treatment with Bevacizumab (Avastin) in patients with secondary neovascular glaucoma, in different stages of the disease.Method: A retrospective study was performed on 67 patients with neovascular glaucoma. The main parameters evaluated were the patients’ history, slit lamp examination, visual acuity, ocular tonometry, fundus examination, gonioscopy, and visual field. Results: It was observed that the pathology had a preponderance in males of the 6th decade, with frequently unilateral damage. Patients were referred to an ophthalmologist when the diseases reached an advanced stage, usually when the visual acuity had no light perception and the intraocular pressure was over 45 mmHg. However, the treatment with Avastin intravitreal showed a good evolution, with regression of neovessels in the first 4-7 days and maintenance of intraocular pressure within normal limits in about 60% of cases, 3 months after injection.Conclusion: The most effective treatment in secondary neovascular glaucoma is the correct therapy of the main disease. The association of Avastin and laser photocoagulation leads to regression in iris and retinal neovessels.Abbreviations: anti-VEGF = anti-Vascular Endothelial Growth Factor, PDGF = Platelet Derived Growth Factor, bFGF = basic Fibroblast Growth Factor  This condition represents the final stage of the retinal ischemic complication [2].Angiogenesis occurs mainly through ischemia, but inflammation and hypoxia are also mechanisms that can lead to neovascular formation. It is known that there is a factor of angiogenesis, which by hypoxia activates the proliferation of neovessels [2].Hypoxia is involved in cases associated with proliferative retinopathy. By compensation, the diffusing part of the oxygen from the aqueous humor to the posterior leads to hypoxia of the iris. This causes the secretion of proangiogenic factors, such as VEGF , PDGF (platelet derived growth factor) and bFGF (basic fibroblast growth factor) [2].VEGF is the best-known proangiogenic factor and has 4 isoforms; variant A is most often involved in iris neovascularization [2].The angiogenesis process begins as follows: small spaces are formed between the cells of the endothelium capillary wall, allowing the passage of proteins and fibrinogen, the latter turning into fibrin and forming a temporary matrix for the neovessel. Then, the endothelial cells are organized, forming the vascular bud, and their proliferation leads to the formation of a thin vascular lumen [2].The neovessels are formed on a fibrovascular support (fibrovascular membrane), which contains inflammatory cells, macrophages, lymphocytes and collagen fibers :There are many causes that can lead to secondary neovascular glaucoma, among which the following should be mentioned .The diagnosis is based on the evaluation of the medical history , slit lamp evaluation and gonioscopy  and angiography with iris fluorescein to observe neovessels that are difficult to see in early stages [6].Positive diagnosis: slit lamp examination, intraocular pressure, gonioscopy, fundus examination, and visual field [1]. In this pathology, treatment efficiency is reduced, sometimes with discouraging results, but it is necessary to be followed because in its absence, the pathology evolves towards progressive deterioration of the visual acuity, followed by atrophy of the eyeball and significant pain. The prognosis is severe [7-9]. In addition to the local treatment with antiglaucoma eye drops and surgery, intravitreal administration of Bevacizumab (Avastin), an anti-VEGF agent, showed optimistic results. Bevacizumab can inhibit the neoformation of vessels, so it is used as a treatment in neovascular glaucoma. The half-life is estimated to be 20 days, and the clearance varies depending on certain constitutional factors. The intravitreal injection may cause side effects such as uveitis, subconjunctival hemorrhage, endophthalmitis, eyelid irritation and retinal detachment [7-9].Differential diagnosis of neovascular glaucoma: uveitic glaucoma, acute glaucoma, chronic closure of the iridocorneal angle, Iridocorneal Endothelial Syndrome, Fuchs heterochromia iridocyclitis  shows that the half-life of Bevacizumab in the vitreous is longer than that of other molecules, due to its small size and significant diffusion through the retinal layers.According to this study, Bevacizumab appears to have very good results, regardless of disease severity. A study by Mordenti et al.  demonstrated in a comparative study that Bevacizumab, Pegaptanib and Ranibizumab have antiproliferative and cytotoxic effects alike.Spitzer et al. [12]. In this study, despite the correct treatment, no improvement was obtained, and two of the patients underwent evisceration of the eyeball.In some cases, the poor control of high intraocular pressure leads to blindness and the eye may be very painful. In this case, the last treatment line represents the evisceration of the eyeball [The etiology of secondary neovascular glaucoma is numerous, but the main cause is represented by diabetes, complicated with proliferative diabetic retinopathy, but also central retinal vein occlusion.In our study, the main cause of neovascular glaucoma was diabetic retinopathy, a complication of diabetes mellitus. The high rate of diabetic retinopathy due to lack of glycemic control is the leading cause of vision loss. Most patients who are referred to a specialist have an advanced stage of the disease, without any treatment prior to presentation.The treatment of the background disease aims to stop the evolution of neovascularization, minimize the impact on the quality of life, functional preservation of vision and preservation of the eyeball.Given the patient’s status and the value of intraocular pressure at presentation, the combination between intravitreal injection of Avastin and retinal laser pan photocoagulation, showed good results in over 50% of the examined patients.Prophylaxis of the background disease is the only effective method to prevent the occurrence of neovascular glaucoma. Conflict of Interest statementThe authors state no conflict of interest.Informed Consent and Human and Animal Rights statementInformed consent has been obtained from all individuals included in this study.Authorization for the use of human subjectsEthical approval: The research related to human use complies with all the relevant national regulations, institutional policies, is in accordance with the tenets of the Helsinki Declaration, and has been approved by the review board of “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania.AcknowledgementsNone.Sources of FundingNone.DisclosuresNone.


Pancreatic cancer has the seventh highest death rate of all cancers. The absence of any serious symptoms, coupled with a lack of early prognostic and diagnostic markers, makes the disease untreatable in most cases. This leads to a delay in diagnosis and the disease progresses so there is no cure. Only about 20% of cases are diagnosed early. Surgical removal is the preferred treatment for cancer, but chemotherapy is standard for advanced cancer, although patients can eventually develop drug resistance and serious side effects. Chemoresistance is multifactorial because of the interaction among pancreatic cancer cells, cancer stem cells, and the tumor microenvironment (TME). Nevertheless, more pancreatic cancer patients will benefit from precision treatment and targeted drugs. This review focuses on the immune-related components of TME and the interactions between tumor cells and TME during the development and progression of pancreatic cancer, including immunosuppression, tumor dormancy and escape. Finally, we discussed a variety of immune components-oriented immunotargeting drugs in TME from a clinical perspective. Pancreatic cancer is one of the deadliest malignancies. Despite substantial improvements in the survival rates for other major cancer forms, pancreatic cancer survival rates have remained relatively unchanged since the 1960s. Pancreatic cancer is usually detected at an advanced stage and most treatment regimens are ineffective, contributing to the poor overall prognosis . Pancreain situ implantation model. Pancreatic duct epithelial cells (Kras G12D-PDECs) originally representing KrasG12D were injected into the pancreas of C57BL/6 wild-type (WT) mice with the same gene by GFP labeling (GFP-KRAS G12D-PDECS). This model Outlines histologically the preinvasive stage of pancreatic intraepithelial neoplasia (PanIN) that develops and induces a similar intrapancreatic immune response , resulting in impaired T cell protein translation, leading to a lack of antigen-dependent proliferation  with diverse and complex functions . After bPSCs are stellate stationary stromal cells that appear as myofibroblast-like cells located in the exocrine region of the pancreas , 74. PSCIn the progression of pancreatic malignant tumors, fibroblasts mainly regulate the invasion of tumor cells, leading to extensive invasion and distant metastasis, but they are also closely related to tumor immunosuppression . ClinicaAt the invasion stage of PDAC, a variety of immune cells participate in the tumor immune response of the body. Like Tregs, B cells also generate regulatory B cells (Bregs) through their own activation and release chemokine CXCL13 to infiltrate the microenvironment , which iPDAC cells can facilitate the colonization of tumor cells by forming a supportive microenvironment at the site of metastasis through a series of cells and cytokines such as circulating tumor cells (CTCs), which is called the premetastatic niche. The formation and stable state of pre-metastatic niche is the key to the progression of PDAC and can also induce tumor dormancy at the metastatic site, and thus relapse at the metastatic site. Intercellular communication is the key to niche formation before metastases, the most common of which is liver metastases . The forWhen PDAC cells spread to distant organs such as liver and lung, due to the limitation of growth microenvironment, tumor cells will be in non-proliferation or equilibrium state, that is, cell proliferation rate is equal to its death rate, which is called tumor dormancy. The mechanisms of PDAC cells dormancy can be divided into three categories: cell dormancy, angiogenic dormancy, and immune-mediated dormancy. The main processes include the cellular mechanisms that push a small number of DTCs (disseminated tumor cells) into a dormant state, the balance between tumor angiogenesis related cell proliferation and death, and the immune system maintaining a constant number of proliferating tumor cells –125. In Immunosuppressive effect in microenvironment is an important factor in the progression of PDAC. In the process of immunosuppression, a variety of cells involved in immune function transmit information through cytokine pathways or intercellular transduction pathways, such as Notch, TGF-β, Wnt, Hippo, Fas/FasL, Hh, et. These signaling pathways are closely related to each other, with extensive mutual interference, and play a communication role in the immune-related microenvironment of tumors  signaling mediates PDAC immune-related behavior, mainly through the regulation of CD44+CD24+epithelial-specific antigen (ESA)+ pancreatic cancer stem cells . The maiTransforming growth factor-Beta (TGF-β) and it signaling pathway are key regulators of PDAC proliferation and differentiation, and their effects depend on TME. Changes in the microenvironment feedback to TGF-β signaling pathway may lead to tumor cells apoptosis or progression resulting in immunosuppression, they are generally viewed as separate fates for TGF-β-stimulated tumor cells, and opposite poles of the duality of TGF-β in cancer, or induce an EMT that promotes tumor invasion and metastasis and promote CSCs heterogeneity and drug resistance , 162. TGvia suppressing the T-cell responses  and transcriptional coactivator with PDZ-binding motif (TAZ) are the two major downstream effectors of the Hippo pathway. YAP and TAZ regulate the behavior of pancreatic stellate cells and the recruitment of TAMs and MDSCs. Among them, YAP plays a dominant role, whose activation is associated with immune dysfunction and induces the recruitment of myelogenous suppressor cells, while counter-inhibition supports the infiltration of antigen-presenting macrophages and the activation of effector T cells, leading to immunosuppression and progression , 178.In today’s medical era, PDAC, as an incurable malignant tumor, escapes from chemotherapy or targeted therapy due to inherent genomic instability . Due to CTLA-4 is an immune checkpoint receptor expressed on Tregs that activates conventional T cells . It’s alin vitro and in vivo and to protect tumor cells from being killed, which unlocked the door of T-cell-based cancer immunotherapy  and natural killer cells (NK cells). These cells recognize and to varying degrees alter tumor cells and their cellular environment with cytokines produced by paracrine and autocrine, which is also named tumor immune microenvironment (TIME). It is particularly important to accurately understand the immune components-related signal transduction pathways, in which the role of the signaling pathways involved in different parts of the TME is a prerequisite for better understanding of preclinical and clinical model data to design better treatments for signaling pathway targets in PDAC. In the treatment of PDAC, traditional chemotherapy has been regarded as an effective method of disease remission, but clinical data show that the effectiveness is only limited to a subset of patients. Therefore, immunotherapy has become a popular direction of clinical research, with its advantages of persistence, universal applicability, and relatively low toxicity. Checkpoint inhibitors have always been a hot spot in clinical research and application. In addition, the following clinical experiments show that PDAC immune checkpoint inhibitors combined with chemotherapy or radiotherapy can enhance the sensitivity of radiochemotherapy, reduce the toxic and side effects of radiochemotherapy, improve the efficacy of radiochemotherapy and reduce the occurrence of treatment resistance. Although immunotherapy is not an all-powerful treatment, it opens a new field in the treatment of PDAC with a very high degree of malignancy. With the progress of drug clinical research, immunotherapy will become one of the main treatment methods for tumors. Given the advances in our understanding of the PDAC microenvironment and emerging strategies, we have reason to be hopeful for the successful treatment of PDAC in the future.JZ completed the main content of the article. SH and RL gave guidance and corrected the mistakes. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cell Therapy, Liver Diseases, and Regeneration, we aimed to gather a series of articles that address issues at the forefront of cell therapy and liver regeneration. Review articles and original research articles were selected for the liver injury models established to evaluate the functions of donor cells, cutting-edge imaging technology which enables tracking donor cells in the host liver in the single-cells manners with a course of time, and the mechanisms of liver cancers and liver regeneration which guide the translation of stem cell therapies from basic research to pre-clinical and clinical applications.The liver is the primary organ maintaining the homeostasis of nutrients and energy metabolism. It plays a role in deoxidation, glucose storage, secretory protein synthesis, and the production of bile required by the digestive system. Due to the complexity and abundance of its functions, the liver constantly is exposed to pathogenic factors that can lead to hepatitis, cirrhosis, and liver cancer, resulting in different forms of acute and chronic liver disease. Despite efforts to understand and treat liver diseases, when liver failure occurs, liver transplantation is still the most effective treatment. Cell-based therapy has become a potential alternative to liver transplantation. In experimental, preclinical, and in a few small clinical trials, hepatocytes and postnatal stem cells  have been reported to effectively rescue or reduce liver injury. Yet, each of these cell or stem cell therapies has its own hopes and challenges, which require more research to become an effective treatment to help patients. In the Research Topic: in vitro with precisely designed growth factor cocktails in a stepwise manner. The functional maturation of these hepatocyte-liked cells can be assessed by their gene expression patterns, functional protein secretion, and capacity for metabolism . However, it is generally believed that in vivo evaluation of cell function has become the experimental gold standard. Due to the genetic and physiological similarities with humans and the capacity for genetic manipulation, mouse models have become the most favored and widely used model in the field of regenerative medicine. In the review by Du et al., key information on current mouse models simulating acute or chronic liver injuries was systematically summarized for studying the aging process and developing cell therapy strategies, and the “Five characters”, including, analogy, reproducibility, reversibility, adequate and appropriately body size to obtain adequate blood and tissue sample, were the suggested standards for an ideal animal model.When it comes to cell therapy or stem cell therapy for liver disease, the quality and quantity of donor cells matter the most. Stem cells, whether pluripotent stem cells or multipotent stem cells, can be gradually induced into hepatocyte-like cells Cheng et al., the authors established a mouse model of spontaneous HCC induced by oncogenes based on the insertion of transposon-mediated oncogene (AKT and NRASV12) into the genome of hepatocytes to induce tumorigenesis. Using this model, they found two novel HCC cell lines with distinct features of lipid metabolism related to cancer stemness and differential interplay with the immune system. This demonstrated that the syngeneic HCC mouse model is a practical tool for studying cancer stemness and discovering new therapies for liver cancers.Properly designed mouse models are also suitable for studying liver diseases or cancers. In the study by ex vivo information and do not allow the dynamic behavior and interaction patterns of immune cells in the inflammatory response to be evaluated in the unique anatomic structure and microenvironment of the liver. Wang and Wang reviewed exciting new findings from research that employs intravital microscopy (IVM), a powerful tool for directly observing cellular events within the intact organ of living animals. IVM can be applied to understand the immune composition and microenvironment in the healthy liver, as well as highly dynamic processes, such as the behavior of immune cells at a single-cell level during the process of inflammation. However, as they concluded at the end of the review, Wang and Wang pointed out that although IVM has been widely used to study the inflammatory response in the liver, most of these studies use artificial models that cannot fully reflect actual liver diseases. The combination of IVM with advanced spatial transcriptomic and proteogenomic techniques may make it possible to obtain comprehensive spatio-temporal information on inflammatory response and provide broad prospects for the diagnosis and treatment of liver disease.Of course, the immune system is involved in the pathogenesis of any form of liver diseases. Understanding the inflammatory responses under physiological and pathological states has attracted the attention of researchers who are focused on liver damaging and regeneration. In recent years, advanced techniques, including single-cell sequencing and spatial transcriptomics, have enabled researchers to better understand the relationship between liver inflammation and disease. However, these approaches only provide static and Yang et al. focused on summarizing and reviewing the progress, challenges, and prospects of mesenchymal stem cell (MSC) therapeutics for treating primary biliary cholangitis. Different from hepatocyte-related liver injuries, primary biliary cholangitis (PBC) is a cholestatic autoimmune liver disease, which is characterized by the gradual destruction of small intrahepatic bile ducts, eventually leading to cirrhosis, liver failure and even hepatocellular carcinoma. Although FDA-approved drugs are available, only some patients respond to treatment. And for patients with end-stage PBC, liver transplantation remains the only effective treatment. By reviewing basic research, preclinical research, and ongoing clinical trials, mesenchymal stem cells are shown to be a new option for treating PBC. By summarizing and discussing the current challenges and prospects of MSC-based therapy in clinical application, the authors hope to accelerate the application of MSCs to clinical practice settings, especially for refractory diseases such as PBC.Lastly,


The ‘plainification of materials’ has been conceptualized to promote the sustainable development of materials. This perspective, for the first time in the field of biomaterials, proposes and defines ‘plain metallic biomaterials (PMBs)’ with demonstrated research and application case studies of pure titanium with high strength and toughness, and biodegradable, fine-grained and high-purity magnesium. Then, after discussing the features, benefits and opportunities of PMBs, the challenges are analyzed from both technical and regulatory aspects. Regulatory perspectives on PMB-based medical devices are also provided for the benefit of future research, development and commercialization.  Metal implants may release ions or particles that contain matrix constituent elements such as aluminum, nickel, cobalt and chromium during service, which may induce adverse tissue reactions and cause health hazards after accumulation to threshold dosage . Given tThe widely used MBs, such as stainless steel, cobalt-based alloys, titanium and titanium alloys, were first applied in the engineering field before being gradually expanded to the medical field. Some elements added to pure metals in the form of alloying are potentially harmful to the human body. At first, biocompatibility as a key property of biomaterials was not given enough attention, because the focus then was on the mechanical and tribological properties and corrosion resistance, which were the big historical issues in the development of MBs. With the emergence of PM processes and technologies, the preparation of biomedical plain metals or alloys with simple compositions has exhibited promising applications.Pure titanium has fine biocompatibility and biomechanical adaptability, which makes it successful candidate in dental and orthopedic implants . HoweverContemporary researchers have attempted to take advantage of the excellent biocompatibility of pure titanium while improving its comprehensive mechanical properties and wear resistance, so as to expand its potential applications . Pure tiPure titanium with ultrafine grain characteristics has many advantages. It can effectively avoid the ion toxicity of alloying elements while keeping the composition simple. Meanwhile, the improvement of its mechanical properties can reduce the size and footprint of the implant. Products currently developed using such material are hopeful and expanding. For example, dental implants made of high-strength pure titanium with nanostructures can be as thin as 2.0 mm in diameter, which can effectively retain the alveolar bone and maintain good osseointegration . Figure Biodegradable magnesium is a recent research hotspot, magnesium is an element in the human body that exists in bones and muscles , 35. Mag2+ ions generated during degradation can promote the osteogenic differentiation of stem cells in the periosteum at the fracture site, which benefits the regeneration of bone tissue around the implant and accelerates fracture healing. As a result, high-purity magnesium has a great potential for orthopedic internal fixation implants [in vivo study results show that high-purity magnesium screws will soon be developed for the treatment of femoral neck fractures [Compared with magnesium alloys, high-purity magnesium has no alloying elements added, which means no secondary corrosion in the matrix to cause micro-galvanic corrosion . The degimplants . For exaimplants , 47. Proractures  and for ractures .et al. [in vivo corrosion of pure magnesium and other degradable materials have been developed, which could provide a convenient and efficient method for future product development [However, the mechanical properties of pure magnesium itself are relatively low. In order to achieve stable fixation, the size of pure magnesium implants is larger than that of conventional titanium implants, thus releasing an increased volume of hydrogen. The gas generated by its rapid degradation may cause complications such as superficial skin necrosis and long-term osteolytic lesions, which need to be reduced in order to achieve successful clinical outcomes . As a reet al.  found thelopment .In addition, Zn metal and alloys, given their degradability, biocompatibility and moderate mechanical properties, have become one of the very promising candidates for biodegradable implants . The Zn At the same time, with the application of novel processing technologies including ECAP, RS and thermo-mechanical treatment in MBs such as titanium, magnesium and zinc, and the research on the deformation mechanism, enhancement of mechanical property and improvement of corrosion resistance of pure metals or simple alloys, the effects of grain refinement, texture and wide stacking faults on materials’ performance have been thoroughly investigated and gradually revealed , 60. In Taken together, we propose the definition of PMBs as pure metals or alloys with relatively simple compositions, which have unique characteristics including precise control of the microstructure through processing technologies, excellent mechanical properties and biocompatibility, and safe for long-term medical services.We have established that PM a significant has positive impact for MBs and their medical devices. Multiple improvements can be made in terms of the composition, mechanical performance, ease of processing, potential of application, impact on health and environment, and cost-effectiveness. The features, benefits and potential opportunities of PMBs are summarized in Although PMBs have outstanding application prospects, they face challenges in the following aspects. The preparation process of new materials needs to be strengthened in terms of stability. Most of the PMBs possess the characteristics of new materials and new processes. The processing methods and follow-up treatments of PMBs are mostly customized. There is a lack data and experience in the structure–property relationships of PMBs. Due to the uniqueness of processing methods such as SPD, there may be uneven structures, high residual stress and obvious anisotropy in different processes. Subsequently, the manufacturing of medical devices is troubled with increased uncertainty and instability. Therefore, it is necessary to continuously explore reasonable methods and technologies to improve the consistency of material properties for the superior performance of medical devices.Standards and evaluation methods for PMBs need to be established urgently. When metals transform from coarse grains to ultrafine grains and nanocrystals, the original method of microstructure evaluation using optical metallographic microscopy is no longer applicable. Novel methods and evaluation standards are required, such as using electron backscatter diffraction for microstructure evaluation . In addiThe challenges brought about by product design merit attention. The vast majority of implants currently on the market are designed based on the mechanical properties of traditional materials. With novel materials such as PMBs, product designs must be adapted. For example, in order to achieve a low-profile design, thinning or reducing the diameter of the implant is needed. Taking the bone plate for the treatment of long bone fractures of the extremities as an example, the use of high-performance pure titanium can meet the needs of tensile stress, but the elastic modulus of the material does not change much after plainification. Thinning of implants will affect the stability of the fracture after fixation, and increase the displacement and dislocation of the fracture, resulting unsatisfactory clinical outcomes. Therefore, the application of each new material should be holistically considered and reviewed in multiple dimensions such as material properties, biomechanics, and clinical applications, rather than simply emphasizing the improvement of performance in one aspect.in vivo studies [In addition to technical challenges, innovative materials, processes, designs and products will pose new regulatory challenges. These pose the challenges of safety and efficacy evaluation and supervision. To enter innovative materials and their medical products into the market, the product development phases such as design verification and validation, production, quality inspection, clinical evaluation, registration and marketing are proven crucial, beyond the basic research in materials science, toxicology, biology and  studies . The proRegulatory science is ‘the science of developing new tools, standards, and approaches to assess the safety, efficacy, quality, and performance of regulated medical products’ . To promIn March 2021, NMPA also issued the ‘Announcement on the Registration Items of Medical Device Master File’ (No. 36 of 2021) . MedicalIn April 2021, the Center for Medical Device Evaluation of NMPA created a biomaterial research innovation and cooperation platform . This plPMBs can significantly improve the comprehensive properties of MBs. While maintaining the simple composition, PMBs have excellent biocompatibility and biosafety that traditional materials do not provide. As a result, new opportunities are created for PMBs in the medical devices market. We have discussed successful case studies where pure titanium can achieve the strength of titanium alloys, and where pure magnesium can improve mechanical properties while reducing corrosion rate. Such approaches provide possibilities for improved design and clinical applications of innovative medical devices. Currently, PMBs are being tested in the fields of oral, orthopedics and cardiovascular implants, and the emergence of these innovative PMB-based medical devices presents regulatory challenges. The development of regulatory science for medical devices could provide scientific evidence to ensure the safety, efficacy, performance and quality of PMB-based medical devices under the premise of both improved benefits and reduced risks.rbac093_Supplementary_DataClick here for additional data file.


H. pylori infection and gastrointestinal microbiota metabolites in functional dyspepsia, focusing on the possible mechanisms by which dysbiosis of the gastrointestinal microbiota contributes to the pathogenesis of functional dyspepsia. Several studies have confirmed that dysbiosis of the gastrointestinal microbiota may cause the occurrence and progression of functional dyspepsia by disrupting the biological barrier of the intestinal mucosa, by disturbing the immune function of the intestinal mucosa, or by causing dysregulation of the microbial-gut-brain axis. Probiotics and antibiotics have also been chosen to treat functional dyspepsia in clinical studies and have shown some improvement in the clinical symptoms. However, more studies are needed to explore and confirm the relationship between dysbiosis of the gastrointestinal microbiota and the occurrence and progression of functional dyspepsia, and more clinical studies are needed to confirm the therapeutic efficacy of microbiota modulation for functional dyspepsia.Functional dyspepsia is a clinically common functional gastrointestinal disorder with a high prevalence, high impact and high consumption of medical resources. The microbiota in the gastrointestinal tract is a large number of families and is one of the most complex microbial reservoirs in the human body. An increasing number of studies have confirmed the close association between dysbiosis of the gastrointestinal microbiota and the occurrence and progression of functional dyspepsia. Therefore, we reviewed the role of dysbiosis of the gastrointestinal microbiota,  Functional dyspepsia (FD) is one of the most common types of functional gastrointestinal diseases (FGIDs) in clinical practice, with a high prevalence affecting 10%–30% of adults and 3.5%–27% of children worldwide . The maiHelicobacter-pylori (H. pylori) infection, post-gastrointestinal infection, diet, genetics, mental and psychological factors , cholic acid, choline metabolites, phenols, lipids, carbohydrates, etc. These metabolites may be harmful or beneficial to the human body, and are closely related to human health and the occurrence and progression of many diseases. Clostridium group of Firmicutes phylum, as well as by Lactobacillus, Bifidobacterium, Eubacteriaceae and Fecal bacteria using some dietary fiber, resistant starch, oligosaccharides and other compounds that are not easy to digest in the intestinal tract (+) and smooth muscle cells (SMCs) through tight intercellular gap junctions , interleukin-1β (IL-1β), interferon-gamma (IFNγ), interleukin-8 (IL-8) and other inflammatory factors are released to disrupt the body’s immune system and induce inflammatory responses , peptidoglycan, and sphingolipids, which are mainly produced by Bifidobacterium, esponses . Sphingoesponses . Indole-tabolism .H. pylori is a Gram-negative that colonizes mainly the stomach and duodenum and interacts with the gastrointestinal microbiota. H. pylori colonization in the stomach and duodenum can cause changes in pH and mucosal damage in the stomach and duodenum, which can further cause colonization by other bacteria, leading to changes in the gastrointestinal microbiota. Sequencing of 16S rRNA genes showed significant differences in the abundance of bacteria on the gastric and duodenal mucosa between H. pylori-positive and H. pylori-negative subjects, with H. pylori- patients having significantly higher levels of Helicobacter, while the relative abundance of the phylum Actinobacteria, Bacteroidetes, Firmicutes and Clostridium were significantly lower , and transforming growth factor-β (TGF-β) levels were increased, consistent with increased T-regulatory cell responses  characterized by an increased concentration of bacteria in the small intestine, is also an important mechanism in the pathogenesis of FD . UsuallyBacteroides, Bifidobacterium, Escherichia coli can also overproduce neurotransmitters such as GABA  inflammation on microglia by increasing LPS ectopic and activating LPS-binding proteins (LBP) activation of the pathway . In anotLactobacillus and Alistipes in the intestine is positively correlated with the concentration of saturated long-chain fatty acids (SLCFAs), and the increased concentration of SLCFAs in the intestine can promote the contraction of intestinal smooth muscle, which causes increased intestinal motility , as an immunomodulatory factor, is present in large quantities in the gastrointestinal tract and is involved in the regulation of gastrointestinal motility and sensation . Dysbiosmotility . The injIn addition, there was a correlation between the gastrointestinal microbiota and the visceral pain pathway, where the presence of inflammatory stimuli in germ-free mice was found to diminish pain perception, while the use of probiotics effectively improved the pain response in mice; indicating that the presence of the gastrointestinal microbiota can influence the onset and progression of gastrointestinal disease processes not only by affecting gastrointestinal myoelectric activity, but also by affecting visceral nociception, among other pathways .Bacillus coagulans MY01 and Bacillus subtilis MY02) versus placebo for the treatment of patients with FD, the efficacy of the treatment group with probiotics was significantly higher than that of the placebo group, but there was no significant difference between the efficacy of patients in the placebo group who were also using proton pump inhibitors and the treatment group  was significantly more effective than the placebo group in improving a variety of clinical symptoms such as eructation, bloating, belching, and acid reflux in patients (Lactobacillus rhamnosus was effective in preventing FGIDs in children with milk allergy (H. pylori and thus may improve H. pylori-associated dyspepsia symptoms, but some studies have also found that probiotics are still effective in improving symptoms in FD patients with H. pylori-uninfected (Since dysbiosis of the gastrointestinal microbiota is closely related to the occurrence and progression of FD, regulation of the gastrointestinal microbiota becomes one of the potential therapeutic modalities for FD. In a randomized controlled trial using probiotics (nt group . In anotpatients . It was  allergy . The Ext allergy . A largeinfected .Firmicutes phylum, which dominates gastrointestinal microbiota and plays an important regulatory role in maintaining immune homeostasis in the intestine. In addition, abnormal metabolites of the gastrointestinal microbiota can also disrupt the intestinal biological barrier and immune barrier. Both dysbiosis and abnormal metabolites of the gastrointestinal microbiota can mediate the occurrence and progression of gastrointestinal diseases by disrupting the intestinal mucosal barrier, disturbing the intestinal immune function, and causing dysregulation of the microbial-intestinal-brain axis (The mechanism of FD due to dysbiosis of the gastrointestinal microbiota mainly includes two situations: on the one hand, the abnormal composition and abundance of the gastrointestinal microbiota itself causes gastrointestinal tract dysfunction, and on the other hand, the change of metabolites due to the alteration of the gastrointestinal microbiota leads to abnormal gastrointestinal tract function. A large number of basic and clinical studies have shown that there are dysbiosis of the gastrointestinal microbiota such as decreased diversity and abundance of the gastrointestinal microbiota in FD patients and animals, especially the decrease of relative abundance of ain axis .H. pylori eradication drugs are commonly used in clinical treatment to treat FD. A large number of studies have confirmed that H. pylori eradication drugs can effectively inhibit the activity of H. pylori in the gastrointestinal tract, while probiotics can effectively regulate the microbiota in the gastrointestinal tract and inhibit the growth of harmful bacteria, thus protecting the barrier function of the gastrointestinal mucosa and immune function and effectively preventing and treating the occurrence of FGIDs (Since dysbiosis of the gastrointestinal microbiota is one of the pathological mechanisms in the pathogenesis of FD, probiotics and of FGIDs . Currentof FGIDs , functioof FGIDs , functioof FGIDs  and otheStreptococcus and Lactobacillus, while after 1 week of life the intestinal tract is dominated by anaerobic bacteria such as Bifidobacterium, Clostridium and Bacteroides, but when they begin to add complementary foods, the gastrointestinal microbiota also begins to become diverse and relatively stable (The gastrointestinal microbiota in human body is influenced by many factors, including daily diet, living habits, drug consumption, genetics, environmental factors, stress factors, etc. These factors have a certain influence on the structure and composition of the gastrointestinal microbiota. And the gastrointestinal microbiota in humans varies with age, and studies have shown that the intestinal tract of newborns is dominated by the colonization of parthenogenic anaerobic bacteria such as Enterobacteriaceae, y stable . Due to


Performance monitoring and evaluation are key to quality improvement in maternal and child healthcare in Sub-Saharan Africa. This study presents the experience of designing and implementing bottom-up and integrated performance evaluation tools for care pathway to monitor and manage maternity healthcare services. The research project involved four health districts and relative reference hospitals, supported by the NGO Doctors with Africa CUAMM in Ethiopia, Tanzania, and Uganda. The maternal and childcare pathway developed consists of 23 indicators, calculated at hospital and district level, relating to pregnancy, childbirth and first year of life phases. The authors developed staves and performance maps, as graphical representation tools, to display longitudinally integrated health services provision performance along the continuum of care. Substantial variation was observed between the phases of each maternal and childcare pathway and across the care pathways of the different analysed settings. The most impressive results across the four settings are: 1) regarding pregnancy phase, that women tend to attend more than four antenatal classes, still with a quite high drop-out rate, and are largely tested for syphilis, 2) with respect to childbirth, that there are varying percentage levels in terms of C-sections, episiotomies and peri/intra-partum asphyxia cases, and 3) as it regards first year of life, there emerges scope for improvement considering the vaccination coverages attained for pentavalent, measles and polio vaccinations. Thanks to the collaboration with local managers and health professionals, the maternal and childcare pathway allowed to monitor the changes in the quality of maternity services provided within the analysed contexts. The benchmarking approach encouraged local professionals to learn from other settings. The use of such tool allowed the development of targeted quality improvement actions, shared among all involved stakeholders.• In collaboration with local professionals, we designed and implemented an integrated pathway for maternal and childcare, covering the phases of pregnancy, childbirth and first year of life.• Benchmarking performance results encouraged collaboration among professionals and allowed the identification of actions to improve the provision of maternal and childcare services.


Metabolic syndrome (MS), a cluster of metabolic risk factors, ranging from abdominal obesity, dyslipidaemia, hypertension, type 2 diabetes and non-alcoholic fatty liver disease , is one Therapeutic approaches to MS emphasize lifestyle changes including dietary interventions associated or not associated with physical activity, especially to reduce body weight and the associated risk of cardio-metabolic diseases. Notwithstanding the well documented beneficial effects of caloric restriction regiments in reducing body weight and improving body composition in both humans and animals ,10,11,12Together with dietary interventions, exercise therapy helps to both prevent and mitigate the impact of MS. Chronic resistance and endurance exercise training, alone or in combination, reduce body weight, blood pressure, and improve lipid profiles, e.g., raising high-density lipoproteins (HDL) and lowering triglycerides ,18,19,20This Special Issue of Nutrients, “Diet, Exercise, and the Metabolic Syndrome: Enrollment of the Mitochondrial Machinery” aimed to further elucidate the biochemical and molecular links between metabolic disturbances, mitochondrial structural/functional changes, and lifestyle interventions which still remain not completely understood and urgently need further investigation.The Special Issue collected three original articles on the topic. These articles allow the reader to focus on whether and how diet, exercise and bioactive compounds impact whole body metabolism and the mitochondrial compartment in metabolically relevant tissues. In particular, the results presented and discussed in the three articles allow the following questions to be answered:Can resistance exercise reverse diet-induced obesity-related deleterious metabolic and inflammatory effects?What are the molecular and biochemical factors involved in the beneficial effects of mild endurance exercise in settings of energy restriction?Can alterations of polyunsaturated fatty acids (PUFA) metabolism impact mitochondrial functions?Pinho and co-workers, with the aim to investigate whether resistance exercise can reverse whole-body and skeletal muscle obesity-induced deleterious metabolic and inflammatory effects, used a new training protocol, consisting of ladder climbing, rarely applied in rodents, to resemble the typical resistance exercise in humans . This prThe beneficial effects of physical activity go beyond skeletal muscles and involve several adaptations in other organs . In the De Lange and co-workers tested a very mild endurance exercise protocol on rats which resulted in positively affecting the brain and skeletal muscles under conditions of energy restriction involving, precisely, BDNF, mammalian target of rapamycin (mTOR) activation and thyroid hormone (T3) . More spLooking inside the cells, an increasing number of studies indicate that the oxidative stress condition strongly contributes to trigger the metabolic diseases with the derangement of the mitochondrial compartment. Mitochondria are the powerhouses of the cell and play a key role in maintaining homeostasis by finely regulating the balance between energy storage and energy expenditure. Their principal function is to synthesize ATP via the oxidative phosphorylation system (OXPHOS) maintained by the oxidation of metabolites through the Krebs cycle and β-oxidation. Moreover, mitochondria represent the main source of cellular ROS. An estimate of about up to 2 percent oxygen consumed can be deviated to the ROS formation .High energy diets increase the flux of substrates to mitochondria, resulting in over activation of OXPHOS that can form excessive ROS as by-products. The ROS, in turn, can directly impair the mitochondrial compartment itself, affecting the cellular redox signalling. Indeed, ROS have been associated in the first place with oxidative damage on lipids, DNA, and proteins . Over thThe evidence of the involvement of mitochondrial dysfunction and related stress pathways has opened the way to research for potential therapeutic purposes in metabolic disorders targeting these organelles .Genetically modified models have been fundamental in understanding the best interventions to improve mitochondrial functionality and health. Studies in both animals and humans have show the ameliorating effects of antioxidants on mitochondrial function in the presence or absence of metabolic disorders ,41. For Among healthy nutrients and foods, it is crucial to pay attention to the so defined “functional foods”, which contain biologically active molecules associated with physiological health benefits for preventing and managing chronic diseases . In receElongation of very long chain fatty acids protein 2 (ELOVL2)—ablated mice offered the possibility to investigate such issues, considering that the ELOV2 gene controls the endogenous PUFA synthesis. Indeed, Elovl2 KO mice display, in liver and serum, substantially decreased levels of omega-3  and omega-6 [docosapentaenoic acid (DPAn-6)] , which aShabalina and coworkers collected evidence about the importance of endogenous long-chain PUFA production for proper mammalian mitochondrial function and metabolism. In the liver, the absence of the Elovl2 enzyme in the endoplasmic reticulum drastically reduced the content of mitochondrial DHA (and of other PUFA) and this was in parallel associated with a decrease in mitochondrial respiration efficiency despite the absence of oxidative damage and preserved content of proteins of the respiratory chain . AlthougOn the basis of the current literature, it is clear that much research has been carried out and now more is known about the effects of diet and exercise on human health, however the pleiotropic effects of lifestyle interventions prompt us to shed further light on a topic of such sensitive interest for public health. Overall, the original contributions included in this Special Issue are examples of new studies that are able to increase knowledge in the field and contribute to the open debate on whether and how healthy diet and exercise are useful approaches to prevent and/or counteract metabolic diseases, with emphasis on the impacts of dietary composition, feeding frequency, exercise training, and bioactive compounds on the mitochondrial compartment. What is expected is that this Special Issue will give new support to translational interventions in the specific targeting of metabolic diseases in the broad spectrum of pathological conditions which generate MS.


Bile acids (BAs) are physiological detergents that can not only promote the digestion and absorption of lipids, but also may be a potential carcinogen. The accumulation of BAs in the body can lead to cholestatic liver cirrhosis and even liver cancer. Recently, studies demonstrated that BAs are highly accumulated in metastatic lymph nodes, but not in normal healthy lymph nodes or primary tumors. Lymph node metastasis is second only to hematogenous metastasis in liver cancer metastasis, and the survival and prognosis of hepatocellular carcinoma (HCC) patients with lymph node metastasis are significantly worse than those without lymph node metastasis. Meanwhile, component of BAs was found to significantly enhance the invasive potential of HCC cells. However, it is still poorly understood how deregulated BAs fuel the metastasis process of liver cancer. The tumor microenvironment is a complex cellular ecosystem that evolves with and supports tumor cells during their malignant transformation and metastasis progression. Aberrant BAs metabolism were found to modulate tumor immune microenvironment by preventing natural killer T (NKT) cells recruitment and increasing M2-like tumor-associated macrophages (TAMs) polarization, thus facilitate tumor immune escape and HCC development. Based on these available evidence, we hypothesize that a combination of genetic and epigenetic factors in cancerous liver tissue inhibits the uptake and stimulates the synthesis of BAs by the liver, and excess BAs further promote liver carcinogenesis and HCC metastasis by inducing immunosuppressive microenvironment. Liver cancer is one of the common digestive system malignant tumors, and its incidence is increasing year by year . The mosBile acids (BAs) are a general term for a class of amphiphilic molecules produced by cholesterol metabolism, and their functions involve lipid digestion, absorption and hormone synthesis . In the The tumor immune microenvironment (TIME) refers to the microenvironment related to immunological components such as T cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor (MDSCs), and NK cells within tumors. TIME can be divided into three types, namely the infiltrated-excluded (I-E) TIMEs, infiltrated-inflamed (I-I) TIMEs, and tertiary lymphoid structures (TLSs) TIMEs according to recent human and mouse data. For detailed classification and explanation, please refer to . TIME isThe disruption of local immune homeostasis is an important reason for the development of many cancers, including HCC . It’s reWe proposed that a combination of genetic and epigenetic factors, including gut microbiota, epigenetic regulation, hepatocyte function, metabolic reprogramming, and more, in cancerous liver tissue inhibits the uptake and stimulates the synthesis of BAs by the liver, and excess BAs further promote liver carcinogenesis and HCC metastasis by inducing immunosuppressive microenvironment Figure 1BAs produced in the liver are metabolized by enzymes from gut bacteria and are essential for maintaining a healthy gut microbiota, balanced lipid, carbohydrate metabolism, and innate immunity . The proBAs receptors play an important role in regulating BAs synthesis, metabolism and transport. There is a high intracellular BAs load during cholestasis, and the main detoxification pathway is to activate BAs receptors to protect liver cells and attenuate BAs toxicity . DisordeBAs are critical components of the gastrointestinal tract that link the gut microbiota to hepatic and intestinal metabolism, and thus influence gastrointestinal motility, intestinal permeability, and carcinogenesis . StudiesEpigenetics refers to a group of genetic mechanisms and phenomena that do not change the genotype but can determine the phenotype of cells . Those hThere are a large number of blood vessels and lymphatic vessels in the liver that can transport cancer cells to distant organs, and subsequently the metastatic tumor cells implant in sites with suitable environment to form new tumors (the ‘seed and soil’ hypothesis proposed by Stephen Paget), which limit the curative effect and prognosis of HCC patients . Cancer BAs play an increasingly prominent role in cancers invasion and metastasis, although the mechanism of action seems unclear. In this regard, BAs are the most widely studied in gastrointestinal cancer and have been shown to drive multiple signaling pathways to enhance the invasiveness and metastatic ability of tumor cells . SeveralInflammation is an immune defense response gradually formed by the body to resist foreign pathogens and respond to tissue damage in the long evolutionary process, mainly manifested in vascular response, immune cell recruitment and cytokine release, and has long been associated with cancer . InflammExternal factors of the tumor, especially stromal and immune cell populations, are equally important determinants of metastatic spread. Tumors actually prepare a supportive and receptive microenvironment for colonization for the arrival of disseminated cancer cells by inducing many systemic molecular and cellular changes, called a premetastatic niche . It is cBAs receptors coordinate with each other to keep BAs levels relatively stable, regulate BAs-mediated signal transduction pathways, and form an effective defense mechanism to inhibit the carcinogenicity of BAs. Although FXR antagonists have been shown to inhibit HCC metastasis , the speTumor metastasis and recurrence are important reasons that hinder the clinical benefit of HCC patients. Sorafenib is the first-line clinical drug for the treatment of liver cancer, but recent studies have pointed out that instead of hindering HCC migration, it further promotes tumor metastasis . TherefoIn recent years, immunotherapy, alone and in combination with conventional therapy, has achieved relatively satisfactory results in clinical trials. However, we still lack a scientific understanding of the immune-related mechanisms that influence metastasis formation and treatment response. The local and systemic immune landscapes of different types of tumors, and even individual tumors of the same type, vary widely . This noThe original contributions presented in the study are included in the article/J-KX and NT systemically analyzed the feasibility of the scientific hypothesis and wrote the paper. H-ZR and X-YW designed and constructed the scientific idea. All authors contributed to the article and approved the submitted version.This work was funded by the National Natural Science Foundation of China (82270646), the Fundamental Research Funds for the Central Universities (0214-14380510), the Nanjing health science and technology development project for Distinguished Young Scholars (JQX19002), fundings for Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (2022-LCYJ-PY-35), the Chen Xiao-ping Foundation for the Development of Science and Technology of Hubei Province, China (CXPJJH121001-2021073).www.figdraw.com).The authors would like to acknowledge the technical assistance provided by the staff of the Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China. The figure of the graphical representation was generated by Figdraw (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


After a systematic literature search corresponding to PRISMA guidelines, eleven studies were included and quantitatively evaluated in this review. The combined data exhibited that the rib cage increases the thoracic spinal stability in all motion planes, primarily in axial rotation and predominantly in the upper thorax half, reducing thoracic spinal range of motion, neutral zone, and intradiscal pressure, while increasing thoracic spinal neutral and elastic zone stiffness, compression resistance, and, in a neutral position, the intradiscal pressure. In particular, the costosternal connection was found to be the primary stabilizer and an essential determinant for the kinematics of the overall thoracic spine, while the costotransverse and costovertebral joints predominantly reinforce the stability of the single thoracic spinal segments but do not alter thoracic spinal kinematics. Neutral zone and neutral zone stiffness were more affected by rib cage removal than the range of motion and elastic zone stiffness, thus also representing the essential parameters for destabilization of the thoracic spine. As a result, the rib cage and thoracic spine form a biomechanical entity that should not be separated. Therefore, usage of entire human non-degenerated thoracic spine and rib cage specimens together with pure moment application and sagittal curvature determination is recommended for future in vitro testing in order to ensure comparability, reproducibility, and quasi-physiological validity.The vast majority of previous experimental studies on the thoracic spine were performed without the entire rib cage, while significant contributive aspects regarding stability and motion behavior were shown in several other studies. The aim of this literature review was to pool and increase evidence on the effect of the rib cage on human thoracic spinal biomechanical characteristics by collating and interrelating previous experimental findings in order to support interpretations of  In case of incomplete information in the abstracts, the respective publications were additionally checked. Using this approach, twelve reports were included in the assessment for eligibility. In a second step, the following two exclusion criteria were applied to the twelve studies: 1) Values of the parameters range of motion, neutral zone, coupled motions, center of rotation, helical axis, stiffness, strength, or intradiscal pressure were not reported, and 2) the boundary conditions of the test setup were not sufficiently described. After this step, the reference sections of the remaining eleven studies were additionally screened for studies potentially not detected by database search. Finally, eleven studies were included in this review.A systematic literature search was performed based on PRISMA guidelines  using a p < 0.05) were adopted from the original sources and are solely termed “significant” in the following.The included studies were scanned for the level of evidence, sample size, age, and sex of the donors, tested and evaluated segmental levels, direction, type, and level of the applied loads, performed transection or resection steps, and biomechanical outcome parameters. The percentage changes of the parameter values were either directly acquired from the publication text, if reported, or calculated from the reported absolute values for the intact or resected conditions. In the case of diagram illustrations, the values were digitized using an open-source tool for image segmentation (Engauge Digitizer 12.1). The collection and post-processing of data were performed using Excel 2019 . For this review, the biomechanical data of the thoracic spine were evaluated and collated regarding the transection or resection type performed on the rib cage, while a loss of spinal stability was defined as an increase in range of motion and neutral zone. Statistically significant differences (in vitro study designs. All of the eleven studies reported the use of at least six specimens. The age ranges were overall comparable with average donor ages of about 60–70 years. Sex distribution varied considerably among the studies, while two studies did not provide information about the sex of their specimens. Ten studies reported usage of a polysegmental test setup, while in nine studies, entire thoracic spine specimens were tested, all three motion planes were investigated, and pure moments were applied. The load levels varied widely, while six studies applied pure moments of 5 Nm in accordance with the recommendations for in vitro testing of thoracic spine specimens (General data on the included studies are summarized in pecimens , and thrpecimens . Range oPassive residual tissue stress of the intercostal muscles was found to significantly stabilize the thoracic spine in all motion planes in one study . The thoThree studies reported significant effects of median sternotomy, which is widely used in cardiac surgery, on both range of motion and neutral zone of the thoracic spine in all motion planes . With reThe sternal fracture at the manubriosternal junction, representing a common injury pattern after blunt chest trauma and being often associated with spinal injuries, significantly reduced thoracic spinal stability in all motion planes in one study . The higOne study reported significant destabilization of the thoracic spine in all motion planes after transverse transection of all anterior intersegmental rib connections . The higTransverse sternal transection at the T5–T6 level combined with bilateral transection of the rib bone-cartilage transition at the T3–T8 level for the potential treatment of spinal sagittal plane deformity, defined as so-called sternal release, significantly reduced thoracic spinal stability in flexion/extension in one study . While sThe anterior rib cage removal up to rib stumps, leaving the costovertebral and costotransverse joints intact, was by far the most frequently reported resection type, being investigated in overall seven studies . Six stuThe effect of complete removal of all rib cage structures, including the stabilizing ligaments of the rib–vertebra connection, on thoracic spinal biomechanical properties was investigated in overall four studies so far . The solin vivo, the rib cage including its ligamentous structures can be seen as the primary stabilizing factor for the thoracic spine. When testing the thoracic spine in a biomechanical in vitro setup with the aforementioned reported initial and boundary conditions, the rib cage, therefore, needs to be considered in order to reproduce the biomechanical properties of the thoracic spine as physiologically as possible, especially with regard to the upper and mid-thoracic regions where the biomechanical effects of the rib cage are strongest. While several past in vitro and in silico studies disregarded large parts of level-related rib cage structures, this review aimed to aggregate the current knowledge on the effect of the rib cage on the biomechanical properties of the thoracic spine in order to expand its evidence and provide an overview for future investigations.The rib cage represents a substantial component of the thoracic complex, connecting adjacent thoracic spinal segments via the stabilizing ligaments of the costovertebral joints and the overall thoracic spine via the rigid costosternal junction. While the cervical and lumbar spinal regions are predominantly stabilized by muscles All rib cage transection and resection types summarized in this review were found to cause a significant range of motion and neutral zone increases. Moreover, thoracic spinal flexibility appeared to be correlated with the number of transections and the amount of resected rib cage material . As a reThe effects of the rib cage on thoracic spinal neutral and elastic zone stiffness were solely investigated for anterior rib cage removal in previous experimental studies, overall showing highly reduced stiffness, especially in axial rotation , which mSagittal plane position of helical axes and centers of rotation during primary flexion/extension were found to be substantially affected by both the testing condition and the test setup. While in the polysegmental test setup, different testing conditions led to significant shifting of the centers of rotation , kinematThe effects of rib cage structures on out-of-plane motions varied considerably among previous investigations. While three studies did not detect any significant changes in the coupled motion characteristics after complete rib cage removal , one stuThe hydrostatic pressure in the thoracic spinal nucleus pulposus exhibited very high dependency on rib cage presence, especially in axial rotation, where the pressure significantly increased together with the range of motion after anterior rib cage removal up to rib stumps , while iin silico studies investigating the effects of the rib cage on thoracic spinal biomechanical properties were not included in this review since these models need to be validated by in vitro data to prove realistic results (in vitro studies should expand knowledge on thoracic spinal stiffness, kinematics, coupled motions, and intradiscal pressure with regard to the rib cage and should use comparable boundary conditions, in particular pure moment application and sagittal curvature determination, to ensure data comparability and reproducibility. Moreover, future investigations should additionally evaluate the effects of single and multiple rib fractures on the biomechanical properties of the thoracic spine. Nevertheless, the collated data of this review enabled a comprehensive overview of the effect of the rib cage on the most relevant biomechanical parameters characterizing the human thoracic spine.Only a few studies investigated the effect of the rib cage on the biomechanical properties of the thoracic spine, while partially exhibiting large variability in testing conditions. Thus, a meta-analysis of data presented in this review was not feasible, except for an overall thoracic spinal range of motion increases after anterior rib cage removal , includi results , thus bein vitro studies on the thoracic spine using the aforementioned reported initial and boundary conditions should, therefore, include the entire rib cage in order to represent the biomechanical properties of the thoracic spine as physiologically as possible, while the disregard of the ribs can be seen as justifiable in isolated testing of lower thoracic spinal levels.The combined data of this review verified that the rib cage stabilizes the thoracic spine in all motion planes, especially in axial rotation and predominantly in the upper thorax half, reducing thoracic spinal range of motion, neutral zone, and intradiscal pressure, while increasing thoracic spinal stiffness, compression resistance, and, in neutral position, intradiscal pressure. More specifically, the costosternal connection was found to be the primary stabilizer and an essential determinant for the kinematics of the overall thoracic spine, while the costotransverse and costovertebral joints predominantly enhanced the stability of the single thoracic spinal segments and did not alter thoracic spinal kinematics. Neutral zone and neutral zone stiffness were more affected by rib cage removal than the range of motion and elastic zone stiffness, thus representing essential parameters for destabilization of the thoracic spine. The experimental