Surgical resection is the first-line curative treatment modality for resectable hepatocellular carcinoma (HCC). Anatomical resection (AR), described as systematic removal of a liver segment confined by tumor-bearing portal tributaries, may improve survival by reducing the risk of tumor recurrence compared with non-AR. In this article, we propose the rationale for AR and its universal adoption by providing supporting evidence from the advanced understanding of a tumor microenvironment and accumulating clinical experiences of locoregional tumor ablation therapeutics. AR may be advantageous because it completely removes the en-bloc by interrupting tumor vascular supply and thus extirpates the spreading of tumor microthrombi, if they ever exist, within the supplying portal vein. However, HCC is a hypervascular tumor that can promote neoangiogenesis in the local tumor microenvironment, which in itself can break through the anatomical boundary within the liver and even retrieve nourishment from extrahepatic vessels, such as inferior phrenic or omental arteries. Additionally, increasing clinical evidence for locoregional tumor ablation therapies, such as radiofrequency ablation, predominantly performed as a non-anatomical approach, suggests comparable outcomes for surgical resection, particularly in small HCC and colorectal, hepatic metastases. Moreover, liver transplantation for HCC, which can be considered as AR of the whole liver followed by implantation of a new graft, is not universally free from post-transplant tumor recurrence. Overall, AR should not be considered the gold standard among all surgical resection methods. Surgical resection is fundamentally reliant on choosing the optimal margin width to achieve en-bloc tumor niche removal while balancing between oncological radicality and the preservation of postoperative liver function. The importance of this is to liberate surgical resilience in hepatocellular carcinoma. The overall success of HCC treatment is determined by the clearance of the theoretical niche. Developing biomolecular-guided navigation device/technologies may provide surgical guidance toward the total removal of microscopic tumor niche to achieve superior oncological outcomes. Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer-related deaths and the fifth most common neoplasm in the world ,2. CuratSurgical methods can be broadly classified into anatomical resection (AR) and non-anatomical resection (NAR) ,10. FirsComparative studies between AR and NAR have indicated that AR provides superior survival benefits by reducing the risk of tumor recurrence and improving overall survival in patients with solitary HCC . A systeAR is one of the strategies for achieving a curative goal and to guide surgical resection. However, facilitating improved survival through curative resection is not limited to AR. Instead of anatomical or non-anatomical, the free  margin is the major concern. Both micro- and macroscopic free margin clearance determines the HCC recurrence rate and survival outcomes.We hypothesize that surgical eradication of HCC should not be AR-restricted. Additionally, surgical eradication of HCC is based on adequate clearance of the tumor together with the surrounding microenvironment niche. Herein, we performed a data-driven debate from an in-depth, focused review.The rationale for AR is theoretically effective for eradicating the intrahepatic metastases of HCC through the removal of tumor-bearing portal territories ,19. HoweAdditionally, HCCs can derive new arterial blood supply from liver segment boundaries ,24,25 anAs HCC neoangiogenesis is not anatomically bound and CTCs can be found in multiple vascular routes other than portal veins, the rationale for performing absolute AR is not sufficiently strong. Surgical eradication of HCC should be flexible and not AR-restricted. The decision of reasonable resection margin clearance should consider the surrounding microenvironment niche .Therefore, HCC neoangiogenesis is not restricted by normal anatomical boundaries.The oncologic benefit is not exclusive to AR. RFA, a non-anatomical tumor ablation treatment performed irrespective of the hepatic blood supply anatomically , has proAnother opposing evidence to the use of universal AR is liver transplantation, which could be considered as AR of the whole liver and a new graft implant. However, post-transplant HCC recurrence can still occur at a rate of 13–27% , even unTherefore, the curative outcome and recurrence mechanism cannot be fully explained by AR alone, and resection margin clearance warrants more implication on recurrence outcome.Recent advancements in the understanding of tumor biology and microenvironment enable reconsideration of the surgical planning strategy from a broader perspective. Cha et al. investigated the interaction between the tumor microenvironment and resection margin in different gross types of HCC and found that patients with expanding and vaguely nodular HCC may safely undergo surgical resection with a narrow resection margin, and patients with gross types, such as nodular with perinodular extension, multinodular confluent, and infiltrative types, should preferably undergo surgical resection with a wider (more than 2 cm) resection margin considering their tumor microenvironment conditions, namely expression of beta-catenin, matrix metalloproteinase 9, and E-cadherin . The priThe key decision in liver resection involves determining the “optimal” amount of non-tumor parenchyma to be removed. Considering the pro-tumorigenic niche heterogeneity in adjacent “non-tumor” parenchyma, which may contribute to future HCC recurrence, the definition of resection margin clearance could additionally be viewed as en-bloc removal of the niche including “HCC will definitely develop” in addition to the infiltration border of current HCC cells. These findings warrant the development of a new surgical planning and navigation strategy.Historically, nodular HCC is round in shape and grows expansively by compressing the noncancerous liver parenchyma; nodular HCC often possesses a fibrous capsule with clear-cut margins instead of infiltrating the non-cancerous parenchyma  Figure . TherefoHoshida et al.  identifiAn ideal “navigation” guiding tool should identify the protumorigenic niche while being cost-effective and convenient to use. Several attempts have been made to determine the “safety” margin of liver resection both clinically and experimentally. Andrea Peloso et al.  proposedAnother attempt in improving surgical guidance has been the development of an intraoperative detection probe similar to the sentinel lymph node detection device during breast cancer surgery. By combining specific markers binding to the HCC receptor, the protumorigenic niche could potentially be identified. In an in vivo xenograft mouse model, Wang et al.  used a cThe limitation of this hypothesis-driven review is that the conclusions proposed in each cited paper are not all supported by hard data. Further studies are invited to confirm these conclusions by presenting the relevant data in the future.In this article, we challenge the rationale for the universal adoption of AR for HCC. Curative resection is not limited to AR, and AR can present its own limitations. The concept of surgical resection should depend on choosing the optimal margin width for achieving en-bloc tumor niche removal, thus balancing oncological radicality and the preservation of postoperative liver function. Overall treatment success of HCC is determined by the clearance of the theoretical niche. Improved knowledge about cancer biology incorporating concepts of neoangiogenesis and micro-tumor biology, an emerging field of tumor microenvironment detection and protumorigenic niche, indicates that a new perspective of redefining resection margin clearance and further refining navigation devices/technologies could provide surgical guidance toward resection radicality for achieving superior oncological outcomes.


Induced pluripotent stem cell-derived cortical and retinal organoids represent a novel class of in vitro three-dimensional models capable to recapitulate with a high similarity the structure and the complexity of the native brain and retinal tissues, thus providing a framework for better mimicking in a dish the patient’s disease features. This review aims to discuss progress made over the years in the field of in vitro three-dimensional cell culture systems, and the benefits and disadvantages related to a possible application of organoids for the study of neurodegeneration associated with Alzheimer’s disease, providing a promising breakthrough toward a personalized medicine approach and the reduction in the use of humanized animal models.Recent progress in tissue engineering has led to increasingly complex approaches to investigate human neurodegenerative diseases  Alzheimer’s disease (AD) is a progressive age-related neurodegenerative disorder characterized by cognitive and psychiatric symptoms, such as memory and cognitive impairments, behavioral abnormalities, disorientation, and circadian rhythms and sensorial disturbances . AlthougAlbeit the genetic factors and individual susceptibilities, the AD brain displays a spread tissue degeneration with dense extracellular deposits of toxic amyloid-β (Aβ) oligomers and hyperphosphorylated tau-enriched intracellular tangles. Mitochondrial dysfunction, diffuse reactive oxygen species, and neuroinflammatory response are, among other, tissue, and cellular manifestations . DiagnosMoreover, visual deficits, including difficulty reading , depth pin vitro disease models, which more comprehensively recapitulate hallmarks of AD, represents an important research strategy that may enable to accelerate the molecule screening process before testing a lead candidate in vivo on transgenic rodent AD models.While more than 200 functional transgenic rodent models have been generated , they onin vitro human disease models  necessary for bringing back to stemness an adult somatic cell  has posed the basis for a revolution in the field of e models . iPSCs re models . These cir usage . Thus, tin vitro to exploit as an alternative and complementary approach to humanized animal models for the study of species- and patient-specific features  culturing approaches  togetherfeatures .in vitro, such as neurons. For instance, in 3D neural constructs, the increased dimensionality allows a more compliant physiological cell-cell interactions between neurons, thus limiting spatial overlap during neurite elongation and branching  may play a pivotal role in designing novel therapeutic approaches . To this process  and how elopment . Hence, elopment . Recentlelopment , howeverin vitro vascularization of retinal organoids using endothelial cells might contribute to foster the identification and development of new molecular targets  alterations are widely reported in AD retina and their investigation as possible diagnostic tools is under evaluation . Retinal targets .in vitro platforms offer promising outlooks that might pave and foster the rapid growth of novel in vitro approaches to tackle AD (In conclusion, although there are yet several gaps to be filled before allowing the use of iPSC-based organoid technology in neurodegenerative and in translational studies, these ackle AD . Moreoveackle AD . Thus, eCB and FC wrote the manuscript with support from LC. AS took the lead in editing the manuscript. AS and SD supervised the writing and conceived the idea. All authors provided critical feedback and helped to shape the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stroke is a life-threatening condition that is characterized by secondary cell death processes that occur after the initial disruption of blood flow to the brain. The inability of endogenous repair mechanisms to sufficiently support functional recovery in stroke patients and the inadequate treatment options available are cause for concern. The pathology behind oxidative stress in stroke is of particular interest due to its detrimental effects on the brain. The oxidative stress caused by ischemic stroke overwhelms the neutralization capacity of the body’s endogenous antioxidant system, which leads to an overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and eventually results in cell death. The overproduction of ROS compromises the functional and structural integrity of brain tissue. Therefore, it is essential to investigate the mechanisms involved in oxidative stress to help obtain adequate treatment options for stroke. Here, we focus on the latest preclinical research that details the mechanisms behind secondary cell death processes that cause many central nervous system (CNS) disorders, as well as research that relates to how the neuroprotective molecular mechanisms of pituitary adenylate cyclase-activating polypeptides (PACAPs) could make these molecules an ideal candidate for the treatment of stroke. Stroke is a potentially fatal interference in the normal blood supply to the brain that precipitates debilitating sequelae including visual deficits, cognitive decline, and paresis. The two major subtypes of stroke are ischemic and hemorrhagic, although the former is profoundly more common and accounts for nearly 85% of total stroke events worldwide . IschemiThe prolonged perfusion deficit in stroke triggers the ischemic cascade, a three-phase series of pathological biochemical reactions that culminates in cellular death. The cascade begins within hours of flow disruption as the acute phase, which is characterized by ionic disorder, microglial activation, and oxidative stress . The locOxidative stress induces secondary cell death through multiple cell death pathways, including mitochondrial dysfunction . MitochoMultiple endogenous mechanisms exist to mitigate damage from oxidative stress. For example, creatine kinase expression is upregulated during ischemia and acts as a temporary energy buffer by regenerating ATP from ADP and creatine phosphate . A seconA less explored endogenous mechanism involves pituitary adenylate cyclase-activating polypeptide (PACAP). PACAP is a neuropeptide that was originally isolated in the hypothalamus but is found extensively in the central nervous system (CNS) and the peripheral nervous system (PNS) . PAC1, t−7 mol/kg, serum levels of oxidative metabolites did not significantly change. Thus, endogenous PACAP plays a critical role in regulating the response to oxidative stress, and a long-term loss of PACAP may culminate in mounting oxidative damage PACAP(1-27) (a compound derived from PACAP) was manufactured and its efficacy was evaluated in vitro and in vivo. Compared to exogenous PACAP, this novel compound showed equal efficacy in protecting SH-SY5Y neuroblastoma cells exposed to MPP(+) neurotoxicity . ImportaN-terminus, culminated in increased resistance to degradation by dipeptidyl peptidase IV [Though PACAP has demonstrated neuroprotective effects preclinically, its clinical use in stroke and neurodegenerative disorders is limited on account of its metabolic instability, poor bioavailability, sparse distribution, and quick clearance from the bloodstream akin to other natural peptides . Pharmacidase IV . In vitridase IV . To bolsidase IV . The modidase IV . VIP is idase IV . A studyidase IV . Among tidase IV . In sum,Before promising preclinical findings can be translated in clinical trials, the optimal route of delivery for PACAP administration needs to be elucidated. Several routes of delivery for PACAP to the brain have been explored including both local and systematic methods . In termWhile the safety and efficacy of PACAP in stroke and neurodegenerative disorders has not been extensively investigated in clinical trials, PACAP-related molecules, such as glucagon-like peptides (GLP-1) and VIP, have been explored. However, the role of PACAP in migraine pathology has been vastly evaluated in the clinical setting. A recent trial among 24 female migraine patients found that PACAP38 administration resulted in a significant increase in migraine attacks . RegardiIschemic-reperfusion injury caused by stroke continues to be a major cause of mortality and morbidity in the United States. Ischemic stroke involves insufficient blood perfusion to vital the brain, which leads to oxygen and nutrient deficiency followed by subsequent cell death. The ischemic cascade, prompted by stroke, causes ionic disruption, metabolic failure, increasing permeability of the blood–brain barrier (BBB) leading to an upregulation of inflammation, and mitochondrial dysfunction ,45,58,59Notably, recent research has elucidated PACAP’s role in ameliorating ischemia-induced mitochondrial impairment and glutamate excitotoxicity . Evidenc


The recent setbacks in efforts to achieve measles elimination goals are alarming. To reverse the current trends, it is imperative that the global health community urgently intensify efforts and make resource commitments to implement evidence-based elimination strategies fully, including supporting research and innovations. The Immunization Agenda 2030: A Global Strategy to Leave No One Behind (IA2030) is the new global guidance document that builds on lessons learned and progress made toward the GVAP goals, includes research and innovation as a core strategic priority, and identifies measles as a “tracer” for improving immunisation services and strengthening primary health care systems. To achieve vaccination coverage and equity targets that leave no one behind, and accelerate progress toward disease eradication and elimination goals, sustained and predictable investments are needed for the identified research and innovations priorities for the new decade. The recent setbacks in efforts to achieve measles elimination goals are alarming . After rMeasles and rubella elimination research priorities have been identified, including operational research and potential game-changing new tools, such as rapid diagnostic tests (RDTs) , 8. EarlMeasles outbreak investigations, case-based surveillance data analysis, vaccination coverage surveys, systematic EPI reviews, vaccine-preventable disease impact assessments, and cost-effectiveness studies provide opportunities for research to generate evidence for refinement of elimination strategies. The published literature is rich with evidence that supports simultaneous EPI strengthening and measles elimination, including the impact of the recently updated Reaching Every District strategy; integration of other public health interventions with immunizations service delivery including supplemental immunization activities (SIAs) –14; incoIn 2009, an accumulation of evidence led to the WHO recommendation that all countries provide two doses of measles-containing vaccine . GloballAdvances in serological surveys, disease mathematical modeling, measles-susceptibility profiles, and measles risk assessments have facilitated identifying measles population immunity gaps and sub-national areas at-risk –30. HowePeriodic nationwide SIAs are a long-established cornerstone of elimination efforts that include special strategies and microplanning for reaching zero-dose and under-vaccinated children previously missed by routine immunization services. Starting in 2016, however, global measles donor funds were redirected toward organizations that focused on health systems strengthening rather than measles elimination ; this waSIA frequency and target age groups should be based on epidemiological analyses, and adequate resources made available to ensure optimal implementation of the indicated target population and SIA timing , 35. PreMeasles epidemiology has changed over time, following decreases in measles incidence in all regions since 2000. Studies have documented this changing epidemiology, including in the AFR , and recSimilarly, a recent review of the measles reproduction number (R0), the measure of transmissibility that drives herd immunity and subsequent vaccination coverage levels needed to interrupt measles virus transmission, showed that R0 estimates vary considerably by setting and more widely than the often-cited 12-18 range, and they are dependent on context-specific factors including population density, birth rates, and age-mixing patterns . Better With changing measles-susceptibility, a recent review of the effect of age at first dose and time since vaccination on measles vaccine effectiveness (VE) was completed. It showed that, in measles-endemic settings, one-dose VE increased by 1.5%  for every month increase in age at first dose and found no evidence of waning VE. More data, however, are needed to answer the question of whether measles VE wanes in measles-elimination settings . Recent Measles virus infection leads to severe viremia and lymphopenia and can cause immunosuppression that can last for months to years ; howeverImportant innovative tools are on the horizon, including a measles rapid diagnostic test (RDT) and a measles-rubella (MR) vaccine microneedle patch that are among the highest priorities for measles and rubella elimination research . A measlWith the decade of vaccines coming to an end in 2020, global immunization partners are establishing the “Immunization Agenda 2030: A Global Strategy to Leave No One Behind” (IA2030)  to be apThe author declares no competing interests.


In this review, we aimed to summarize the current function and mechanism of ncRNAs in regulating the sensitivity of HNC cancer cells to radiotherapy and comprehensively described the state of the art on the role of ncRNAs in the prognosis prediction, therapy monitoring, and prediction of response to radiotherapy in HNC.Head and neck cancers (HNCs) rank as the sixth common and the seventh leading cause of cancer-related death worldwide, with an estimated incidence of 600,000 cases and 40–50% mortality rate every year. Radiotherapy is a common local therapeutic modality for HNC mainly through the function of ionizing radiation, with approximately 60% of patients treated with radiotherapy or chemoradiotherapy. Although radiotherapy is more advanced and widely used in clinical practice, the 5-year overall survival rates of locally advanced HNCs are still less than 40%. HNC cell resistance to radiotherapy remains one of the major challenges to improve the overall survival in HNC patients. Non-coding RNAs (ncRNAs) are newly discovered functional small RNA molecules that are different from messenger RNAs, which can be translated into a protein. Many previous studies have reported the dysregulation and function of ncRNAs in HNC. Importantly, researchers reported that several ncRNAs were also dysregulated in radiotherapy-sensitive or radiotherapy-resistant HNC tissues compared with the normal cancer tissues. They found that ectopically elevating or knocking down expression of some ncRNAs could significantly influence the response of HNC cancer cells to radiotherapy, indicating that ncRNAs could regulate the sensitivity of cancer cells to radiotherapy. The implying mechanism for ncRNAs in regulating radiotherapy sensitivity may be due to its roles on affecting DNA damage sensation, inducing cell cycle arrest, regulating DNA damage repair, modulating cell apoptosis, etc. Additionally, clinical studies reported that  Head and neck cancers (HNCs) rank as the sixth common and the seventh leading cause of cancer-related death worldwide, with an estimated incidence of 600,000 cases and 40–50% mortality rate every year . HNCs arRadiotherapy is a common local therapeutic modality for cancers mainly through the function of ionizing radiation. Ionizing radiation could kill cancer cells via directly damaging the DNA strands of cancer cells to end the infinite proliferative capacity, and also indirectly causes DNA damages in cancer cell through ionizing water to generating highly reactive oxygen species. Radiotherapy is one of the major treatment modalities for HNC, with approximately 60% of patients treated with radiotherapy or chemoradiotherapy . Many hiNon-coding RNAs (ncRNAs) are newly discovered functional RNA molecules that are different from messenger RNAs, which can be translated into a protein . Over thMany previous studies have reported the dysregulation and function of ncRNAs in HNC . NcRNAs Since the discovery of ionizing radiation in 1895, the concept of radiation-based therapy modality was prompted and has been regarded as a major treatment for many types of cancers , b. Espe5 ionizations per cell, which cause about 40 DSBs, 2,000 single-strand breaks (SSBs), as well as other types of damages in the DNA  and non-homologous end joining NHEJ; .The HR repairing system depends on homologous DNA sequences from sister chromatids. This makes HR restricted to phases of the cell cycle where homologous sister chromatids coexist . In contThe sensitivity of cancer cells to radiotherapy depends on the intensity of DNA damage with cells, the cells’ ability to balance the expression of genes associated with apoptosis promotion and inhibition, the expression level of genes regulating the induction of cell cycle arrest, and the DNA repair system. Radiotherapy-resistant cancer cells showed obvious tendency to inhibit cell apoptosis and augment the DNA damage repair rate. This adaptation to radiotherapy is closely related with the dysregulated expression of genes in resistant cells. NcRNAs, especially the regulatory ncRNAs, are characterized for their role on regulating gene expression in cancer cells. Because of such an important role, ncRNAs are believed to be associated with radiotherapy resistance and could regulate cell sensitivity to radiotherapy.The role of ncRNAs on cancer development and progression is one of the hottest topics in cancer cell biology research. With the help of microarray and next-generation technology, researchers have discovered an amount of ncRNAs, such as miRNAs, lncRNAs, and circRNAs, which was differently expressed in cancer tissues compared with the normal tissues. Many of these ncRNAs could significantly regulate the biological behaviors of cancer cell. In HNC, many researchers also focus on the expression and function of ncRNAs. Numerous ncRNAs are reported to modulate HNC proliferation, invasion, metastasis, apoptosis, and other biological processes . RecentlThe efficacy of radiotherapy depends mainly on the sensitivity of cancers to radiotherapy. Many factors are found to influence the radiotherapy sensitivity of cancers. Several research groups have discovered that some differentially expressed ncRNAs in HNC could affect radiotherapy sensitivity. Meanwhile, several research groups also discovered differentially expressed ncRNAs in radiation-sensitive and radiation-resistant HNC cancer cells by sequencing and microarray analysis. Some high-regulated ncRNAs in radiation-resistant cancers could decrease the sensitivity of cancer cells to radiotherapy, whereas ncRNAs down-regulated in radiation-resistant cancers could enhance the sensitivity of cancer cells to radiotherapy. These ncRNAs modulate the response of cancer cells to radiotherapy mainly through regulating different genes involved in important processes that are tightly associated with radiotherapy sensitivity. In this section, we mainly describe the effect and molecular mechanisms of ncRNA on regulating radiotherapy sensitivity of HNC cells by DNA damage sensing, cell cycle progression, DNA damage repair, and cell apoptosis of HNC cells.In response to DNA damage, cells have evolutionarily developed a mechanism to sense the DNA damage and initiate DNA damage repair. In the process, the ataxia telangiectasia mutated (ATM), an important Ser/Thr kinase, plays a major role in sensing DNA DSBs and initiating a series of cascade responses leading to cell cycle checkpoint activation and DNA repair , 2. AfteWhen DSBs, the most harmful type of DNA damage, occurred in cells, the histone variant H2AX was immediately phosphorylated by the activated ATM . TherefoWhen the radiation-induced DNA damage is too lethal to be repaired, cells would initiate an automatic death program like apoptosis to protect genome stability. Therefore, the flexibility of cancer cells to initiate cell apoptosis sometimes may determine whether cancer cells are sensitive to radiotherapy or resistant to it. Some ncRNAs have been reported to influence the sensitivity of cancer cells to radiotherapy through modulation of cell apoptosis. Mechanistically, these ncRNAs mainly function through regulating the expression of anti-apoptosis or pro-apoptosis members .In vitro and in vivo studies illustrated that miR-29c could promote cell apoptosis, through which ectopic restoration of miR-29C substantially enhanced the sensitivity of NPC cells to radiotherapy  family including myeloid cell leukemia 1 (MCL1) and Bcl-2 itself in human NPC. otherapy . MiR-378otherapy . Overexpotherapy .On the other hand, several miRNAs were reported to inhibit cell apoptosis and enhance HNC cancer cell resistance to radiotherapy. Evidence has shown that PTEN is involved in regulating cell response to radiation-induced cell apoptosis and cell cycle arrest through inhibiting radiation-induced activation of the PI3K-Akt signaling pathway. Several miRNAs including miR-205 and miR-96-5p were reported to be elevated in HNC tissues from NPC patients after radiotherapy, and target PTEN to mediate resistance of HNC cells to radiotherapy . MiR-504Recently, evidences have discovered that several lncRNAs and circRNAs could influence cancer cell sensitivity to radiotherapy through modulation of cell apoptosis. These lncRNAs and circRNAs function mainly through alleviating the role of certain miRNAs, which could be bound by lncRNAs and circRNAs through the base-pairing principles. For example, lncRNA ANRIL could function as a miR-125a sponge and negatively modulate miR-125a expression. ANRIL could reverse the inhibited proliferation, induced apoptosis, and enhanced radiosensitivity triggered by miR-125a overexpression . PTPRG-AIonizing radiation activates the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which, in turn, can initiate PI3K/AKT or MAPK pathways.Through activating the PI3K-AKT pathway, EGFR signaling can prevent radiation-induced apoptosis. EGFR signaling can also promote cancer cell growth by inducing cell cycle progression, which was driven by activation of the retrovirus associated DNA sequence (RAS)–rapidly accelerated fibrosarcoma (RAF)–mitogen/extracellular signal-regulated kinase (MEK)–ERK pathways. Published data demonstrate that miR-203 is a critical determinant of NPC cells’ response to radiotherapy, and reduced miR-203 could promote NPC cell radioresistance by activating IL8/AKT signaling . In addiEpithelial-to-mesenchymal transition (EMT) is a phenotypic change in which the epithelial cancer cell acquired a fibroblastoid-like morphology. Such transition could result in enhanced tumor cell motility and invasiveness, increased metastatic potential, as well as resistance to radiotherapy or chemotherapy . ZEB1, aIt is well known that ncRNAs have shown an important regulatory role on the sensitivity or resistance of HNC cells to radiotherapy. Based on this, ncRNAs expression can be useful biomarkers to identify HNC patients who will be sensitive to radiotherapy and to predict the survival outcomes of HNC patients receiving radiotherapy in clinical practice. One study explored the associations between miR-200b and miR-155 expression in HNC tissues and outcome, and confirmed the prognosis predictive value of candidate miRNAs . AdditioCirculating biomarkers in the peripheral blood, such as the biomarker HSP70 in HNSCC patients , could pRadiotherapy is a common therapeutic modality for HNC. Decreased sensitivity or resistance to radiotherapy is still a significant challenge in clinical practice and a barrier to improve the prognosis of HNC patients. With the development of molecular biology and sequencing technology, mounting evidence revealed the important role of ncRNAs on the carcinogenesis, development, and therapy resistance of HNC. These findings were of great significance for the following reasons. First, they highlight the potential of intervening relevant ncRNAs to overcome resistance and re-sensitize cancer cells to the effects of radiotherapy. Second, the ncRNAs could be used as circulating biomarkers in the peripheral blood to predict therapy response and survival outcomes, as well as monitor the therapy.In the future, more studies are required to further elucidate the potential roles of novel ncRNAs like circRNAs and piRNAs in regulating the sensitivity of HNC cells to radiotherapy. Furthermore, it is still unclear and less studied whether the tumor cells or tumor mesenchymal cells-derived exosomal ncRNAs confer HNC cell resistance characteristics to radiotherapy. These topics will be very interesting and meaningful because circRNAs were more stable than linear RNAs due to their covalently closed loop structures, and exosome encapsulated ncRNAs were less easy to be degraded in the circulation system. These characteristics make circRNAs and exosomal ncRNAs potential biomarker candidates for prognosis prediction and therapy response monitoring. Additionally, with the development of bioinformatics, the integration of ncRNA signature profiling into HNC screening algorithms may help in increasing the specificity of screening patients who will benefit from radiotherapy and improving the prognosis of HNC patients.JY designed the review. XZ and JY collected the related manuscript. XZ and JY drafted and revised the manuscript. Both authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tumor progression depends primarily on vascular supply, which is facilitated by angiogenic activity within the malignant tissue. Non-small cell lung cancer (NSCLC) is a highly vascularized tumor, and inhibition of angiogenesis was projected to be a promising therapeutic approach. Over a decade ago, the first anti-angiogenic agents were approved for advanced stage NSCLC patients, however, they only produced a marginal clinical benefit. Explanations why anti-angiogenic therapies only show modest effects include the highly adaptive tumor microenvironment (TME) as well as the less understood characteristics of the tumor vasculature. Today, advanced methods of in-depth characterization of the NSCLC TME by single cell RNA sequencing (scRNA-Seq) and preclinical observations enable a detailed characterization of individual cancer landscapes, allowing new aspects for a more individualized inhibition of angiogenesis to be identified. Furthermore, the tumor vasculature itself is composed of several cellular subtypes, which closely interact with other cellular components of the TME, and show distinct biological functions such as immune regulation, proliferation, and organization of the extracellular matrix. With these new insights, combinational approaches including chemotherapy, anti- angiogenic and immunotherapy can be developed to yield a more target-oriented anti-tumor treatment in NSCLC. Recently, anti-angiogenic agents were also shown to induce the formation of high endothelial venules (HEVs), which are essential for the formation of tertiary lymphoid structures, and key components in triggering anti-tumor immunity. In this review, we will summarize the current knowledge of tumor-angiogenesis and corresponding anti-angiogenic therapies, as well as new aspects concerning characterization of tumor-associated vessels and the resulting new strategies for anti-angiogenic therapies and vessel inhibition in NSCLC. We will further discuss why anti-angiogenic therapies form an interesting backbone strategy for combinational therapies and how anti-angiogenic approaches could be further developed in a more personalized tumor-oriented fashion with focus on NSCLC. Angiogenesis is regulated by the balance of pro-angiogenic and anti-angiogenic factors present in a tissue, if vascular remodeling is required this balance shifts to an activating state, called the “angiogenic switch” . In progThe transient combinational efficacy of anti-angiogenic agents and chemotherapy  could possibly be attributed to a “vascular normalization” phenotype. Nevertheless, the time window of vessel re-organization and normalization is not well understood in the clinical setting but could play a major role in the transmission of chemical agents directly to the tumor, thereby enhancing anti-cancer efficacy . AdditioPhysiological angiogenesis has already been characterized in detail and previously reviewed elsewhere . The proAdditionally, the tumor stroma, which consists of a mix of resident fibroblasts and pericytes as well as bone-marrow derived tumor infiltrating leukocytes , regulates angiogenesis. M2 polarized tumor associated macrophages can either directly activate angiogenesis by releasing VEGF, bFGF and PlGF or indirectly via the release of matrix-metalloproteinases (MMPs), which in turn remodel the extracellular matrix for an enhanced endothelial migration . FibroblIn vitro studies of glioma cells suggest that tumor cells that facilitate vessel co-option are dependent on the endoplasmic reticulum based stress sensing protein IRE1  assay, matrigel plugs, and tumor xenograft models. CAM assays, which have already been in use for decades, utilize chorioallantoic membranes of fertilized chicken eggs to evaluate angiogenic processes. While this method is cost effective, highly reproducible and the outcomes are easily visualized, it must be taken into consideration that vessel growth is evaluated during developmental stages, which can affect studies investigating mechanisms in mature vasculature. Matrigel plug assays enable the use of an in vitro tool in an in vivo setting. Here, vascular growth is evaluated by injection of matrigel, a synthesized substrate resembling basement membrane matrix, into an animal model which allows easy stimulation, subsequent excision, and investigation of the plug with, for example, immunohistological stainings. Compared with CAM assays, the matrigel plug can be used in more analytical methods and provides a fast and reliable representation of angiogenic processes in a biological system. Nevertheless, this method may require more replicates due to higher variability of results and is therefore more expensive. Lastly, transplantation xenografts represent the most advanced method to investigate tumor angiogenesis in a living organism. Tumor cells, mostly of human origin, are injected into immunodeficient mice to induce formation of a cancer mass that can be further treated and monitored for changes regarding tumor angiogenesis. This method most suitably reflects the pathological mechanism of vessel growth in vivo in the presence of blood circulation, as well as diverse environmental factors. Furthermore, it enables the long-term study of diverse processes associated with angiogenesis that are observed in a biological system such as tissue invasion, distant metastasis formation as well as non-angiogenic processes like vessel co-option and VM, which are known to promote resistance mechanisms in various cancers. Aside from the ethical aspect, a considerable disadvantage of this method is the incomplete or lacking representation of the immune system due to immunosuppression of the study organism.For more accurate information regarding angiogenic processes upon treatment in a biological system or to perform long-term studies, Examining which experimental assay is most suitable for investigating a chosen angiogenic process under certain conditions, necessitates extensive deliberation with the desired endpoint, required technical equipment, level of experimental throughput, cost, and ethics kept in mind. Additionally, the complexity of angiogenesis cannot be unraveled using a single analytical method but the thought-out application of multiple overlapping analyses, ranging from cellular to physiological levels, are necessary to obtain robust findings worth testing in the clinical setting.In 2004, the first VEGFA-inhibiting antibody, bevacizumab, was approved for use in advanced colorectal cancer in combination with chemotherapy and was followed in 2006 in NSCLC . Since tAs previously mentioned, there is a great need for biomarkers to predict and track anti-angiogenic therapy efficacy, to help overcome innate and acquired resistance as it is still the main obstacle that restrains clinical success . So far,In a recent study, it was demonstrated that immunohistochemically confirmed TTF-1 expression in advanced non-squamous NSCLC samples, which is a known prognostic biomarker of lung adenocarcinomas, could be linked to therapy success of bevacizumab in combination with pemetrexed plus platinum derivatives . TTF-1 pFurthermore, despite the previous results of the IMpower150 study, where significant clinical benefits of bevacizumab in combination with ICI and chemotherapy were shown, regardless of PDL-L1 expression, a phase 1b study by Herbst et al. observed contrary results. They reported a beneficial efficacy of ramucirumab in combination with the PD-L1 inhibitor pembrolizumab especially in patients with a PD-L1 expression above 50% . AccordiIn vitro assays demonstrated that exogenous CXCL2 could recover anti-angiogenic-induced inhibition of migration and invasion and prevent apoptosis of anlotinib-resistant cells. Furthermore, in a retrospective analysis, anlotinib-induced decrease of the inflammatory cytokine CCL2 in serum correlated with prolonged PFS and OS  which limit analytical accessibility.Several studies suggested a potential prognostic value of VEGF in NSCLC but so far investigations into circulating VEGF levels have not yielded consistent results . In the However, confirming if any of these molecular markers indeed exhibit adequate predictive features necessitates further investigation. New aspects of processes which promote tumor angiogenesis, and a better understanding of the endothelium as driving force can help identify reliable biomarkers and overcome therapy failure in NSCLC.There are several mechanisms on both the cellular and environmental levels which can promote vessel formation in human tumors, many of which are not yet been completely elucidated. Although angiogenesis may represent the most important part of tumor vascularization, other processes that result in perfusion of the tumor tissue should be investigated in more detail and considered when designing new anti-angiogenic approaches in NSCLC.In the following part we summarize various levels of tumor vascularization that may represent new targets for vessel inhibition in NSCLC. All mentioned mechanisms are summarized in The endothelium is postulated to be a large contributor to the therapeutic efficacy of anti-angiogenic therapies, and therefore represents a possible source of therapy response or failure. It is well known that the process of angiogenesis is comprised of different EC phenotypes which execute distinct functions. During the elongation of the sprouting vessel VEGF-sensitive tip ECs migrate into avascular tissue regions, thus leading the proliferating trailing stalk ECs, which built up the growing vessel. Newly formed vasculature finally adapts a mature and quiescent phenotype referred to as phalanx ECs . The EC In order to develop new angiogenesis-inhibiting therapies, the molecular differences between physiological and pathological ECs will need to be elaborated. Genetically TEC and NEC phenotypes significantly differ in gene expression affecting diverse cellular mechanisms such as proliferation, migration, inflammation, and angiogenesis . Previoude novo biosynthesis of proline; and squalene epoxidase (SQLE), the rate-limiting enzyme in cholesterol biosynthesis. Silencing of ALDH18A as well as SQLE impaired EC proliferation, migration and vessel sprouting in in vitro assays. Summarized, targeting endothelial metabolism in cancer is an interesting therapeutic option that could possibly assist an anti-angiogenic approach for treating NSCLC.Focusing on endothelial metabolism in cancer, a recent study could identify at least two metabolic signatures which are highly upregulated in angiogenic endothelium and TECs. One for proliferation, which includes gene sets associated with biomass production e.g., glycolysis, TCA cycle, fatty acid oxidation, cholesterol biosynthesis and amino acid metabolism, and one for remodeling of the extracellular matrix including gene sets for collagen biosynthesis in particular proline synthesis . These rAnother key feature of TECs in lung cancer is the downregulation of inflammatory responses thus contributing to tumor-associated immune escape. Single-cell analysis of NSCLC samples identified the most downregulated genes of the tumor endothelium in connection to inflammation, which included CCL2, CCL18, and IL6, essential for immune cell recruitment; MHC I and II, essential for immune cell activation; and ICAM, required for immune cell homing . As the As mentioned above, in some cases tumor vascularization can be facilitated by non-ECs which adapt certain properties to sustain access to the circulation, which may support anti-angiogenic drug resistance. During tumor progression, processes that lead to vascularization of the malignant tissue can vary locally as well as temporarily and involve angiogenic as well as non-angiogenic mechanisms even in the same lesion . In lungin vitro, furthermore, sunitinib, a multi targeting anti-VEGFR inhibitor, even promoted VM in breast cancer mouse models  via the angiopoetin/tie signaling pathway, an associated ligand for the less studied tie1, has not yet been identified . In contin vitro when internalized by HUVECs. Additionally, miR-23a transfection increased permeability and transendothelial migration of cancer cells in vitro by downregulation of the tight junction protein ZO-1 and stimulated neovascularization and tumor growth in vivo in CL1-5 xenograft mice, proposing it to be an appealing target for anti-angiogenic therapy. Upregulation of miR-195 in squamous lung cancer cells in vitro on the other hand could be associated with impaired VEGF expression and hampered migration and invasion, thereby facilitating a tumor-suppressive function. Additionally, overexpression of miR-195 in HUVECs was observed to inhibit tube formation and reduced the expression of VEGF, which hampered their angiogenesis activity in vitro (Another considerable strategy of anti-angiogenic therapy could include targeting micro RNAs (miRNAs) as they represent a new paradigm in molecular cancer therapy. The impact of miRNAs in post-transcriptional regulation has already been associated with pathways involved in cancer and vascular disease as summarized in in vitro .in vitro by downregulation of TIMP-2. Additionally, it could be observed that miR-130b was significantly upregulated in tumor tissue of NSCLC patients with vascular cancer cell invasion  which are inhibited under physiological conditions by tissue inhibitors of metalloproteinases (TIMPs). miR-130b could be identified as a promotor of MMP-2 activity and invasion of NSCLC cancer cells invasion . AccordiUribesalgo et al. suggested targeting the apelin signaling pathway to inhibit tumor vessel formation in lung cancer . Apelin in vitro. When injected into lung cancer mouse models, autologous generated anti-peptibody antibodies inhibited tumor progression and angiogenesis and decreased expression of bFGF, VEGFA and PDGF in the tumor tissue. Targeting angiogenesis with fusion proteins exhibited potent anti-tumor efficacy in murine models and may represent a new approach for vessel inhibition in NSCLC, especially in combination with other therapy agents aimed at important angiogenic factors, previously discussed potential TEC specific markers or cellular mechanisms  conjugated to a VEGFR2-inhibiting peptide showed increased internalization in HUVECs in vitro as well as potent activity against breast cancer models in vivo  impedes drug delivery to the local lesion. Although anti-angiogenic therapy can temporarily restore tissue perfusion and drug delivery by vascular normalization, treatment withdrawal often results in vessel hyper-permeability and can even induce a rebound effect of tumor angiogenesis . As cont in vivo . Althoug in vivo . Further in vivo . In 2018 in vivo . A more  in vivo . It is sSo far, the main obstacles of anti-angiogenic therapy in NSCLC are evading- or intrinsic resistance mechanisms which still remain elusive. We have discussed a wide array of possible therapies and therapy systems that could improve anti-angiogenic efficacy when combined with standard treatment. The principal goal would be to expand the therapeutical effect of angiogenesis-inhibiting drugs on vessel normalization and render the tumor more vulnerable to additional agents such as chemotherapy or immunotherapy. In a recent study, Hosaka et al. could show that dual angiogenesis inhibition could sensitize resistant off-target tumors to therapy. Therefore they created mouse models of breast cancer or fibrosarcoma, both resistant to anti-VEGF and anti-PDGF treatment due to increased tumor associated expression of bFGF, a molecule which modulates the vasculature via pericyte recruitment in a PDGF-dependent process . NeitherAngiogenesis is a main therapeutic concept in oncology, especially in NSCLC, where three approved agents are available in combination with chemotherapy or immunotherapy. Nevertheless, the therapeutic efficacy of the current anti-angiogenic therapies is not satisfying and needs a more personalized/individualized approach. Increasing knowledge in angiogenic processes and non-angiogenic processes that contribute to tumor vascularization, provide precise targets for novel therapy strategies and pave the way for developing new anti-angiogenic treatment concepts that target e.g., TEC metabolism, TEC specific factors, tumor vessel normalization and combinational approaches with CAR T-cells. These therapeutic concepts need to be evaluated for synergistic effects as, in our view, modern anti-angiogenesis represents the concept of shaping the TME rather than being a direct anti-tumor therapy itself. However, these therapeutic strategies are very promising in preclinical setting and the translation into a clinical setting is not only warranted but highly desired. Furthermore, a new horizon of targeted and functional TEC characterization was opened by scRNA-Seq studies, which proved that the tumor vasculature is highly heterogenous and differs from the normal adjacent vasculature more than primarily assumed in terms of metabolic activity, immune suppression and heterogeneity for example. In addition, new synergistic effects of TECs in their role of immunomodulation were identified and induction of HEV formation for immune priming is suggested to be a new therapeutic strategy. Next the organ specific context of the vasculature plays an important role and has to be further studied for better therapy allocation.In conclusion the concept and goal of anti-angiogenesis in NSCLC in the future can be reshaped by abolishing the traditional vessel priming concept and moving toward a side specific molding of the TME, using the tumor vasculature as a tool, like a trojan horse.SD, HH, EN, AP, and DW developed the concept of the review. SD, HH, and EN drafted the review. DW and AP corrected and reviewed the review. 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. The handling editor LT declared a past co-authorship with one of the authors AP.


Autologous platelet concentrates such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) have gained overwhelming popularity in regenerative endodontics. Clinical evidence reveals the lack of a particular advantage of using PRP or PRF over an evoked blood clot in promoting canal wall thickening and/or continued root development in immature necrotic teeth. Moreover, despite stimulating tissue repair and repopulating the root canals of immature and mature permanent teeth, the new vital tissue may not possess the functional activity of the native pulp tissue.To better understand the origin, nature, and long-term fate of the tissue types found within the pulp space, we critically examine all available histo-/morphological evidence for pulp–dentine complex regeneration using PRP and/or PRF, alone or together with an evoked blood clot, specialised or unspecialised primary cells, and other biomaterials.Histological data from clinical studies is scant. Reportedly, the inner dentinal surface supports cementum-like tissue formation, but this interface likely deviates in structure and function from the native cementodentinal junction. Presence of bone-like tissue within the pulp space is intriguing since de novo osteogenesis requires closely coordinated recruitment and differentiation of osteoprogenitor cells. Compared to untreated necrotic teeth, an evoked blood clot (with/without PRF) improves fracture resistance. Tooth regeneration using PRF and dental bud cells is unreliable and the constituent neoformed tissues are poorly organised.PRP/PRF fail to demonstrate a significant advantage over an induced blood clot, alone. The true nature of neoformed tissues remains poorly characterised while their response to subsequent insult/injury is unexplored. Regenerative endodontics has evolved over the years as a treatment option for not only immature permanent teeth with open apices  but also for apical periodontitis in mature permanent teeth –3. Basedument}β) , 8, automentclass2pt{minimument}β) .neoformed tissues).In a systematic review of clinical studies, the lack of a particular advantage of using PRP or PRF compared to an evoked blood clot, alone, in promoting thickening of the canal walls and/or continued root development in immature necrotic teeth is strongly underscored . Current®/PubMed®, Scopus, Google Scholar, and ScienceDirect, the following search term  was applied: “platelet rich AND pulp”. All non-English language publications were disregarded. Publications that deviated from the theme , review papers, in vitro studies, and in vivo studies that did not report histological, morphological, or immunohistochemical data were excluded. Based on these criteria, a total of 21 publications were shortlisted , continued root development (or root lengthening), narrowing or closure of the root apex, formation of reparative dentine, presence of neoformed vital  tissues, ingrowth of bone-like and cementum-like tissues, presence of islands of bone-like and cementum-like tissues, deposition of mineralised tissue on the inner canal/dentinal wall, thickening of the canal wall, and presence/absence of inflammatory cells Fig. .Fig. 1Ne2, mineral trioxide aggregate (MTA), and enamel matrix derivative (EMD). However, in terms of reparative dentine thickness and odontoblast density, the use of PRP does not offer a significant advantage for intracanal tissue regeneration [Among the most basic endodontic applications of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are iatrogenic pulp exposure, pulpotomy in immature teeth, and pulpectomy in mature teeth. For direct pulp capping, a protective, circumferential layer of reparative dentine along the root canal walls can be achieved with PRP, Ca(OH)neration .In partial- and high-level pulpotomy procedures where hydroxy(l)apatite (HAp) is used in combination with PRP, root growth retardation and reparative dentine bridge formation occur as frequently as when HAp is used alone. It is noteworthy that, regardless of therapeutic modality, high-level pulpotomy tends to afford a less favourable outcome than partial pulpotomy .Pulpectomy entails complete extirpation of the pulp. The canal is subsequently irrigated and dried using sterile paper points. Using PRP, ingrowth of bone-like tissue is observed throughout the canal—from the coronal third to the apex of the root. Such islands of bone-like tissue are dispersed between neoformed connective tissue comprised of blood vessels and fibroblasts, and populated with osteoblasts in addition to some osteoclasts on their surface. Canal walls tend to be thinner than normal teeth and there is no physiological narrowing of the apex through deposition of new dentine .Intriguingly, the histological picture is no different when PRF is used. The neoformed vital tissues also include islands of bone-like tissue scattered between connective tissue made up of blood vessels and fibroblasts. In addition, dentine-like tissue exhibiting an irregular tubular structure forms along the inner dentinal wall but apparently discontinuous from the native dentinal tubules. Not surprisingly, odontoblasts associated with this dentine-like tissue do not display the typical polarised and palisade appearance .Even in combination with dental pulp stem cells (DPSCs), PRP shows no enhancement in new tissue formation compared to an evoked blood clot into the root canals . The incExperimentally, pulp necrosis and apical periodontitis can be induced by intentionally exposing the pulp to the oral environment or by placing a suspension of oral plaque within the pulp for an extended period of time. In necrotic teeth, the outcome of PRP is largely unremarkable compared to that of an evoked blood clot. The incidence of vital tissue regeneration and severity of inflammation are generally comparable . The per2 paste does not lead to an improvement in the efficacy of PRF compared to that in aseptically extirpated pulps [2 in its ability to support mineralised tissue deposition on dentinal walls of previously contaminated canals [Canal disinfection using a preparation containing metronidazole, cefixime, and ciprofloxacin in equal parts significantly increases the occurrence of apical closure and mineralised tissue deposition on dentinal walls in response to PRP . Canal ied pulps . Howeverd canals . Periapid canals .Fig. 3CeAn evoked blood clot, both alone and together with PRF, facilitates resolution of plaque induced apical periodontitis. Radiographic measurements indicate greater increases in root length and thickness, while histological observations reveal very high incidence of neoformed mineralised tissue, characterised as islands of bone-like tissue dispersed within the canal space and cementum-like tissue at the apex and along the inner surface of the canal wall . FurtherOne study reports an increase in the area fraction of canal space occupied by vital tissues with the use of PRP, either alone or in combination with dental pulp cells (DPCs), compared to an evoked blood clot. The neoformed tissues comprised of fibrous connective tissue and vasculature, in addition to cementum-like and bone-like tissues. Interestingly, the fraction of unmineralised tissue in response to PRP with DPCs was only 30% that of PRP without DPCs, suggesting the potential of DPCs to stimulate mineralised tissue formation . The comThe possibility to regenerate an entire tooth has been demonstrated using a PRF and fibrin glue scaffold seeded with dental bud cells, which was subsequently implanted into the alveolar socket . Tooth rAmong clinical studies, one instance of a necrotic pulp and immature/open apex of a premolar tooth that was accidentally extracted and immediately replanted, radiographic examination revealed evidence of continued root development following the use of PRP . The regIn a molar tooth presenting with a large carious lesion, periapical radiolucent areas, and apical resorption, an evoked blood clot with PRP  or without PRP  resulted in obliteration of the canal space by cementoid- or osteoid-like mineralised tissue and fibrous connective tissue Fig. . HoweverIt is hardly surprising that the histological data reported by presumably successful clinical studies using autologous platelet concentrates for pulp–dentine complex regeneration is scant. The observations are contradictory and the evidence in favour of using platelet-rich plasma (PRP) and platelet-rich fibrin (PRF), therefore, remains less than persuasive. With the exception of one study exploring the efficacy of PRP against an induced inflammatory plug , no studWhich tissue type(s) should ideally occupy the pulp space after regenerative procedures? This question is not a new one . HoweverLimited attempts have been made to characterise the composition  or structure  of the extracellular matrix in neoformed mineralised tissues. Many of these questions can be addressed using analytical tools commonly applicable for characterisation of bone , includiCurrently, the longest follow up durations among experimental and clinical studies are 6- and 25 months, respectively. In a simian model of pulpotomy , the preNevertheless, the presence of ectopic mineralised tissue brings up questions regarding long-term fate and consequences for functioning of the local environment. What factors modulate the formation of neoformed mineralised tissues? How does their architecture compare to the native tissues they appear to resemble? Would disproportionate amounts of neoformed mineralised tissue occlude the apex, as has been reported in the case of the combined application of PRP and dental pulp cells , and res2, CaO, MgO, K2SO4, and Na2SO4. This trend is unsurprising since as many as 85% of all clinical regenerative endodontic procedures use MTA as the coronal barrier [®, claimed to be similar in composition and comparable in efficacy to MTA [success perceived from radiographic observation of continued root development, apical closure, and resolution of periapical lesions must not be attributed to the use of an autologous platelet concentrate. The in vitro osteogenic potential [in vivo bone-bonding ability [It is noteworthy that, without exception, all studies reporting the presence of neoformed bone-like tissue have used mineral trioxide aggregate (MTA) as the pulp capping material, which is composed of Portland cement as the major component , 41, bis barrier . Other cy to MTA , have aly to MTA  and PRF y to MTA , clinicaotential  and in v ability  of MTA aA common feature among descriptions of the various neoformed tissues is the morphological likeness attributed to different cell types identified. Examples include osteoblast-like, osteocyte-like, cementoblast-like, cementocyte-like, odontoblast-like, and fibroblast-like cells. It is unclear whether such terms serve to imply similarities or deviations in function compared to the corresponding cell types in native tissues.The available quantitative data also presents shortcomings: discrete data  is less precise than continuous data . For this reason, it is highly probable that differences (or similarities) between the various treatment groups may have been lost to detection.formed in response to autologous platelet concentrates is strongly misleading.Given the dearth of convincing data, the application of autologous platelet concentrates such as PRP or PRF in pulp–dentine complex regeneration does not appear to offer a significant advantage over an induced blood clot, alone. Many key questions arise and remain unanswered. It is apparent that the true nature of neoformed tissues, as reported by experimental and clinical studies employing autologous platelet concentrates, is far from the native tissue types and remains poorly characterised. In fact, it is highly likely that the types and relative proportions of neoformed tissues are primarily a function of canal preparation procedures. But to describe these as having


In the present article, an overview of advanced analysis of coronary atherosclerosis by coronary computed tomography angiography (CCTA) is provided, focusing on the potential application of this technique in a primary prevention setting. Coronary artery calcium score (CACS) has a well-demonstrated prognostic value even in a primary prevention setting; however, fibro-fatty, high-risk coronary plaque may be missed by this tool. On the contrary, even if not recommended for primary prevention in the general population, CCTA may enable early high-risk atherosclerosis detection, and specific subgroups of patients may benefit from its application. However, further studies are needed to determine the possible use of CCTA in a primary prevention setting. The prevalence of traditional risk factors and overall cardiovascular mortality have been constantly decreasing in the last two decades, as recently reported by the European Society of Cardiology (ESC) ; howeverAccording to primary prevention ESC guidelines, systematic cardiovascular risk assessment may be considered in men >40 years old and in women >50 years old, and adoption of risk estimation systems  is recommended . HoweverIn the last few decades, coronary computed tomography angiography (CCTA) gained wide diffusion in the clinical field for noninvasive evaluation of coronary artery disease. This important role is supported by an extensive amount of literature demonstrating a good diagnostic performance vs. invasive coronary angiography ,4, togetRecent data may suggest a potential role of CCTA in the setting of primary prevention, thanks to the capability of this technique to provide an extensive evaluation of coronary atherosclerosis, improving early and accurate identification of patients at a higher risk of future coronary events, even if asymptomatic and without known cardiovascular disease.When compared to a traditional thoracic CT scan, the evaluation of cardiac structures needs the intrinsic heart movement and related artifacts to be considered and incorporated. In the last two decades, technological advances, especially with the wide clinical application of the multidetector CT scan, allowed the advent of ECG-gated cardiac CT. More precisely, thanks to ECG-gated CT, only the diastolic phase of the cardiac cycle is selected and acquired, enabling the obtainment of images of coronary arteries despite their physiological movement. Coronary calcium burden is evaluated without the need for contrast medium, and the prognostic value of the coronary artery calcium score (CACS) has been widely validated. With the adjunct of intravenous contrast medium, coronary artery lumen can be noninvasively evaluated with ECG-gated contrast CT scans acquired at the early angiographic phase, paving the way to the first noninvasive tool for coronary anatomy evaluation. This type of acquisition was performed with a non-negligible increase of radiation dose  that has been progressively reduced to less than 3–5 mSv after the advance of whole-heart coverage CT scans .2 above a threshold of 130 HU; a CAC score is then obtained by taking into consideration both the total area and the attenuation values of coronary atherosclerosis [CACS can be considered the first approach to coronary atherosclerosis evaluation by cardiac CT beyond coronary lumen stenosis quantification. Performed on noncontrast cardiac CT images, coronary artery calcium (CAC) is defined as a hyperattenuated lesion of at least 1 mmclerosis . CACS evclerosis . Similarclerosis . Subsequclerosis , while aclerosis . Moreoveclerosis . More reclerosis  , the segment-stenosis score (SSS), and CT-adapted Leaman score (CT-LeSc) ,29. SIS CT-LeSc  is of paAdvanced coronary plaque analysis by CCTA can be performed by qualitative/semiquantitative methods in the evaluation of a single specific lesion or by more comprehensive methods enabling patient-based atherosclerosis volume quantification .Qualitative/semiquantitative plaque characteristics are lesion-based findings that can be evaluated together or alone in a single coronary plaque and that are defined as outlined in Postprocessing tools recently implemented have resulted in the accurate quantification of coronary artery luminal area, atherosclerotic plaque area, and plaque volume when compared with measurement by IVUS ; both lop = 0.40) and patients with ACS did not differ significantly from control subjects in total plaque volume and in calcified plaque volume but had significantly higher fibro-fatty volumes . In 2019, results from a SCOT-HEART trial subanalysis showed that adverse coronary plaque characteristics are associated with a three-fold higher risk of coronary heart disease death or nonfatal myocardial infarction. Of interest, in patients with a CACS <100, those with adverse plaque features had an increased risk of coronary events compared to patients without [Several studies have suggested the important prognostic role of advanced atherosclerosis analysis by CCTA beyond lumen stenosis severity. In 2009, Motoyama et al. demonstrated that acute coronary sindrome (ACS) was independently predicted by postive remodeling (PR) and/or low attenuation plaque (LAP) in a consecutive cohort of patients who underwent CCTA for suspected CAD  after 27 without . These r without ; more prPrevious data showed the prognostic value of CACS over traditional risk factors in asymptomatic patients, supporting the possible role of CACS in primary prevention , even ifIn 2016, Hun-Kang et al. enrolled 591 consecutive asymptomatic patients with type 2 diabetes mellitus who underwent CCTA. The event-free survival rates for a composite endpoint of cardiac death, nonfatal myocardial infarction, and unstable angina requiring hospitalization or late coronary revascularization, at 6 years follow-up, were 99.3% in patients with normal coronary arteries, 96.7% in patients with nonobstructive CAD, and 86.2% in patients with obstructive CAD . Of noteMore recently, Halon et al.  enrolledThus, the potential benefit of CCTA over CACS in selected asymptomatic patients may be supported by CCTA’s capability to directly identify location and severity of fibro-lipidic coronary plaque that may be underestimated by CACS but have independent prognostic roles beyond traditional risk factors and coronary lumen stenosis, as recently demonstrated ,28,34,35Assuming that CCTA cannot be performed in the entire population and that it is presently not recommended, it is of utmost importance to identify subgroups of patients that may potentially benefit from CCTA as screening tests targeting healthy people. Among others, asymptomatic diabetic patients may benefit from early CCTA, at least for the excellent prognosis if normal coronary arteries are found ,39,40,41Senior athletes are a potential subgroup that may benefit from CCTA even if asymptomatic and without previous cardiovascular disease. Indeed, cardiac CT  may hold the best potential for use in preparticipation screening of older athletes with several cardiovascular risk factors, among which sudden cardiac death is most frequently due to unknown CAD .After appropriate selection of subjects that should undergo CCTA, the absence of therapeutical consequences of nonobstructive but high-risk CAD identification may limit the clinical application of CCTA, as no drug directly acting on atherosclerosis plaque regression or stabilization is currently available for appropriate secondary prevention treatment after early identification of subclinical high-risk coronary atherosclerosis. However, preliminary evidence of atherosclerosis-targeted therapy after CCTA comes from a SCHOT-HEART trial subanalysis: statin and aspirin prescription rate significantly increased after CCTA when compared with a functional test-arm, possibly justifying the prognostic benefit demonstrated in the CCTA arm . Statin Despite recent data providing evidence for early identification of subclinical coronary atherosclerosis, it should be underlined that several limitations exist regarding the use of CCTA in asymptomatic patients. One of the main criticisms is the absence of clear-cut evidence supporting the prognostic role of CCTA in this setting and, of most importance, the absence of atherosclerosis-targeted therapy that could be introduced in patients at higher risk; moreover, the risk of potential adverse events associated with the use of intravenous contrast medium  cannot be underestimated.The prognostic role of CACS, even among asymptomatic patients, has been extensively supported by several previous studies and across different ethnic groups ,53, and Overall, these data are shifting the paradigm of coronary artery disease treatment from a lumen- and ischemia-centered view to a more comprehensive approach reflecting the natural history of atherosclerosis progression. As suggested by J. Min in 2016 , “the di


Idiopathic rapid eye movement (REM) sleep behavior disorder (iRBD) is increasingly recognised as an important precursor disease state of alpha-synucleinopathies. This parasomnia is characterized by a history of recurrent nocturnal dream enactment behaviour, loss of skeletal muscle atonia, and increased phasic muscle activity during REM sleep. Neuroimaging studies of striatal dopamine transporter uptake tracer signaling suggest increasing dopaminergic deficit across the continuum of the alpha-synucleinopathies, with early sleep dysfunction suggestive of early caudate dysfunction. Henceforth, we set out to investigate the relationship between early sleep changes and the striatal dopaminergic availability in iRBD.Twelve patients with iRBD, who had undergone a video polysomnography and a neuroimaging assessment of striatal dopamine transporter (DaT) uptake tracer signaling, and 22 matched controls who had similarly undergone a video polysomnography were retrospectively identified. Data were statistically analyzed to identify altered sleep parameters and correlate them with striatal dopamine transporter uptake tracer signaling.P=0.001), compared to 22 age-matched healthy subjects. In addition, several significant links were found between regional DaT-uptakes and sleep architecture. Correlational analyses suggested a strong positive association between sleep fragmentation and dopamine deficiency in left caudate , whilst an increased uptake in the whole striatum was strongly linked to the sleep efficiency, and to a lesser degree to the length of sleep duration.The iRBD patients exhibited an increased number of periodic limb movements during sleep (To the best of our knowledge, this is the first demonstration of a close relationship between dopaminergic availability in striatum and the quality of sleep in iRBD. Taken together, our exploratory findings suggest that subtle but functionally significant striatal changes in early stages of iRBD may contribute to the further shaping of sleep architecture. Idiopathic rapid eye movement (REM) sleep behaviour disorder (iRBD) was originally described as a rare, dream re-enacting primary parasomnia.3Over the past few decades, neuroimaging studies of PD have consistently demonstrated uneven dopaminergic deficit within the striatum, with more severe involvement of the posterior putamen and a relative sparing of the head of caudate nucleus.10123I-ioflupane-SPECT data in de novo iRBD patients, as well as in comparison with polysomnographic (PSG) data from healthy controls.To date, surprisingly little is still known about macroscopic and microscopic sleep structure in iRBD,123I-ioflupane at a large tertiary sleep centre between 2009 and 2019 was conducted  classification,onducted A. The stOnce the iRBD cohort was defined, simple demographics  were used to select matched control subjects from a subset of patients entered into the same database, over the same period, with unremarkable PSG . Control15All subjects had undergone overnight PSG, and the subsequent sleep scoring and video-analysis were performed in accordance with AASM by two experienced sleep technologists.16123I-ioflupane, as previously described.17SPECT imaging was done following an overnight PSG, and the SPECT images were acquired 3-hours after intravenous bolus injection of P < 0.05. 123I-ioflupane SPECT data were analysed using the BRASS software  following a semi-quantitative approach.123I-ioflupane were calculated for each striatum, including caudate and putamen values, relative to the non-specific uptake measured in the occipital cortex.123I-ioflupane uptake values, was calculated for both hemispheres. Similarly, the average binding for ROI was calculated per individual as the mean uptake value for both hemispheres, as previously defined.9Statistical interrogations were performed using the Statistical Package for the Social Sciences V.21 . Data were assessed for normal distribution using the Kolmogorov–Smirnov test. Fisher’s exact test of independence  and Mann–Whitney U-test (non-parametric) were conducted; all tests used a threshold of 123I-ioflupane tracer uptake and sleep parameters was tested, using Spearman’s rank correlation. Due to the low number of subjects receiving SPECT and the exploratory nature of the DaT analysis, we did not apply correction for multiple comparisons.For the purposes of SPECT-analysis, images were further normalised to age and gender-matched healthy controls from the European-Database-of-DaTSCAN-of-healthy-controls (ENC-DAT)P=0.001). A longer period of time needed to initiate sleep was similarly recorded for the iRBD group, although it did not reach statistical significance (SL: P=0.056).The patients and the controls were well matched according to age and sex, and it is notable that three patients and controls were prescribed selective serotonin reuptake inhibitors (SSRIs) antidepressants at the time of study . Of the Reduced striatal uptake, reflecting dopamine deficiency, was initially reported for nine iRBD patients (75%) following visual (unquantified) investigation; subsequent semi-automatic quantification analysis confirmed reduced DaT-uptake in eight patients . Conversely, increased uptake in the whole striatum was strongly linked to the sleep efficiency , and to a lesser degree also to the length of sleep duration . The positive association between the dopamine availability and the period of sleep that patients spent in REM stage (REM%) was demonstrated in the left caudate  and putamen region . Moreover, the correlations appeared strongly driven by the presence of pathology as determined via DaT-uptake z-scores (>±2) for that region . No other significant links were found between regional DaT-uptakes and sleep architecture.Further correlational analysis of links between sleep parameters and regional DaT-uptakes  suggesteOur data converge on two key findings, that suggest a close relationship between dopaminergic availability in striatum and the quality of sleep in iRBD. Firstly, in this study, the link between sleep efficiency and sleep duration and dopaminergic availability in the striatum is demonstrated. Next, an important modulatory role for caudate in arousals, sleep fragmentation and REM mechanisms is intimated by the results , Table 3The importance of appreciation of links between dopaminergic functionality of caudate region and sleep issues, as suggested by these results, may indeed be of particular clinical note. Lately, in opposition to traditional views, nigro-caudate dopaminergic deafferentation has been argued as a principal biomarker of iRBD.Historically, unrelated to RBD disease process, both caudate nucleus and REM sleep have been linked to “orienting and alerting responses” in early animal experiments.3In this study, we were unable to demonstrate macrostructural sleep differences between our iRBD and control subjects. This is in some agreement with previous reports of a relatively preserved sleep architecture in iRBD patients.28Finally, in this exploratory study, iRBD patients were recorded with a higher number of PLMs than control subjects. This result is in keeping with previous reports and may be reflective of shared basal ganglia etiology, which we were unable to demonstrate in this study.Lastly, it should be noted that depression and mood changes have been increasingly recognised amongst the important prodromal non-motor symptoms of an α-synucleinopathy process, albeit their specificity and the exact underlying mechanisms remain elusive.39In summary, to best of our knowledge, this is the first study that has linked early changes in sleep quality in iRBD with dopaminergic availability in the striatum. However, there are several important limiting factors to this exploratory study , which will need to be rectified in future larger, prospective multimodal neuroimaging studies of iRBD, in order to truly authoritatively address the causality and directionality of implied associations. Nonetheless, we believe that the findings presented here raise some fundamental questions. They also suggest subtle but functionally significant alterations in the striatal region of brains of patients with iRBD that might represent the early expression of the supposed neurodegenerative processes already taking place at this stage of the disease, and that might be the target of better and effective future therapeutic strategies for this condition.


Vaccinating women in pregnancy  has emerged as a promising tool to tackle infant morbidity and mortality worldwide. This approach nurtures a ‘gift of nature,’ whereby antibody is transferred from mother to fetus transplacentally during pregnancy, or postnatally in breast milk, thereby providing passive, antigen-specific protection against infections in the first few months of life, a period of increased immune vulnerability for the infant. In this review, we briefly summarize the rationale for maternal immunization programs and the landscape of vaccines currently in use or in the pipeline. We then direct the focus to the underlying biological phenomena, including the main mechanisms by which maternally derived antibody is transferred efficiently to the infant, at the placental interface or in breast milk; important research models and methodological approaches to interrogate these processes, particularly in the context of recent advances in systems vaccinology; the potential biological and clinical impact of high maternal antibody titres on neonatal ontogeny and subsequent infant vaccine responses; and key vaccine- and host-related factors influencing the maternal-infant dyad across different environments. Finally, we outline important gaps in knowledge and suggest future avenues of research on this topic, proposing potential strategies to ensure optimal testing, delivery and implementation of maternal vaccination programs worldwide. To improve maternal and neonatal health remains a focus of international investment in global health, given that Millenium Goals 4 and 5 were not achieved . DespiteThere is increasing momentum to develop and implement vaccination of women during pregnancy  to prevent specific infections of particular relevance to pregnancy and the newborn . This apTwo broad aims can be achieved: (1) Pregnant women can be protected against a number of infectious diseases with more severe outcomes in pregnancy  and (2) their newborn babies are protected by high titres of antigen-specific IgG antibody against diseases of particular importance in the newborn  due to their associated susceptibility  or severity  and until they develop protective antibody after receiving their own vaccines or following natural exposure.Depending on geographical location, maternal immunization is already routinely recommended to prevent tetanus, pertussis and influenza with proven safety, immunogenicity and efficacy, but uptake and coverage vary widely . New vacin utero setting to the new reality of an environment full of germs: some hostile, some not. The principle of maternal immunization essentially represents the augmentation of specific antibodies against organisms of high pathogenicity to the newborn, throughout this period of significant immune vulnerability; actively immunizing a mother during her pregnancy capitalizes on the indirect protection that will already be conferred to her newborn, thereby combining nature with nurture , the villous stroma and the fetal capillary endothelium. Signaling across the placenta is now considered to be bidirectional, involving both active and passive mechanisms, with a potentially significant role played by extracellular vesicles and seeding of genetically foreign maternal and fetal cells, closely regulated throughout pregnancy to ensure optimal fetal growth and development .Maternal immunoglobulin G (IgG) is transported across the placenta from around 13-weeks’ gestation and provides passive protection to the infant in the early, most vulnerable postnatal period, prior to initiation of the primary immunization schedule . It is aUnlike syncytiotrophoblast cells, stroma or fetal endothelium appears not to express FcRn and, therefore, the mechanisms underlying the onward transport of IgG beyond the syncytiotrophoblast require further exploration; alternative non-canonical Fcγ receptors, such as FcγRI/RII/RIII, are known to be expressed by these final placental layers and may play a key role .ex vivo human placental model demonstrated that the functionality of CMV antibody may play a significant role: weakly neutralizing compared to potently neutralizing monoclonal IgG facilitated placental CMV infection  . Congeninfection .After birth, the placental barrier protection is replaced by the mammary gland barrier. The specific antibodies generated are also secreted into breast milk (primarily colostrum) with secretory IgA (sIgA) as the predominant antibody class, and transferred orally to infants during lactation . IgA is By preventing epithelial adhesion and neutralizing toxins or virulence factors, sIgA inhibits invasion and damage from pathogens at mucosal surfaces . InteresIn general, given the usual half-life of immunoglobulins, higher maternal and cord antigen-specific IgG concentrations are associated with subsequently longer, more effective protection in the infant in early life . The aimHaemophilus influenzae type B (HiB), Neisseria meningitidis C and varicella zoster virus (VZV)  . Specifius (VZV) . One conus (VZV) . It has us (VZV) .Placental transfer is distinctly influenced by different antigen-specific IgG populations . Vaccinein vitro  is important albeit inconsistent between studies . CompariMoreover, HIV-exposed uninfected (HEU) infants have up to fourfold higher rates of morbidity and mortality from diarrhoeal and respiratory infections compared to uninfected unexposed (HUU) infants, in part explained by lower maternal production and reduced placental transfer of protective IgG . Vaccina197-conjugated trivalent GBS vaccine was found to be lower in HIV-infected pregnant women compared to HIV-uninfected women, irrespective of CD4+ T-cell counts , may alter IgG production, damage placental villi, decrease FcRn expression and/or compromise transplacental transfer rate . Of noteSimilar factors may contribute to maternal antibody transfer in breast milk, although there is paucity of data. One example is the impact of vaccination timing and gestational age; significantly more sIgA (and IgG) was measured in the colostrum and mature breast milk of women who delivered preterm, waning more gradually than in term women . This maN-glycans compared to IgA1, which dominates in serum  antigen neutralization of live replicating viral vaccines (2) epitope masking preventing antigen binding by infant B-cells, thereby limiting their priming, the most popular theory (3) inhibition of infant B-cell activation by FcγRIIB-receptor-mediated signaling, and (4) removal of maternal antibody-vaccine antigen immune complexes by Fc-dependent phagocytosis .Although a blunting response has been demonstrated, this data is generated from immunogenicity studies and, to date, there is no evidence of relevance to clinical outcomes (disease incidence or severity) ; howeverB. pertussis growth inhibition assay) was overall better in wP-vaccinated infant sera, and after maternal immunization. These data suggest that maternal antibodies may boost the production of antibodies with distinct biophysical features to achieve effective pathogen control in the infant  and/or a boost in uptake and antigen presentation, mediated by immune complexes in a Fc glycosylation–dependent manner . Most ree infant .Finally, many have argued that blunting is short-lived and reversible, especially as maternal antibodies decline over time, and does not remain significant following booster vaccination . LongituTo date, vaccine-induced CoPs are predominantly defined by the development of serum antibody responses using quantitative assays, with less focus on cell-mediated immunity (CMI), despite the significant induction of cellular responses post-vaccination by some vaccines, such as pertussis, and the important role CMI plays in disease protection . This exA lowered antibody titre, caused by the blunting effect, does not necessarily imply reduced protection; a recent comprehensive review concluded that in the majority of both human and animal studies, priming of CMI after infant vaccination occurred even in the presence of high maternal antibody titres, with minimal or no blunting effect on cellular responses reported . FurtherIn contrast to other blunting theories, a recent study by in utero, both qualitatively and quantitively, to vaccine antigens to which the mother has encountered during pregnancy, either as free antigens or antigen-antibody complexes . Moreover, it may be relevant in cases where the pediatric primary immunization course is too late to prevent severe disease, for example, with GBS or RSV infections in early life  of vaccines, including heterologous lymphocyte effects and induction of innate immune memory (‘trained immunity’). The latter is thought to be mediated by epigenetic and metabolic reprogramming which elicits long-term functional upregulation of innate immune cells . NSE areIn addition to preventing disease, maternal immunization may be a strategy to protect young infants from early bacterial carriage at the mucosa, via either breast milk- or transplacentally derived antibody . This isSimilarly to transplacental antibody, we must consider whether interference from vaccine-derived antibodies in breast milk may impede immunogenicity of mucosal vaccines; it has been speculated that this may contribute to the poor performance of live oral rotavirus immunizations given to infants in LMICs, where breastfeeding rates are high . A recenWe have already discussed factors that may affect transfer of maternal antibody to infants, but adequate production of antibody in the first place is a crucial determinant of subsequent infant antibody titres. Further maternal, neonatal and environmental factors also shape the early immunological milieu and, therefore, the success of maternal immunization . A detaiIt is well established that age and sex affect vaccine-induced immunity, particularly quantitative antibody responses, relevant to both the pregnant woman and her developing infant .Neisseria meningitidis serotypes. This includes variants in the genes regulating both innate and adaptive compartments, encoding Toll-like receptors, HLA molecules, cytokines, and cytokine receptors. However, very few data are available from studies conducted in infants  is inversely correlated with long-term vaccine-induced antibody responses . By contGeographical setting, both defined by development status or urban/rural location, is known to play a role in immune responses at all ages and might also affect responses to vaccines in pregnancy . EquallyActinobacteria and Firmicutes was consistently correlated with both higher antibody and cellular responses to several vaccines, including BCG, Hepatitis B, IPV, OPV, and tetanus immunizations; an inverse correlation was found for Proteobacteria and Bacteroidetes , so far shown to be safe and effective in seasonal/pandemic influenza vaccines in non-pregnant human adults, as well as in pregnant animal models  and increasing evidence is emerging on oil-in-water adjuvants, such as MF59, which has shown to improve both epitope breadth and binding affinity of antibody responses if used in hemagglutinin -based influenza vaccines . Anotherl models .in vitro and in vivo  transplacental transport.•Impact of maternal antibody avidity on placental transfer and the impact of timing on avidity.•Quantitative and qualitative changes in Fc glycosylation throughout pregnancy and across different antigen-specific populations, as well as the effect of pregnancy, disease and specific vaccine factors on glycosylation patterns.•Biological and clinical implications of distinct glycosylation profiles, including potential association with Fc-mediated maternal antibody interference.•Detailed mechanisms underlying breast milk antibody transfer and key regulating factors.•Effects of vaccination during pregnancy on the composition of breast milk, particularly the presence of pathogen-specific sIgA antibodies.•How vaccine design, delivery, dosing and timing may determine efficacy of antibody transfer, in the context of different types of vaccines.•Robust experimental models to interrogate the materno-fetal interface, specifically utilizing new technologies and imaging techniques.•Definitive  correlates of vaccine-induced protection for key maternal vaccines.•Application of novel systems biology tools to investigate placental, maternal, fetal, and neonatal immune compartments.•Standardized qualitative and quantitative assays to fully characterize the short-, intermediate- and long-term effects of maternal immunization on fetal and neonatal immunity.•Targeted studies on the maternal, neonatal (both genetic and acquired) and environmental co-factors shaping early neonatal quantitative and qualitative immune responses.•Mechanisms explaining blunting and its potential impact on functional responses.•in utero priming, plus the impact on subsequent infant cell-mediated responses to primary vaccines, including heterologous effects.Mechanisms underlying potential maternal vaccine-induced microchimerism and •Interactions between IgG with other Fc receptors, particularly on fetal or neonatal innate cells.•The role of vaccine-induced mucosal cellular immunity and its interaction with mucosal humoral responses and/or microbiota communities.•Impact of breast milk immune factors induced by maternal immunization on infant immune responses.•Biological and clinical relevance of immune phenomena induced by maternal vaccination in the context of infant infection and immunity.Conversely, a systems vaccinology approach can help to understand the effect of pregnancy itself on vaccination, including the specific acute inflammatory pathways induced and markers of immunogenicity/safety. Using Boostrix vaccine , Therefore, it is hoped that newer ‘omics’ assays may elucidate key mechanisms controlling vaccine immunity, particularly functional responses, define molecular signatures, identify novel CoP and predict clinical end-points, including safety. It is time to direct these tools to the maternal/infant dyad during clinical trials.These methods must be applied within a convincing model of the materno-fetal interface. Paired maternal–cord samples, specifically the ratio of cord:maternal antibody concentration, are often used as a surrogate for placental transfer, although this fails to provide mechanistic insights.Animal models have provided ‘biologically complete’ insights into the possible mechanisms of FcRn-mediated IgG transfer, overcoming ethical and practical limitations of human studies. Nevertheless, the morphological, developmental, physiological and immunological characteristics of the placenta often vary between humans and animals, including the expression of FcRn and mechanisms of IgG transport .Similarly, animal models of breast milk production/transfer are limited; differing from humans, IgG is the primary immunoglobulin class in milk throughout many animal species , predominantly transported across the duodeno-jejunal epithelium into the neonatal circulation  . The proex-vivo or in-vitro and performed at any stage of gestation, unlike trophoblast cells or placental perfusion models, enabling interrogation of early placental function  to optimize our existing models and/or apply novel technologies to more accurately recapitulate the diverse cell types and complex interactions at the materno-fetal interface. Key examples include co-cultures cultivating both trophoblastic and endothelial cells on a membrane; three-dimensional trophoblast organoids; computational modeling of tissue dynamics and blood flow; and bio-engineered tissue constructs .With the considerable international interest in vaccination in pregnancy by academic researchers, industry and community stakeholders alike, it has become apparent that in addition to the multitude of biological challenges summarized above, considerable implementation challenges also need to be addressed . These rOver the last 2 years, IMPRINT, the international IMmunising PRegnant Women and INfant NeTwork has brought together investigators and stakeholders in 50 countries across the globe to conduct research into specific biological and implementation challenges. The Pregnancy Research Ethics for Vaccines, Epidemics, and New Technologies (PREVENT) team has set out a framework for ethical consideration of inclusion of pregnant women in clinical trials of new vaccines ; more spIn As we have summarized in this review, significant progress has been achieved to understand how mother and child are immunologically linked, thereby optimizing vaccine design to effectively and safely target this materno/fetal dyad. Much more remains to be done in this exciting area of vaccinology, which has already achieved unprecedented and laudable multi-stakeholder engagement.BK developed the concept for the review and provided senior support. AS conducted the literature review and wrote the first draft. Both authors contributed to writing the manuscript and reviewed the final draft.BK’s institution receives funding from a variety of donors, including industry for the conduct of vaccine-related research in women and children. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.