The lymphatics play important roles in chronic diseases/conditions that comprise the bulk of healthcare worldwide. Yet the ability to routinely image and diagnose lymphatic dysfunction, using commonly available clinical imaging modalities, has been lacking and as a result, the development of effective treatment strategies suffers. Nearly two decades ago, investigational near-infrared fluorescence lymphatic imaging and ICG lymphography were developed as routine diagnostic for clinically evaluating, quantifying, and treating lymphatic dysfunction in cancer-related and primary lymphedema, chronic venous disease, and more recently, autoimmune and neurodegenerative disorders. In this review, we provide an overview of what these non-invasive technologies have taught us about lymphatic (dys) function and anatomy in human studies and in corollary animal studies of human disease. We summarize by commenting on new impactful clinical frontiers in lymphatic science that remain to be facilitated by imaging. We then focus on the clinical use of NIRF-LI/ICG lymphography for interrogating and treating chronic conditions for which lymphatic dysfunction is known or suspected. Next, we review chronic conditions for which the lymphatics are not commonly associated as part of clinical disease etiology, but which NIRF-LI/ICG lymphography suggests could provide a new, potentially a more effective target of treatment to improve outcomes. We then describe research on the role that lymphatics play in both the cause and abatement of neuroinflammation and how the lymphatics can be better harnessed to improve immunotherapies. Because of the limited tissue penetration provided by fluorescence technique, this review will not cover lymphatics in the trunk or the gastrointestinal tract that are described elsewhere , nor do The visualization of the lymphatics has been challenging since the mesenteric lacteals were first described as “arteries containing milk” around 300 BC . Largely99mTc. Lymphoscintigraphy is considered the “gold standard” of lymphatic imaging. In this technique, radionuclide is indirectly administered to the lymphatics via intradermal or subcutaneous injection for uptake by the lymphatic plexus, a dense network of lymphatic capillaries located below the epidermis and surrounding all organs of the body. Typically, 20–45 min after injection, the subject is placed under a gamma camera and, because only one gamma photon is released for each radionuclide decay, several minutes are required to acquire a single lymphoscintigram detailing a low resolution image of the lymphatics. Sequential lymphoscintigrams are used to determine the transit time of the radionuclide from the injection sites to the draining nodal basin as a measure of lymphatic function. While lymphoscintigrams provide images of major lymphatic trunks, deep lymph nodes, and, in the case of disease, may indicate areas of blockage and/or the dermal backflow of the radionuclide, the poor spatial and temporal resolutions limit the ability to visualize smaller lymphatic vessels or active lymphatic propulsion within conducting vessels. In addition, the use of ionizing radiation poses risk to patient and clinician alike, limiting its use in routine and longitudinal studies.Sometimes used in the United States as a diagnostic for lymphatic dysfunction and more frequently so in Europe, lymphoscintigraphy is based on the collection of gamma rays emitted from the decay of radionuclide via cannulation of a lymphatic vessel or intranodal injection. For a more comprehensive description of x-ray lymphography see  is frequently referred to as MR lymphangiography or lymphography (MRL) and may be performed with or without contrast depending on the desired image set. Non-contrast MRL is most commonly used to image the central conducting vessels or dilated lymphatic vessels based upon the high signal intensity of static or low flow lymph in heavily weighted T2 sequences that attenuate signal from background tissues. The technique typically does not resolve thin lymphatic vessels. Contrast-enhanced MRL can resolve smaller lymphatic vessels when sufficient contrast is taken up following intradermal, subcutaneous, or intranodal injection of a gadolinium (Gd)-based contrast agent. By acquiring sequential image sets, the transport of the contrast through the lymphatics over time can also be assessed. However, one of the major challenges to contrast-enhanced MRL is that low molecular weight Gd-based agents can rapidly egress into the blood vasculature causing background signals that obscure lymphatic vessels. In addition, the fate of Gd into organs, in particularly the brain, have raised concerns over the long-term safety of Gd-based contrast agents and some regulatory guidelines (such as those in Japan) encourage the use of macrocyclic over linear Gd-based agents, as less deposition is observed with these structures . For morPhotoacoustic imaging is an emerging imaging modality that is being translated for clinical lymphatic imaging. In this technique pulsed laser light is scanned across the surface of the skin. As the pulsed laser light travels deeply through tissues, it is absorbed by molecules (such as hemoglobin or an exogenous contrast agent) and converted to heat which causes transient thermoelastic expansions within the tissues and produces pressure waves that can be detected using an ultrasonic transducer . The detThe routine uses of these conventional and emerging techniques in clinical lymphatic research and in the diagnosis of lymphatic dysfunction is limited by substantial instrumentation requirements, expense, patient risk, ability to conduct longitudinal measurements to monitor treatment response, and/or the temporal and/or spatial resolution limitations of the modality. Fluorescence imaging modalities offer point-of-care approaches that do not involve ionizing radiation, offer unprecedented temporal resolution, and when compared to lymphoscintigraphy, provide improved spatial resolution for evaluating peripheral lymphatics.Fluorescence is a phenomenon where a molecule absorbs excitation light and, upon relaxation, reemits light of lower energy . After a fluorescent molecule has relaxed to the ground state it can be repeatedly excited thereby providing greater photon counts than possible with the single emission events from decay of radionuclides as measured with lymphoscintigraphy. Lymphatic imaging of preclinical models using fluorescent contrast agents or dyes has been demonstrated over the years. For a more comprehensive review of these approaches and more particular of the various fluorescent contrast agents used preclinically,  see .et al. visualized the lymphatics in the feet and ankles of human subjects using florescence microscopy following subepidermal injection of FITC-labeled dextran  and ICG lymphography are closely related imaging technologies that collect a NIR fluorescent signal emanating from the lymphatics following the off-label administration of ICG. Using this approach, the peripheral lymphatic vessels can be visualized in health and disease. As shown in While no imaging device has been formally approved by the United States Food and Drug Administration (FDA) for the indication of ICG-based lymphatic imaging, several commercially available devices, indicated for blood perfusion imaging based on ICG-fluorescence, have been used off-label to image the peripheral lymphatics . In the 2 to mimic light scattering and quantum dots with small fluorescence cross sections in the NIR wavelengths, provide a means to assess device performance as well as to calibrate registered signals from CCD/sCMOS detectors into standardized international units that are required for all medical devices . For example, the GaAs image intensifier in NIRF-LI devices amplify the collected fluorescence signal and the integrating CCD or sCMOS detector zero-averages noise, resulting in an increased SNR. As shown in  devices . These p devices .The contrast agent used for ICG lymphography/NIRF-LI is ICG, a dye that has been used safely in human studies for approximately 60 years and is approved for i.v. administration, typically in boluses of 5 mg with a total dose ≤ 2 mg/kg. Clinical uses include determination of cardiac output and hepatic function as well as fluorescence angiography for surgical guidance. The fluorescent properties of ICG depend on its concentration and the solvent, with peak absorption and fluorescent emission being 780 nm and 830 nm in a dilute aqueous solution and 805 nm and 835 nm in whole blood. Because ICG is a planar molecule, at concentrations greater than 25 μM, it can “stack” together forming oligomers that quench fluorescence . Once inDepending on lymphatic uptake rates, an epidermal depot of ICG can drain into the lymphatics for multiple hours, enabling ample time to visualize lymphatic function and its responses to physiotherapy or other medical intervention without the need for additional injections. The rapid image acquisition times possible with ICG lymphography/NIRF-LI enable real-time viewing of lymphatic pumping that is the hallmark of the unique physiology. ICG-laden lymph moves progressively to upstream lymphangions, or lymphatic vessel segments that are bounded by valvular structures depicted . These vvia the subclavian vein, ICG is exclusively cleared by the liver  and secreted into the bile, providing a means to evaluate peristaltic movement in rodents and potentially in infants , sometimes referred to as NIR-II imaging, in which fluorescence signals of wavelengths ranging from about 900 to 1,700 nm are collected using InGaAs scientific cameras. Significant advantages of imaging at these wavelengths are the reduced scattering of photons in the tissues and a greater, wavelength separation between excitation and fluorescent light, i.e., Stoke’s shift, that results in reduced light leakage through the optical filters. Thus, SWIR potentially allows for the acquisition sharper images of anatomic structures beneath the skin. However, an increase in water absorption at SWIR wavelengths may offset this advantage. In addition, the advantages associated with the amplification of signal and reduction of noise offered by intensified CCD or sCMOS Si detectors are not offered by SWIR systems. Perhaps the biggest hurdle however, is the lack of efficient fluorescent contrast agents approved for clinical use. While the emission spectra of ICG extends beyond 1,000 nm, the number of photons emitted in this portion of its spectra is a small fraction of that emitted at 830 nm . IncreasThe remainder of this review will focus upon what clinical research applications of non-invasive investigational NIRF-LI/ICG lymphography has taught us about the role of peripheral lymphatics in chronic conditions that are, and are not, traditionally associated with lymphatic dysfunction.In the developed world, the chronic condition most commonly associated with lymphatic dysfunction and the first condition probed with NIRF-LI and ICG lymphography , is cancThe hands and arms of breast cancer-related lymphedema subjects are characterized by the loss of straight lymphatic vessels that normally pump lymph proximally to draining axillary lymph node basins  without In early stages of both upper and lower extremity lymphedema, NIRF-LI shows that manual lymphatic drainage improves lymphatic pumping , consistIn addition, NIRF-LI has shown lymphatic dysfunction and dermal backflow in the unaffected contralateral arms of subjects diagnosed with unilateral breast cancer lymphedema , a findiEmerging treatments of cancer-related lymphedema involve microsurgeries, including i) lymphovenous anastomoses (LVAs) in which lymphatics are surgically connected to veins for drainage directly into the regional venous system, and ii) vascularized lymph node transplants (VLNTs) wherein lymph nodes from a normal part of the body are excised and transplanted into the effected lymphatic watershed. While ICG lymphography is used intraoperatively to guide microvascular surgeries, there have been few studies to longitudinally follow restoration of lymphatic function . While NRecently, Aldrich and coworkers used NIRF-LI to longitudinally image the lymphatics of advanced breast cancer patients during their treatment journey of axillary lymph node dissection, radiation treatment, and across 6-month follow-up periods . As depiNonetheless, these longitudinal studies support other studies that suggest ICG lymphography/NIRF-LI may provide a more accurate screening tool of cancer-related lymphedema as compared to lymphoscintigraphy .As described above, cancer-related lymphedema following lymph node dissection and radiation treatment for breast, prostate, melanoma, bladder, and gynecological cancers is associated with upper and/or lower extremity lymphedema which is clinically diagnosed by limb volume changes. However, head and neck cancer survivors experience lymphatic dysfunction in ways that do not always result in measurable changes in tissue volumes. Cervical lymph node dissection and radiation treatment typically cause internal lymphatic dysfunction that leads to difficulties with swallow, speech, and breathing or external lymphatic dysfunction manifesting as fibrosis, often without the obvious swelling that occurs in with upper and lower extremity lymphedema. Because microdoses of ICG do not leave permanently green-stained tissues, NIRF-LI can be used to longitudinally evaluate cranial lymphatic watersheds following ICG injections on the face and mandible. In contrast to the recent longitudinal study of breast cancer patients described above, in a longitudinal study of head and neck cancer patients, imaged before and after surgery, radiation, and during 6 months follow-ups , ALL patBecause objective diagnosis of head and neck lymphedema can be challenging, dermal backflow may provide the earliest indicator of lymphatic dysfunction. However, once diagnosed, the treatment of head and neck lymphedema presents additional challenges due to limited access to rehabilitation specialists offering physiotherapy for head and neck cancer survivors. Even with specialist involvement, the inability to access and evaluate internal cervical lymphatics, and the inability to identify functional lymphatic watersheds to manually direct drainage can complicate treatment. For example, therapists are traditionally trained to direct manual lymphatic drainage away from surgical scars due to a longstanding belief that lymphatic vessels cannot regrow across scar tissue. In the case of head and neck lymphedema subjects, this often means directing massage towards the back of the neck into a functional lymphatic watershed [In a pilot study of 10 head and neck cancer patients, we used NIRF-LI to assess the impact of early pneumatic compression intervention on lymphatic recovery about 4 weeks after completion of radiation therapy . Eight oex vivo show impaired lymphatic vessel contractile pumping   and othe pumping , indicat pumping  and by N pumping . Impaire pumping  with der pumping  , at puberty or shortly thereafter (praecox), or after the age of 35 (en tarda). Currently, there are a handful of known gene variants associated with syndromic and non-syndromic lymphedema  to find causative gene variants in families that harbored members with non-syndromic, congenital, function . Using t testing .Proteus syndrome (PS) all involve germline, genetic mutations in the RAS signaling pathway. In transgenic mouse models of these conditions, causative gene mutations generate lymph vessel hyperplasia and hypoplasia, chylothorax lymphangiectasia, chylous ascites, and absence of lymphatic valves. NIRF-LI/ICG lymphography can be used to characterize both mouse and human phenotypes associated with these gene mutations. For example, we demonstrated a unique phenotype of lymphatic vessel hyperplasia in a transgenic animal model of CM-AVM in which conditional knock-out of RASA1 in lymphatic endothelial cells caused prolonged MAPK and PI3K signaling to lymphangiogenic growth factors, namely, VEGF-C  is especially impactful in the lower extremities where both venous and lymphatic return must overcome gravitational forces. CVI is descriptively classified using the clinical section of the CEAP  classification system. There are seven main classifications: C0, no visual evidence of venous insufficiency; C1, telangiectases (spider veins) and/or reticular veins; C2, varicose veins; C3, edema; C4, skin changes such as pigmentation (hemosiderin staining), eczema, lipodermatosclerosis, atrophieblanche, or corona phlebectatica; C5, healed venous ulcer; and C6, active ulceration. Because edema is a feature of both lymphedema and CVI, primary lymphedema can be misdiagnosed as CVI . While tThe adventitia of blood vessels contains initial lymphatic capillaries known to be essential in the arterial wall for removal of cellular waste as well as reverse cholesterol transport by macrophages to maintain or “cleanse” it of cholesterol . When lyWhether dermal backflow in C0- C4 disease portends the future development of an ulcer remains to be investigated in longitudinal studies. Indeed, the relationship between lymphatic dysfunction and the etiology of CVI is unclear. A better understanding of how inflammation, lymphatic load, high interstitial fluid pressures, fluid statis, or some combination of these impact lymphatic health and disease progression could be impactful to disease management. Associated with an aging population, venous leg ulcers are difficult to heal and frequently reoccur with substantial impact on the healthcare system .Fluorescence techniques have demonstrated CVI treatments impact lymphatic function. Because lymph contains pro-inflammatory tissue waste products, their accumulation may be responsible for ulcer formation. Using ICG lymphography, Suzuki et al., demonstrated improved lymph transport from the foot to the knee after vein stripping in patients with varicose veins . PhysiotWhile preliminary NIRF-LI results suggests lymphatic insufficiency occurs in peripheral arterial disease , more imChy mouse model that lacks dermal lymphatics  and human immunodeficiency virus (HIV).The most common infectious cause of lymphatic dysfunction is filarial nematodes such as backflow , consistMtb infection  following a thumb needlestick injury. After successful anti-TB medication, the subject presented with neutropenia, severe pain, and prominent lymphatic cording connecting to a fibrotic axillary lymph node later found to be PCR positive for Mtb. NIRF-LI was used to image lymphatics and indicated dermal backflow and dysfunctional lymphatic draining from the site of injury, and the lack of drainage through the thrombosed, cord-like structure similar to axillary web syndrome (AWS)  . AWS is nfection . In the + T-cells and immune suppression that reduces immune responses and increases susceptibility to TB and other infections. Antiretroviral therapy can eliminate detectable viral burden in plasma, but fails to do so in lymph nodes   or, in the case of the brain, CSF reabsorption.Under normal conditions, cerebrospinal fluid (CSF) i) is predominantly generated in the cerebral ventricles, ii) flows from the lateral ventricles to the third and fourth ventricles, and iii) exits the fourth ventricle ce (SAS) . From thThe structures for absorption and drainage of CSF into the extracranial cervical lymphatics and lymph nodes include the i) lymphatic vessels near the cribriform and olfactory bulb junction , ii) thebefore the onset of amyloid deposits and reduced cervical lymph node drainage . Without protein binding and at sufficiently high concentrations, planar ICG molecules can stack together, resulting in fluorescence quenching as well as oligomer formation that, with much controversy, has been attributed to neurotoxicity . ToczyloThe immune status of tissues within a lymphatic watershed is mediated by lymphatic vessels and lymph nodes. Whether regional inflammation in the synovium of RA patients or the immune tolerance permitting cancer metastasis to regional lymph nodes, most targets of immune modulating drugs lie within the lymphatic vasculature. Except for the initial lymphatic capillaries that surround all organs, (including the epidermis) or those that are formed through the process of lymphangiogenesis, there is no other route of entry into the lymphatics. Entrance into the intact, high endothelial venules of lymph nodes is privileged for neutrophils and naive T and B cells arriving from the thymus. Because lymph empties into blood and blood does not empty into the lymphatics, conventional routes of dosing may be inadequate to maximize relevant drug targets in the lymphatics. As a result, drugs administered intravenously have a limited opportunity for maximum exposure to their targets in the peripheral lymphatics. While i.v. administered drugs extravasate through a leaky blood vasculature present in inflamed or cancer tissues, the majority of the drug is carried to liver, gut, and other tissues causing significant off-target side-effects. Subcutaneous administration below the epidermal space  resultsattenuate immune responses regionally, as opposed to systemically, susceptibility to infections as well as other adverse events associated with systemic dampening immune responses may possibly be alleviated. In normal study subjects, hollow microneedles delivered ICG at volumes clinically relevant for drug administration to draining lymph nodes, suggesting feasibility for clinical translation  and programmed death protein 1 (PD-1), we have shown that lymphatic delivery at equivalent doses of anti-CTLA-4 or anti-PD-1 , also results in greater anti-tumor responses including reduced or eliminated metastasis  function until the advent of point-of-care, real-time imaging. Because chronic conditions typically do not have a clinically relevant animal model, and lymphatics in quadrapedals are not as susceptible to the same gravitational effects as bipedals (humans), it is essential that discoveries arise from clinical investigations. NIRF-LI and ICG lymphography may be used as an accurate measurement of the peripheral lymphatic anatomy and function to identify when lymphatic dysfunction plays a significant role in the etiology and progression of chronic conditions. These imaging techniques may also provide early surrogate measures of treatment outcomes, resulting in more efficient clinical trial design of new treatment strategies without the cost and infrastructure burden associated with radiological procedures or the long timeframes required for clinical outcomes. Finally, the recent recognition that (g) lymphatics serve as a “garbage truck” of the brain  could beIt is highly likely that our understanding of lymphatic dysfunction and clinical strategies to manage it will evolve dramatically with the application of point-of-care technologies that facilitate clinical research. In addition, it is highly likely that instrumentation and drug advances will add additional capabilities than currently achievable. For example, the ability to probe tissues deeper than 3–4 cm requires new advances in optical filtering and detectors. While ICG provides a safe imaging agent, the ability to image following microdosing  opens up opportunities for using brighter and molecularly targeting dyes that could provide a molecular basis of lymphatic dysfunction.In summary, the opportunities to advance lymphatic science, lymphatic imaging technologies, and the treatments of clinical conditions they affect represent the next frontier for interdisciplinary collaboration between engineers, physiologists, clinicians, and the patients they treat.


In this review, we discuss the advantages and disadvantages of each system, and how studying axon growth in human neurons can provide species-specific knowledge in the field of CNS axon regeneration with the goal of bridging basic science studies to clinical trials. Additionally, with the increased availability and quality of ‘omics datasets of human cortical tissue across development and lifespan, scientists can mine these datasets for developmentally regulated pathways and genes. As there has been little research performed in human neurons to study modulators of axon growth, here we provide a summary of approaches to begin to shift the field of CNS axon growth and regeneration into human model systems to uncover novel drivers of axon growth.Axon regeneration is limited in the adult mammalian central nervous system (CNS) due to both intrinsic and extrinsic factors. Rodent studies have shown that developmental age can drive differences in intrinsic axon growth ability, such that embryonic rodent CNS neurons extend long axons while postnatal and adult CNS neurons do not. In recent decades, scientists have identified several intrinsic developmental regulators in rodents that modulate growth. However, whether this developmentally programmed decline in CNS axon growth is conserved in humans is not yet known. Until recently, there have been limited human neuronal model systems, and even fewer age-specific human models. Human  SCI is a devastating injury that results in life-long disability with high rates of morbidity and mortality, resulting in reduced quality of life, and a lifetime economic burden of approximately 2–4 billion dollars . The lacof birth . This laof birth . Despiteof birth . The lac2.It has been well-established that developmental age alters the response of rodent CNS neurons to extrinsic and intrinsic factors that limit adult but not embryonic axon growth and regeneration . ExtrinsKlf4 and 9 can increase axon growth in adult corticospinal tract (CST) neurons and retinal ganglion cells  were attractive targets because they can regulate an abundance of genes. For example, Krüppel-like factors (KLF) 6 and KLF7, which promote embryonic axon growth, are downregulated postnatally , whereasls RGCs; . KLF9’s ls RGCs; . KLF4 acls RGCs; . In contls RGCs; . These sIn addition to developmentally regulated TFs, cell signaling proteins such as insulin-like growth factor, cytochrome P450, and cyclic adenosine monophosphate (cAMP) levels are all downregulated during development . cAMP isPten) and tuberous sclerosis 1 (Tsc1), promotes robust axon regeneration in RGCs and CST neurons  resulted in a strong regenerative phenotype, the same deletion in middle-aged animals (12–18 months) resulted in limited CST neuron axon regeneration  pathway is a central pathway that promotes growth through translational regulation . During  neurons . Howeverneration , 2017. T animals . Thus, Pr singly  or in cor singly , is hindr singly .in vitro  technology which includes both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), and direct reprogramming technologies, we have seen an increase in human disease and therapeutic models that can now be applied to studies of axon growth and regeneration.3.1.in vitro, and differentiated into a wide variety of cells, including neurons , for example, there has been great progress with midbrain dopaminergic progenitor transplantation to replace the endogenous degenerated neurons in animal models , and nowTransplantation has also been used in efforts to treat SCIs. Early studies transplanted rodent embryonic cortical neurons with high intrinsic growth potential into the adult rodent cortex, and observed neuron grafts extending long-range projections and forming synapses with cortical and subcortical structures . With th3.3.To ask if developmental and age-specific regulators drive changes in human CNS axon regeneration requires the use of a human neuronal model system that maintains age. Direct reprogramming is the conversion of one somatic cell type into another, or transdifferentiation . This prin vitro directly reprogrammed neurons have become a powerful tool to study the molecular underpinnings of aging in neurons, and to model neurodegenerative diseases . Using bulk RNA sequencing (RNA-seq) and microarrays, multiple studies have published developmentally-regulated transcripts and their expression trajectories across time in various brain regions such as the dorsolateral prefrontal cortex , prefronTranscription can be regulated at the epigenetic level through controlling DNA accessibility. It is well known that during development and aging, the chromatin landscape can reflect and control gene regulation . DNA metAll the datasets described above have been used primarily to understand neurodevelopmental disorders, however, they are also suitable for mining developmentally-regulated genes for human axon growth and regeneration studies .4.in vitro system has its unique positive attributes and limitations suitable for answering specific types of questions. While in vitro studies can provide a wealth of knowledge, the inability to study human neurons in an intact in vivo nervous system, which has a dynamic endogenous environment and age-specific signaling, cellular, and structural changes, has limited the adaptation of human model systems for studies of axon growth. However, developments in chimeric transplantation of human cells into the rodent CNS to dynamically visualize adult human axon growth in a systemic environment could take us one step closer to an improved human in vivo model. Harnessing the knowledge gained from these diverse human model systems and datasets may reveal novel human-specific, cell type-specific regulators of axon growth, as well as potentially identifying developmentally-regulated factors that could influence this growth. Ultimately, adding human systems to our studies of CNS axon growth and regeneration may accelerate and increase the success of the transition of preclinical studies to clinical trials.The establishment of numerous techniques to make human CNS neurons allows us to ask species-specific questions about regulators of human axon growth. Each 4.1.Permission must be obtained for use of copyrighted material from other sources (including the web). Please note that it is compulsory to follow figure instructions.BL and DM co-designed, interpreted the relevant literature, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors thank our funding sources: 1F30NS122478 (to BL), 1R21NS111192–01 (to DM), T32GM140935 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nurses play an essential role in patient safety. Inadequate nursing physical assessment and communication in handover practices are associated with increased patient deterioration, falls and pressure injuries. Despite internationally implemented rapid response systems, falls and pressure injury reduction strategies, and recommendations to conduct clinical handovers at patients’ bedside, adverse events persist. This trial aims to evaluate the effectiveness, implementation, and cost–benefit of an externally facilitated, nurse-led intervention delivered at the ward level for core physical assessment, structured patient-centred bedside handover and improved multidisciplinary communication. We hypothesise the trial will reduce medical emergency team calls, unplanned intensive care unit admissions, falls and pressure injuries.A stepped-wedge cluster randomised trial will be conducted over 52 weeks. The intervention consists of a nursing core physical assessment, structured patient-centred bedside handover and improved multidisciplinary communication and will be implemented in 24 wards across eight hospitals. The intervention will use theoretically informed implementation strategies for changing clinician behaviour, consisting of: nursing executive site engagement; a train-the-trainer model for cascading facilitation; embedded site leads; nursing unit manager leadership training; nursing and medical ward-level clinical champions; ward nurses’ education workshops; intervention tailoring; and reminders. The primary outcome will be a composite measure of medical emergency team calls , unplanned intensive care unit admissions, in-hospital falls and hospital-acquired pressure injuries; these measures individually will also form secondary outcomes. Other secondary outcomes are: i) patient-reported experience measures of receiving safe and patient-centred care, ii) nurses’ perceptions of barriers to physical assessment, readiness to change, and staff engagement, and iii) nurses’ and medical officers’ perceptions of safety culture and interprofessional collaboration. Primary outcome data will be collected for the trial duration, and secondary outcome surveys will be collected prior to each step and at trial conclusion. A cost–benefit analysis and post-trial process evaluation will also be undertaken.If effective, this intervention has the potential to improve nursing care, reduce patient harm and improve patient outcomes. The evidence-based implementation strategy has been designed to be embedded within existing hospital workforces; if cost-effective, it will be readily translatable to other hospitals nationally.Australian New Zealand Clinical Trials Registry ID: ACTRN12622000155796. Date registered: 31/01/2022.The online version contains supplementary material available at 10.1186/s12912-023-01439-x. Improving patient safety in hospital settings is an ongoing challenge . Among hPatient harm is a significant burden for healthcare systems internationally . For AusNurses play a central role in patient safety by conducting physical assessments and communicating findings to the multidisciplinary team, and implementing and evaluating the effect of nursing clinical interventions. Minimal vital signs datasets inform physiological tracking systems that determine the need for urgent patient interventions , and abeComplementary to improving nursing assessment practices, effective nurse-to-nurse and nurse-to-medical officer communication is essential for patient safety . ClinicaIn response to these patient safety issues, the Assessment and Communication Excellence for Safe Patient Outcomes (ACCELERATE) feasibility study was conducted in 2021 consisting of a stepped-wedge cluster randomised trial and process evaluation . The intBuilding upon the ACCELERATE feasibility study, the aim of the ACCELERATE Plus Trial is to evaluate effectiveness, implementation, and cost–benefit of an enhanced intervention for nursing core physical assessment, structured patient-centred bedside handover and improved multidisciplinary communication. The ACCELERATE Plus Trial will be delivered and facilitated remotely by the research team using a train-the-trainer model, emphasising integration with existing hospital structures, personnel, and resources.The trial methods are reported according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) , and theA pragmatic, hybrid type 1, stepped-wedge cluster randomised trial design will be conducted  with a cThe trial intervention will be sequentially implemented over three periods of 17 weeks, totalling 51 weeks. A total of 24 wards will be enrolled to the trial, with eight sites each contributing three wards . ExclusWards will be stratified by hospital and randomly allocated to one of the three sequences. Planned stratified allocation is shown in Fig. Intervention components are reported against the Template for Intervention Description and Replication (TIDieR)  in SupplAll registered and enrolled nurses will be required to conduct a comprehensive and systematic physical assessment for their allocated patients at the beginning of each shift. This consists of 16 core physical elements structured in the ‘A-E’ format  protocol , and patBedside nurses will be encouraged to attend and actively participate in medical ward rounds and other multidisciplinary meetings by ‘speaking up’ on concerns identified from the core physical assessment, and collaboratively discuss plans of care and escalation requirements. ‘Speaking up’ has been defined as ‘assertive communication of patient safety concerns through information, questions or opinions where immediate action is needed to avoid patient harm’ .The intervention will be delivered using proven implementation strategies for changing clinician behaviour, consisting of: nursing executive site engagement; a train-the-trainer model for cascading facilitation , 44; embThe ACCELERATE Plus Logic Model  at the participating sites. Led by the trial clinical principal investigator , this process involves advocacy for the key facilitative role of nursing executive support to drive the intervention locally.Delivery and facilitation of the intervention will ‘cascade’ through stakeholders from all hospital managerial levels, from both nursing and medical disciplines, to ward clinicians and patients. The planned cascading facilitation is informed by the integrated Promoting Action on Research Implementation in Health Services Framework (i-PARIHS) , 56, andEach participating hospital will nominate an internal nursing staff member to act as the site lead responsible for implementing the trial at their local site. Site leads will be nurses in an existing higher-grade position or senior clinical nurses who have high influence amongst the nursing discipline, with experience in nurse education and facilitating clinical practice change. Responsibilities will include: (i) engaging with nursing and medical management teams and presenting trial overviews, (ii) delivering nurses’ education workshops and additional top-up training as required, and (iii) supporting nursing unit managers to iteratively co-design their ward action plan with the ward clinicians to implement and embed the intervention.A one and a half-day in-person train-the-trainer workshop with the site leads will be conducted two months prior to the start of the trial. Site leads will be briefed on the overall trial design, the intervention components, and their role within the trial, in particular, proposed engagement processes and training for delivering the nurses’ education workshops. The research team will remotely provide training, support, and resources for site leads to deliver the intervention and engage with site-specific stakeholders. Resources will include the workshop teaching material , action plan templates, posters and lanyard cards to use as reminders on the ward, and intervention guides for optional internal audits.Prior to and during the trial, all site leads will meet fortnightly with the research team via videoconference as a learning collaborative group to share progress, report risks, and discuss strategies for navigating barriers. Determined per site, regular internal meetings will be organised by the site lead to maintain engagement and support nursing unit managers .One month prior to each step, nursing unit managers of the participating wards in that step will participate in a one-day in-person workshop facilitated by an external consultant with extensive nursing leadership and education experience. The workshop aims to equip nursing unit managers with leadership and change management skills to lead and embed the intervention on their wards. A session of the workshop will be for nursing unit managers’ direct line managers to be informed of the trial and their role in providing operational oversight, addressing organisational barriers and serving as a liaison to other nursing and medical stakeholders.Site leads will support nursing unit managers and their ward leadership team to identify and support clinical champions from both nursing and medical disciplines . ChampioThe previous ACCELERATE feasibility study involved medical heads of department during the initial engagement prior to each step and medical engagement at the ward team level. To strengthen interprofessional collaboration, the ACCELERATE Plus Trial will augment medical engagement by requesting each site to nominate a medical lead . This will assist in engaging with participating wards’ medical heads of department to identify medical champions and provide operational support. With the support of the site lead, nursing unit managers will collaborate with medical heads of department to facilitate the attendance of nurses in medical ward rounds.A fortnight prior to the commencement of each step, site leads will conduct a two-hour workshop for nurses on the participating ward. The workshop will be repeated over five consecutive days to maximise attendance, with additional education sessions as required. These workshops will cover the three intervention components and facilitate discussion around anticipated ward-specific factors, specifically enablers, barriers, and proposed solutions.The core physical assessment and medical engagement components of the trial may be tailored to the ward specialty and context. Wards with clinical specialties will be encouraged to tailor the core physical assessment by incorporating clinically specific elements in addition to the core 16 elements (e.g. cardiology wards may decide to incorporate arrhythmia assessment). To improve medical engagement, site leads and nursing unit managers will collaboratively determine any tailored approaches for facilitating nurse participation at medical ward rounds according to the multidisciplinary workflow of the ward.The posters, lanyard cards and demonstrative videos provided by the research team can be used as reminders on the ward of the intervention components.Involvement of consumers is outlined in Table The primary outcome will be a composite measure of: (i) medical emergency team calls (consisting of: a) rapid response system calls, i.e. patients identified as potentially/actually clinically deteriorating, and b) ‘Code Blue’ calls, i.e. medical emergency and cardiopulmonary arrest ), (ii) uIndividual measures from the primary composite measure above will be secondary outcomes: medical emergency team calls , unplanned intensive care unit admissions, in-hospital falls  and hospital-acquired pressure injuries (separated by staging ).Patient reported perceptions of safety and overall hospital experience will be calculated using the following validated tools (i) Patient Measure of Safety Questionnaire  and (ii)Registered and enrolled nurses’ perceptions of safety attitudes, organisational readiness to change, barriers to physical assessment, staff engagement and multidisciplinary collaboration will be quantified using the following validated tools: (i) Safety Attitudes Questionnaire Short Form  , (ii) OrMedical officers’ perceptions of safety attitudes and multidisciplinary collaboration will be measured using the following validated tools (i) Safety Attitudes Questionnaire Short Form  and (ii)All patients who are present on participating wards for 48 h or longer during the pre/post intervention survey periods will be invited to participate in the patient survey. Patients will be ineligible if they are: less than 18 years old, non-English speaking, or too unwell or cognitively impaired to participate (as determined by the primary nurse or site lead). Patients will be approached a maximum of three times to participate in the survey. If present, visiting family members and carers may assist patients to complete the survey.All registered and enrolled nurses (including nursing unit managers) who have worked on participating wards for two weeks or more will be invited to complete the nurse survey, including part-time, temporary contract and casual staff who work 4 shifts or more per month. Agency staff employed by an external provider and assistants in nursing will not be eligible to participate.All medical staff  who have been based on participating wards for 2 weeks or longer will be invited to participate in the survey.Primary outcome data of medical emergency team calls , unplanned intensive care unit admissions, in-hospital falls, hospital-acquired pressure injuries, and patient demographic data  will be retrieved from routinely collected hospital data. Data will be collected for the trial period and the preceding three years (i.e. January 2020 – December 2023). Where routinely collected data does not clearly identify the location where a patient experienced an outcome event, medical record chart audits may be undertaken.Prior to each step and at the conclusion of the trial, patient, nurse, and medical officer experience measures will be obtained via paper-based self-administered surveys. Surveys will be distributed to intervention ward participants by site leads or ward-level clinical champions. For patients who have vision or physical impairments, but otherwise are eligible and willing to participate, site leads or caregivers may provide assistance in completing the survey. Return of completed surveys will constitute informed consent. Surveys will also contain some brief demographic questions: patient surveys will ask time on ward, age, sex, previous admission history and the 0–100 overall health visual analogue scale from the EuroQol 5-Dimensions Questionnaire ; nurse aWe expect to collect data from 48,960 patient episodes of care representing 244,800 bed days. With this sample, and with assumptions based on unpublished data for falls occasioning harm, pressure injuries and rapid response calls  we will be powered to detect difference in the rate of incidents of 12–28% from pre- to post-intervention .The data analysis period will extend across 68 weeks, comprising 51-week trial period and an additional 17-week control period prior to the first step Fig. . All patPatient outcome data will be modelled as counts using Poisson regression with an offset for time on ward. Using a Generalised Linear Mixed Model (GLMM) approach, we will use fixed effects for secular trend, and a random effect for ward. The primary outcome will be a composite measure of: (i) medical emergency team calls ; (ii) unplanned intensive care unit admissions; (iii) in-hospital falls; and (iv) hospital-acquired pressure injuries.Patient, nurse and medical officer experience measures will be reported as means and standard deviations of overall component scores, and changes from pre- to post-intervention will be tested using a GLMM with a Poisson link function adjusting for respondent age, tenure (if appropriate) and correlation within ward. We will conduct secondary analyses: i) to investigate changes in outcomes over time, ii) perform the above with additional fixed effects for patient demographics , and iii) model differences in primary outcome components by level of comorbidity. A statistical analysis plan pre-specifying all analyses will be formulated and finalized prior to the study data lock.All analysis will be conducted in the R language for statistical computing, using the lme4 package for generalised linear mixed models .Economic evaluation of the ACCELERATE Plus intervention compared with usual care will be assessed using a cost–benefit analysis. Resource use and cost of intervention will incorporate the involvement of staff, equipment hire and other materials for clinician training and related activities. The intervention’s effect on the primary outcome  will be valued in monetary terms using the Australian unit costs obtained from Australia’s Independent Health and Aged Care Pricing Authority ) and previous published/unpublished work . ResultsA process evaluation will be undertaken at the conclusion of the trial to understand the factors that influence intervention uptake and future sustainability by exploring the first-hand experiences of key stakeholders and participants. As outlined in Table Data collection and analysis will be guided by the Normalisation Process Theory. Concepts from this theory cover individual, collective, and contextual factors that are associated with changing practices in complex healthcare settings, including: coherence (i.e. meaning and sense-making by participants), cognitive participation (i.e. commitment and engagement by participants), collective action  and reflexive monitoring  . MembersThe ACCELERATE Plus Trial aims to evaluate the effectiveness, implementation, and cost–benefit of an intervention for improving patient safety in acute care general ward settings consisting of nursing core physical assessment, structured patient-centred bedside handover and improved multidisciplinary communication. Current physical assessment practices predominantly consist of nurses completing minimal sets of vital signs which serve as criteria for activating medical emergency team calls . Such moBuilding on the findings from the ACCELERATE feasibility study , the curMultidisciplinary collaboration between nurses and medical officers is an important factor for intervention uptake and adherence. The ACCELERATE feasibility study involved medical heads of department during the initial engagement prior to each step. For the current trial, engagement with medical teams will be enhanced so that all organisational levels  for both nursing and medical disciplines will be informed of the trial and potential areas for support.The characteristics of the eight participating sites  increases the likelihood that, if effective, the findings will be generalisable and can be readily translated to other similar hospitals for upscale. Culturally and linguistically diverse patients however will likely be underrepresented, as this trial will not have adequate resourcing for interpretive services. A notable consideration is that intervention adherence will not be quantitatively measured due to the scale of the trial. Rather, the trial includes a process evaluation and cost–benefit analysis that will inform implementation factors and considerations for future translation. Another consideration will be the accuracy and consistency of routinely collected hospital data that comprise the primary outcome. However, using such data sources is also a strength as it provides a low-cost method of evaluating the effects of the trial, and can be accessed again at a later date to evaluate the long-term effects. The use of routinely collected hospital data, in combination with an externally facilitated implementation strategy and embedded site leads, aligns the ACCELERATE Plus Trial with the movement towards learning health systems for improving healthcare .If found to be effective, the ACCELERATE Plus Trial presents a model of care that will improve patient safety, nursing care, and both clinician and patient experience measures in a cost-effective manner. The addition of a trial process evaluation will allow the intervention and implementation strategy to be evaluated for readiness of translation to hospitals nationally. Findings from this trial may inform the design and evaluation of future trials that will aim to scale and/or sustain the intervention in different contexts.Additional file 1:Supplemetary S1. ACCELERATE Plus Trial Intervention, reported using the TIDieR Checklist.Additional file 2:Supplementary S2. ACCELERATE Plus Trial Logic Model [ic Model .


Multiple biological processes rely on direct intercellular interactions to regulate cell proliferation and migration in embryonic development and cancer processes. Tumor development and growth depends on close interactions between cancer cells and cells in the tumor microenvironment. During embryonic development, morphogenetic signals and direct cell contacts control cell proliferation, polarity, and morphogenesis. Cancer cells communicate with cells in the tumor niche through molecular signals and intercellular contacts, thereby modifying the vascular architecture and antitumor surveillance processes and consequently enabling tumor growth and survival. While looking for cell-to-cell signaling mechanisms that are common to both brain development and cancer progression, we have studied the infiltration process in glioblastoma multiforme (GBM), which is the most malignant primary brain tumor and with the worst prognosis. Cell-to-cell contacts, by means of filopodia-like structures, between GBM cells and brain pericytes (PCs) are necessary for adequate cell signaling during cancer infiltration; similarly, contacts between embryonic regions, via cytonemes, are required for embryo regionalization and development. This GBM–PC interaction provokes two important changes in the physiological function of these perivascular cells, namely, (i) vascular co-option with changes in cell contractility and vascular malformation, and (ii) changes in the PC transcriptome, modifying the microvesicles and protein secretome, which leads to the development of an immunosuppressive phenotype that promotes tumor immune tolerance. Moreover, the GTPase Cdc42 regulates cell polarity across organisms, from yeast to humans, playing a central role in GBM cell–PC interaction and maintaining vascular co-option. As such, a review of the molecular and cellular mechanisms underlying the development and maintenance of the physical interactions between cancer cells and PCs is of particular interest. Intercellular communication is a key process in cell decision-making during the development and progression of cancer. The microenvironment is regulated by cell signals that influence other cells through paracrine mechanisms, facilitating cancer cell proliferation and migration. Moreover, cell-to-cell contact is necessary to polarize cells and regulate morphogenesis and cell motility. Here, we aim to review the developmental mechanisms of intercellular communication which are reproduced in glioblastoma multiforme infiltration, resulting in vascular co-option and immune system conditioning.Gliomas are glial tumors of the central nervous system. They are classified as: oligodendrogliomas, astrocytomas, and glioblastomas. Glioblastoma multiforme (GBM) is a high-grade infiltrative neoplasm, it is the most aggressive of all brain cancers with a low life expectancy of no more than 15 months after diagnosis . The pooCaspani et al. (2014) demonstrated that GBM cells target the PC cells in the vascular wall and are necessary for cancer infiltration into the edge of the tumor mass and tumor survival . PericytPCs form perivascular layers that support the vasculature and participate in the structure and function of the blood–brain barrier (BBB). Adherens junctions of endothelial cells and PC coverage are responsible for the correct function of the BBB. In fact, if pericyte coverage is lost or damaged, the BBB is compromised as its permeability increases, resulting in an accumulation of plasma-derived proteins in the extracellular space and neuronal inflammation . PCs, thThe perivascular location of PCs in the Virchow–Robin space, where they are in contact with the cerebrospinal fluid and vascular feet of astrocytes, means they are perfectly positioned to control several aspects of the CNS immune response. Pericytes constitute a new class of cell-mediating immunological defense in the brain ,19. FurtIn addition to contributing to innate immunity, PCs may also modulate adaptive CNS immune functions. Pericytes express major histocompatibility complex (MHC) class II molecules, which are type I membrane glycoproteins that bind peptide fragments derived from exogenous protein sources, including viral and bacterial pathogens, and transport them to the cell surface for recognition by helper T cells. All of this suggests that pericytes can present antigens to T cells ,28. FinaGlioblastoma cells use PC contacts to exploit and migrate along preexisting blood vessels ,8. They It has been suggested that direct interaction between proliferating neural progenitors and vascular cells is the fundamental process behind neurogenesis and cell migration in CNS development and adult neurogenesis ,37. In tMost tumors induce angiogenesis in order to grow. However, some cover their developmental needs using preexisting vessels, which they can also migrate along, as occurs in vascular co-option . Co-optiThe infiltration of GBM cells into surrounding tissues is a key factor in tumor recurrence. Tumor cells often reoccur within a 2–3 cm radius around the primary tumor ,46 becauSeveral studies using different live microscopy approaches have shown that GBM cells use preexisting brain microvessels as scaffolding for migration ,6,8,49. During brain development, secretable morphogenetic signals that code positional information into developmental fields regulate the proliferation of cell progenitors and organ morphogenesis. The precision and temporal resolution of positional information during a specific timeframe requires contact-dependent signaling between cells. Cytonemes and tunneling nanotubes are specialized filopodia that are mainly composed of actin filaments which establish physical contact between cells ,53. ScafCell–cell contact between tumor cells and pericytes changes the latter’s behavior. In vitro experiments on PCs cultured for 2 days on deformable silicone substrates, which were covered with human laminin to reproduce basal lamina, showed that the PCs generated compression forces around local nodes corresponding to areas with more contractile activity. Interestingly, when PCs were co-cultured with GBM cells, they started producing new wrinkles and destabilizing existing ones. These results show that GBM cells modify the contractile activity of pericytes, affecting the tone of co-opted vessels and subsequently modifying the vascular morphology, resulting in glomeruloid body formation at the GBM infiltrating edge . AlthougFurthermore, changes in the GBM extracellular matrix produce increased amounts of fibulins, among other substances, which are believed to facilitate GBM cell invasion. Studies suggest that PCs are one of the cells responsible for this change in extracellular matrix composition. In fact, the matricellular protein fibulin-7, which is crucial for the formation of aberrant blood vessels in GBM, was overexpressed in the glioblastoma microenvironment, particularly in perivascular cells such as pericytes .The production of GBM–pericyte hybrids is another effect of the contact between tumor cells and PC. Notably, some of the hybrids were found on altered vessels and associated with oxidative/nitrative stress, indicating that the hypercontractility could be linked to the stress induced by the cancer cells. Caspani et al. proposed that fusion cells expressing nitrotyrosine could be a source of reactive oxygen and nitrogen species that corrupt pericyte contraction . In factFinally, the GBM cell–PC interaction produces important changes in the immune response of PCs, as will be explained later.The molecules involved in GBM cell co-option were recently reviewed by Seano and Jain (2020) . Some ofCell division cycle 42 (Cdc42) GTPase regulates cell polarity across organisms, from yeast to humans, playing a central role in the morphogenesis of neuroepithelial cells. As Cdc42 regulates the actin–myosin cytoskeleton, it plays an important role in cell adhesion, vesicular trafficking, cell migration, and cytokinesis during embryonic development . In factBlood vessels also produce molecules that promote the interaction between tumor and vascular cells. Bradykinin is expressed in endothelial cells of co-opted vessels and is related to the chemotaxis of GBM cells and favors GBM invasion . SDF-1α Flectopodia-dependent cell–cell interactions between GBM cells and PCs are also connected to the immune response, as explained later. Interestingly, when Cdc42 is inhibited in tumor cells, PCs transform into macrophage-like cells capable of phagocytizing the cancerous cells. This indicates another important role of the Cdc42 signaling pathway as it can be used to prevent PCs from transforming into macrophage-like cells and, subsequently, favoring tumor survival over its clearance .Immune cells play a key role in host defenses against foreign antigens and unhealthy cells, including tumor cells. When they encounter signs of danger, immune cells are activated and modulate their immune functions. However, cancers have developed different strategies to suppress the antitumor immune response.Studies indicate that glioma cells may interact with PCs, transferring malignant properties to and affecting the function of the latter ,69. In tThe cytoneme-mediated interaction between PCs and GBM is known to produce in vitro changes in pericyte cytokine expression levels. The analysis of cytokines secreted from actin-GFP transgenic mice PCs (GFP-PC) co-cultured with a GBM human cell line revealed a significant increase in the production of anti-inflammatory cytokines IL-10 and TGF-β . MoreoveIn vivo studies with grafts of co-cultured human GBM cells and GFP-PCs grafted into the brain cortex of an immunocompetent C57Bl/6 mouse model  have expActivated PCs present properties of macrophages, expressing macrophage markers and acquiring phagocytic activity ,71. An iThe immunosuppressive ligand of PD-1 (PD-L1) is a negative regulator of T cell activation and has been associated with glioblastoma progression ,72,73. P+ T cells [+ T cells were defective in proliferation and IL-2 cytokine production when co-cultured with antigen-loaded antigen-presenting cells in the presence of GBM-conditioned PCs [T cells are activated when their antigen-specific T cell receptor (TCR) interacts with a specific ligand. Effective T cell activation requires the engagement of two separate T cell receptors. The antigen-specific T cell receptor (TCR) binds foreign peptide antigen–MHC complexes, and the CD28 receptor binds to the B7 (CD80/CD86) co-stimulatory molecules expressed on the surface of antigen-presenting cells (APCs). In general, T cells do not recognize native protein antigen, but rather only antigen that has been physically altered  and subsequently presented in association with MHC class II (Ia) molecules by APCs. MHC II molecules are type I membrane glycoproteins that bind peptide fragments derived from exogenous protein sources, including viral and bacterial pathogens, and transport them to the cell surface for recognition by helper T cells. The immunogenic peptide derived from chicken ovalbumin, Ova323-339, has been used extensively to study the nature of MHC II–peptide binding and T cell activation . SeveralTranscriptome modification in the expression of these molecules and/or alterations in the actomyosin cytoskeleton due to Cdc42 transfer may interfere with the formation of adequate immune synapses and, therefore, reduce the expression and clustering of MHC II molecules in conditioned pericytes. As is the case with leukemia cells, GBM cells may be able to modify actin cytoskeleton dynamics by increasing Cdc42 in pericyte cytoplasm, thus introducing adhesion and motility defects .GBM cell proliferation and enhanced tumor growth is facilitated by PCs in GBM cells co-cultured with pericytes . GBM celConversely, PCs did not increase proliferation in an in vitro model when co-cultured with GBM cells, which indicates that the underlying signal of cell proliferation was unidirectional and that cell specificity exists between GBM and PCs.GBM cell interaction with PCs induces chaperone-mediated autophagy (CMA) in the PCs in response to the oxidative stress in GBM. CMA is a lysosomal process that selectively degrades intracellular proteins . OxidatiAlthough CMA and LAMP2A overexpression has been detected in several types of cancer , its impWhen GBM cells interact with PCs they produce more ROS which leads to an upregulation of CMA receptor expression (LAMP-2A) in PCs ,85. LAMPOther studies found that LAMP-2A upregulation protected GBM cells from apoptosis by degrading nuclear receptor co-repressor (N-CoR) and inhibiting the unfolded protein response (UPR) downstream .A recent study reported that CMA plays a role in glioblastoma stem cells (GSCs). Proteomic and transcriptome analyses have revealed that CMA and LAMP-2A play an intrinsic role in maintaining GSC activity by modulating multiple pathways and processes .The PC secretome comprises various functional molecules, including inflammatory modulators, angiogenic and trophic factors, and extracellular matrix proteins ,89. An iAlthough the possible role of CMA in embryonic development has not been studied, alterations in macroautophagy have been associated with developmental abnormalities, such as neuronal migration, dendritic differentiation, and synapsis formation and pruning, which are often considered to be causes of autism spectrum disorder, tuberous sclerosis, and fragile X syndrome . CMA andThis abnormal increase in CMA activity in PCs is responsible for the switch in PC immune function and for promoting more stable interactions with GBM, all of which increases tumor survival and prevents the secretion of proteins with antitumor activity . GBM celCMA activity in PC is required to stabilize PC–GBM interactions, which help promote tumor survival. The co-culture of GBM with impaired CMA activity PCs  resulted in a higher percentage of GBM cell death and a significant loss of adherence in GBM . GranuloThe physical interaction between GBM cells and PCs can produce GBM–pericyte hybrids, which alters pericyte contractility, prevents pericytes from transforming into macrophage-like cells, and inhibits the inflammatory response ,33,57,95As explained above, PCs develop immunosuppressive characteristics after coming into contact with GBM cells. Tumor cells induce an abnormal increase in CMA in pericytes that acquire an anti-inflammatory phenotype, thus inactivating the T cell response ,57. The Pericytes are located in a strategic position between blood vessels and the surrounding tissue, so they are the first cells to sense environmental changes. They respond to environmental stimuli by secreting molecules that act over both short and long distances . In contCells can secrete molecules directly or through microvesicles, small membrane-enclosed vesicles that detach from the plasma membrane . Human bPericytes are involved in multiple processes through their secretome. The in vitro study of pericyte secretome showed that pericytes are vital for tumor immune response, inflammatory reaction, and immune evasion .As explained previously, pericytes are known to assist tumors in tumor cell infiltration and immune evasion, which are the main obstacles in GBM treatment. In addition to changing membrane expression proteins, GBM-contacted pericytes secrete a group of factors that help suppress the immune response . They alThe interaction between tumor cells and PCs changes the pericyte secretome to an immunotolerant secretome rich in factors that impair the immune response and tumor clearance. This reaction is apparently due to an increase in CMA induced by tumor cell–pericyte contact. In fact, autophagy-ablated pericytes do not suppress immune function .-/- mice were used as autophagy-ablated pericytes. Although these PCs contacted GBM cells, they did not increase CMA activity, and the immune and inflammatory response pathways were found to be upregulated, as was the phagosome pathway. Interestingly, genes related to cell adhesion molecules (CAMs) were also upregulated in autophagy-ablated pericytes [Pericytes are considered the first line of defense against GBM because they are associated with the vasculature, they act as macrophages, and they can present antigens, which initiates a proinflammatory response ,71,104. ericytes . CAMs, wericytes . CAMs weericytes .Genes related to angiogenesis, actin and adherens junctions, on the other hand, were downregulated in autophagy-ablated PCs, which is consistent with the pericytes’ antitumoral behavior observed previously when CMA is blocked ,96.In one of those studies, Molina et al. analyzed the differential expression of protein secretion (secretome) from GBM and control or autophagy-ablated pericyte co-cultures . They foOn the other hand, an analysis of the secretome of autophagy-ablated PCs cultured with GBM cells demonstrated that actin cytoskeleton regulators were not differentially expressed . HoweverSecretome analysis reveals that cell–cell contact is a key element in promoting tumor progression through angiogenesis, co-option, and by generating an immunosuppressed microenvironment that induces abnormal CMA in pericytes. This subsequently causes changes in the actin cytoskeleton that probably affect pericyte–T cell interactions.The physical interactions mediated by cell protrusions (filopodia or flectopodia) are fundamental for understanding the intercellular communication processes underlying cell polarity. These interactions are an essential component of embryonic development processes (cell proliferation and migration) and tumor growth (cell infiltration and vascular co-option). The flectopodia-mediated physical interaction between GBM cells and PCs, that is, perivascular cells in the tumor microenvironment, produces important functional changes in the PCs’ response to tumor cells. Specifically, these are vascular co-option, which mediates vascular malformation, and PC transcriptome, which modifies the PC secretome and leads to the development of an immunosuppressive phenotype that promotes tumor immune tolerance.Cdc42 gene production, e.g., the Cdc42 GTPases, both represent interesting strategies for preventing PC–GBM cell contact and PC corruption. ARN22089 is a novel compound that has been reported to block the interaction between Cdc42 GTPases and their effectors in mouse melanoma models and in patient-derived xenografts in vivo [Due to the important role of tumor cell–pericyte contact in the progression of GBM, some authors have proposed PCs as therapeutic targets for different approaches, such as tracking tumor progression by radioactively labeling PCs, inhibiting pericyte proliferation, and blocking pericyte–GBM cell interactions via Cdc42 inhibition . Targeti in vivo . This laWe have shown that the increase in CMA in GBM-conditioned PCs underlies PC transformation and flectopodia stabilization, which suggests that CMA regulation could be another therapeutic target in peritumoral PCs. While CMA activity is greater in cancer cells and pericytes, it would seem the anti- or pro-cancer function of CMA depends on GBM cell transformation and PC expression. This highlights the importance of more research into context-dependent therapy .These advances hold a lot of promise for the future treatment of GBM. However, our knowledge of PC–GBM cell interactions is limited, and further studies are needed to fully understand the molecular mechanisms underlying GBM–PC communication and the changes in PC behavior so that we may move closer to a definitive treatment for this devastating disease.


Due to anatomical complexity, large rectal gastrointestinal stromal tumors (GISTs) in the pelvis at the anterior aspect often require extended abdominal surgery to obtain clear surgical margins. Here, we show our trans-anal minimally invasive surgery combined with a robotic anterior approach for a huge low rectal GIST that was widely in contact with the prostate and urethra. By performing lateral dissection first, we can identify the orientation of critical organs such as the prostate, urethra, and neurovascular bundles, facilitating anterior anorectal dissection without urethral injury. Although the combination with a transabdominal robotic approach was required because of firm inflammatory adhesion between the tumor and prostate, the preceding trans-anal dissection plane facilitated the robotic anterior dissection and contributed to achieving complete dissection with negative resection margins. Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and originate from interstitial cells of Cajal, which are known as gut pacemaker cells. GISTs rarely occur in the low rectum, which accounts for <1% of the total . RecentlThe patient was a 66-year-old man with a huge low rectal GIST at the anterior aspect that measured 70 mm and was widely in contact with the prostate and urethra Figure . No distAfter 12 months of imatinib mesylate therapy, the tumor had shrunk remarkably to 45 mm, but it remained in extensive contact with the prostate and urethra, though without any signs of tumor invasion (Figure StrategyOn sagittal MRI, the caudal end of the tumor was located at the level of the urethra, where the dissection line was obscured by the complex anatomy, particularly, the rectourethral muscle Figure . TherefoWe planned to start with lateral dissection and identify the surface of the prostate, the key extraluminal landmark Figure . After dOperative procedureFollowing circumferential marking of the mucosal incision line, we first made a full-thickness incision dividing the rectum on both sides of the tumor. Then, the mesorectum was divided, and neurovascular bundles and the surface of the prostate, the key anatomical landmark in this operation, were identified Figure .Next, we moved on to anterior-caudal dissection. Anteriorly, the complex anatomy made identification of the dissection line difficult, but prior identification of the prostate helped us identify it just outside of the tumor. Traction of a urinary catheter helped to identify the prostate. In this way, safe dissection of the anterior anorectum was achieved while keeping the tumor capsule intact Figure .Although surgery combined with a transabdominal robotic approach was required because of firm inflammatory adhesion between the tumor and prostate probably due to effects of neoadjuvant therapy, the preceding trans-anal dissection plane facilitated the robotic dissection and contributed to achieving complete dissection with negative resection margins.Histopathological findingsFinal histopathological findings revealed a GIST measuring 42 mm that was positive for DOG-1 and C-kit, with Ki-67 index 23% and a mitotic index of 5 per 80.Video OutcomesThe better trans-anal visualization and preceding trans-anal dissection contributed to achieving complete dissection with negative resection margins. There were no intraoperative complications. At the 18-month follow-up, the anal sphincter and urogenital function were well preserved with no recurrence.Huge rectal GISTs are a challenge even for the specialists of colorectal surgery. As rectal GISTs do not metastasize to lymph nodes, anterior resection has little benefit. Trans-anal excision is inadequate for large, extra-rectal growing tumors and abdominoperineal resection is overtreatment for the tumor with intact anal canal. So, in this case, we planned complete local excision using trans-anal minimally invasive surgery keeping the tumor capsule intact while preserving the anal sphincter and urogenital function.The optimal surgical approach depends on multiple factors including tumor size, location, and presence of local invasion. Various surgical techniques have been described for rectal GISTs, including conventional trans-anal resection, trans-sacral approach , trans-vBecause of the difficulty of locating the tumor’s extraluminal component through a trans-anal approach, a trans-sacral approach in the prone position has been reported to obtain better visualization of the anterior surgical field, but this approach involves large skin incisions even when skin is spared with tumor invasion. This approach is also associated with a higher rate of fistula formation . TEM is Recently, the efficacy of trans-anal endoscopic surgery in rectal cancer surgery has been demonstrated -5. The tThe effect of neoadjuvant imatinib mesylate therapy has been reported ,13, thouTrans-anal minimally invasive surgery for a huge rectal GIST at the anterior aspect is safe and feasible. This approach helped us to identify the prostate, urethra, and neurovascular bundles, the important extramural structure, and safe anterior anorectal dissection was achieved without urethral injury or damage to the tumor capsule. Preceding trans-anal dissection can facilitate robotic or laparoscopic transabdominal anterior dissection and may improve oncological and functional outcomes.


Diabetic nephropathy (DN) is a major cause of end-stage renal disease worldwide, resulting from uncontrolled diabetes. Oxidative stress plays a critical role in the pathophysiology of DN, leading to cellular damage and disease progression. Magnesium, an essential mineral, has emerged as a potential therapeutic agent due to its antioxidative, anti-inflammatory, and antifibrotic properties. An extensive literature search was conducted on Medline using the keywords “Diabetic nephropathy,” “Magnesium,” and “Chronic Kidney Disease,” and the results published after 2000 were exclusively studied to build this review. This review aims to summarize the role of magnesium in DN and explore its therapeutic potential. Magnesium acts as a cofactor for antioxidant enzymes, directly scavenges reactive oxygen species, and enhances the expression of antioxidant proteins. Furthermore, magnesium exhibits anti-inflammatory effects by suppressing pro-inflammatory cytokine production and inhibiting inflammatory signaling pathways. Magnesium supplementation has been shown to reduce oxidative stress markers and improve antioxidant enzyme activities in clinical studies. Additionally, magnesium has been found to mitigate renal fibrosis, maintain tubular integrity and function, improve endothelial function, and modulate renal hemodynamics. Although limited clinical trials suggest the renoprotective effects of magnesium in DN, further research is needed to determine the optimal dosage, duration, and long-term effects of magnesium supplementation. Despite existing drawbacks and gaps in the literature, magnesium holds promise as adjunctive therapy for DN by targeting oxidative stress and preserving renal function. Diabetic nephropathy (DN) is a progressive kidney disease and a serious consequence of uncontrolled diabetes that continues to be a significant cause of end-stage renal disease (ESRD) globally. DN is diagnosed if persistent albuminuria (>300 mg/day or >200 μg/minute) is confirmed on at least two occasions within three to six months, along with a progressive decline in the glomerular filtration rate . The patStudies have been investigating potential therapeutic ways to slow the progression of DN and improve patient outcomes for many years . MagnesiThis review aims to summarize the current knowledge on the role of magnesium in DN and shed light on its potential as a treatment strategy to reduce oxidative stress damage and preserve renal function in diabetic patients. In addition, the review explores how magnesium exerts its renoprotective effects, including its antioxidant, anti-inflammatory, and antifibrotic capabilities. Finally, it discusses the drawbacks and potential gaps of using magnesium in DN. We can potentially pave the road for more effective and personalized treatments for DN patients if we address these information gaps.Diabetic nephropathyBoth people with type 1 and type 2 diabetes are susceptible to DN, a progressive kidney condition caused by poorly treated diabetes mellitus. Surprisingly, DN is one of the main causes of ESRD globally. DN is diagnosed when there is consistent albuminuria (>300 mg/day or >200 μg/minute) on two separate occasions, with a gap of three to six months between the tests, along with a gradual decrease in the glomerular filtration rate . PersistAside from oxidative stress, the renin-angiotensin-aldosterone system (RAAS) is important in the development of DN. Chronic hyperglycemia and hemodynamic alterations promote angiotensin II production, a strong vasoconstrictor and pro-inflammatory mediator . AngioteFurthermore, chronic low-grade inflammation contributes significantly to the development of DN. The infiltration of immune cells into renal tissues, such as macrophages and T lymphocytes, causes the release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) . These cRole of Oxidative StressThe pathophysiology of DN, a progressive kidney disease that arises as a consequence of diabetes mellitus, is heavily influenced by oxidative stress. An imbalance between the generation of ROS and the antioxidant defense system causes oxidative stress, which causes cellular damage and contributes to the development and progression of DN , as reprROS can harm a variety of biological components, including lipids, proteins, and nucleic acids. Lipid peroxidation is a prominent consequence of ROS activity that causes oxidative damage to cell membranes, resulting in structural and functional degeneration of renal cells . FurtherOxidative stress serves a dual role in DN, contributing to its initiation while also acting as a critical mediator of its downstream effects. Renal cells, including podocytes, endothelial cells, and tubular cells, are damaged by oxidative stress, resulting in malfunction and cell death, which contributes to renal function loss in DN ,33. FurtRole of magnesiumMagnesium, an essential mineral with various physiological functions, has been recognized for its potential role in reducing oxidative stress damage. Magnesium’s ability to mitigate oxidative stress is attributed to its involvement in multiple cellular and enzymatic processes that regulate antioxidant defense mechanisms . One of In addition to its direct antioxidant actions, magnesium influences the expression and activity of several proteins involved in the regulation of oxidative stress. Magnesium has been demonstrated to increase the expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which is important for activating the antioxidant response element (ARE) pathway . When thFurthermore, magnesium exhibits anti-inflammatory properties, which indirectly contribute to the reduction of oxidative stress damage. Inflammation and oxidative stress are interconnected processes, with oxidative stress triggering and perpetuating inflammatory responses. Magnesium has been shown to suppress the production of pro-inflammatory cytokines, such as IL-6 and TNF-α, and inhibit the activation of NF-κB, a key transcription factor involved in the inflammatory response ,41. By aIn another study, magnesium supplementation enhanced redox balance and decreased oxidative stress in hemodialysis patients . These dMoreover, magnesium has been shown to have vasoprotective benefits in CKD patients. CKD is characterized by endothelial dysfunction and vascular calcification, both of which lead to cardiovascular problems. Magnesium supplementation has been found to improve endothelial function by increasing the generation of nitric oxide (NO) and decreasing vascular calcification ,49. MagnAs an additional token, clinical investigations have found that magnesium supplementation has renoprotective effects in CKD patients. Magnesium supplementation enhanced renal function and decreased urine protein excretion in CKD patients in a randomized controlled experiment . In anotApart from the above-mentioned mechanisms, magnesium has other effects which show renal protection. Very limited studies are available in the literature, and further studies are needed for a concrete opinion. The effect of magnesium on blood pressure and endothelial function is notable. Higher serum magnesium levels in CKD patients have been linked to better endothelial dysfunction, as seen by greater flow-mediated dilation of the brachial artery . Higher Drawbacks and potential gapsThe literature on using magnesium as a therapy for DN has notable gaps. Limited research trials with small sample sizes have explored magnesium as a potential treatment option. All studies mentioned above focused on the relationship between magnesium and DN. To establish its superiority as a therapy option, larger human-based clinical trials comparing magnesium to other available treatments are essential.Although some studies suggest a beneficial relationship between magnesium and DN, data are insufficient to establish magnesium as a superior therapy option. More research is needed to fully understand its medicinal potential. The optimal dosage and duration of magnesium supplementation and its long-term effects as a therapeutic remain uncertain. Conducting well-designed trials is crucial to address these concerns and evaluate the feasibility and safety of incorporating magnesium into DN treatment.To summarize, the pathophysiology of DN is complex and multifaceted, and oxidative stress is important in its genesis and progression. Magnesium appears to be a promising therapeutic agent for reducing oxidative stress. In addition to its role in lowering oxidative stress, magnesium demonstrates various renoprotective mechanisms in the context of CKD to improve DN, such as anti-inflammatory qualities, antifibrotic properties, tubular integrity maintenance, vasoprotective effects, modulation of renal hemodynamics, and overall renal function preservation.Clinical investigations have shown that magnesium supplementation positively affects CKD patients with DN. However, further well-designed clinical trials are needed to assess its true potential as an additional therapy for DN and to inform evidence-based clinical guidelines for its use. As research in this area progresses, a better knowledge of magnesium’s mechanisms of action and its appropriate role in the management of DN may lead to better patient outcomes and a higher quality of life for people suffering from this debilitating condition. Furthermore, research is needed to identify the therapeutic dose of magnesium and the long-term effects of magnesium supplementation as an additional therapy in the treatment of CKD.


Periodontitis is a multi-faceted inflammatory disease that impacts the gingiva and the structures that support our teeth, and may eventually increase tooth mobility and the risk of tooth loss. Inflammation is a viable therapeutic target of periodontitis for both biologic (dietary) and host modulatory agents/drugs. Conventional therapeutic approaches for periodontitis, including nonsurgical or surgical periodontal therapy as well as occasional adjunctive antimicrobial therapy, have been only marginally effective. Malnutrition, or at least poor dietary habits, can be highly prevalent among patients with periodontal diseases. As several food nutrients can aid in periodontal healing and regeneration, there is a critical need to evaluate natural dietary sources and supplement ingredients that can counterbalance the inflammatory processes and improve the periodontal status of our patients. Here, we reviewed the current state of knowledge  on the anti-inflammatory actions of food ingredients and supplements in clinical studies of patients with periodontal diseases. A diet that includes fruits and vegetables, omega-3 polyunsaturated fatty acids, and supplements of vitamins and plant-derived compounds seems to counteract gingival inflammation and has a promising therapeutic impact in patients with periodontal diseases. Despite the positive indications that several nutrients can be used as an adjunct to periodontal therapy, additional studies with bigger sample sizes and longer follow-up periods are needed to elucidate their therapeutic benefits and the most effective doses and administration. Periodontitis is the sixth most common health condition in the world and the primary reason for tooth loss in adults . At leasPeriodontitis is a multifaceted chronic disease involving constant interactions between bacteria and the host immune/inflammatory response, which are modified by genetic and environmental factors. Although dental bacterial plaque has been mainly considered the crucial triggering factor for periodontal diseases, patients do not all have the same susceptibility and the same response to treatment; the host immune response is ultimately what largely guides the pathological process . PeriodoDental cleaning, subgingival scaling/root planning (SRP), and meticulous oral hygiene are the main traditional therapeutic approaches for treating periodontal diseases. These approaches attempt to reduce the levels of pathogenic bacteria in periodontal pockets . Our staInflammation is a viable therapeutic target of periodontitis, for both biologic (dietary) and host modulatory agents/drugs . PeriodoAn electronic database search on MEDLINE (PubMed) and Web of Science was conducted to identify empirical human clinical studies on the anti-inflammatory effects of different nutrients on periodontal diseases published between 1 January 2010 and 31 December 2022. The electronic search using the combination of keywords that represent periodontal diseases  and those that represent the nutrients  allowed us to identify 553 possible eligible studies. After excluding duplicate articles, the number of studies decreased to 303. The 303 articles from the electronic search were further filtered by selecting only randomized clinical trials, case-control studies, and cohort studies published in English, as well as studies conducted only in human subjects . We further manually searched all available studies and excluded any formulations of different nutrients in oral health products  that could be used for the management of periodontal diseases, as we would like to focus our review only on the oral intake of nutrients. The total number of articles that were included in this review were 46 clinical studies, which are summarized into different nutrient categories and presented in tables throughout the manuscript.A well-balanced daily diet plays a crucial role in our general health and in supporting our defense against infections, including those from periodontal pathogens and bacterial plaque. Nutrients derived from our diet are necessary for our health as they provide a vital energy source (macronutrients) as well as essential cofactors required for enzyme function, structural elements, and transport (micronutrients) . StudiesA recent randomized controlled trial (RCT) evaluated the impact of the Mediterranean diet on gingival inflammation . The expn = 2187 subjects) using a validated food frequency questionnaire showed that the Nordic diet, which is rich in fruits, vegetables, cereals, and fish and low in unsaturated fats, was inversely associated with gingival bleeding and periodontal pocket depth (PPD) in patients with poor oral hygiene [A large population-based survey in Finland /d of vitamin D3, 1000 IU/d of vitamin D3, or placebo for 3 months . VitaminVitamin C deficiency has been strongly associated with severe gingivitis, periodontal disease, and generalized gingival bleeding. In a case-control study, periodontitis patients were shown to have lower plasma vitamin C levels than individuals without periodontal breakdown . In anotVitamin E has important antioxidant and anti-inflammatory properties. Deficiencies in vitamin E have controversial associations with periodontitis . The admB. serrata, vitamins, mineral salts, and fish oil, did not show any significant advancement in clinical periodontal parameters compared to SRP and olive oil [n = 15), and this small sample size does not allow us to draw definite conclusions yet. Collagen peptides are very popular anti-inflammatory supplements, especially for the prevention of arthritis. Periodontal recall patients with a history of treated periodontitis who received collagen peptides after professional mechanical debridement showed significantly lower gingival bleeding and periodontal inflamed surface area (PISA) compared to patients who received professional mechanical debridement and placebo, decreasing the risk of re-activation of periodontitis [®, Enfarma SRL, Misterbianco, Italy) with SRP in patients with moderate periodontitis significantly decreased PPD and BoP but also pain after SRP, compared to SRP and placebo [Combined micronutrient supplements have also been investigated in patients with periodontal diseases. Patients with severe periodontitis who adhered to the Mediterranean died and received SRP with multi-micronutrient food supplement administration, including the co-enzyme Q10 multicomposite, docosahexaenoic acid (DHA), live oil . Howeverdontitis . The adj placebo . More stOmega-3 (n-3) polyunsaturated fatty acid (PUFA) dietary supplementation has been promoted for chronic inflammatory disorders such as RA and cardiovascular disease , diabeteA recent systematic review that assessed omega-3 PUFAs as an adjunct to nonsurgical periodontal treatment in patients with periodontitis showed that the use of omega-3 PUFAs resulted in a significant PPD reduction and CAL gain of 0.42 mm and 0.58 mm, respectively, when compared to nonsurgical periodontal treatment alone . SeveralPlant-derived compounds have been potential therapeutic agents recognized for their vigorous antioxidant and anti-inflammatory properties. Polyphenols are beneficial ingredients in many plant foods and can be classified into flavonoids, proanthocyanidins, phenolic acid, polyphenolic amides, and other polyphenols, such as resveratrol and curcumin . The typn = 60) evaluated the impact of an adjunctive phytonutrient supplement after SRP [A new nutritional supplement made of oligomeric proanthocyanidins (OPCs) was evaluated in an RCT using a 21-day established protocol of experimental gingivitis by quitting oral hygiene in dental students in Seville, Spain . The Silfter SRP . The usefter SRP . Resveratrol, a polyphenol present in various foods derived from fruits and vegetables, has been considered a promising natural drug for the prevention and therapy of various inflammatory diseases due to its ability to inhibit the oxidative damage and mitochondrial dysfunction in cells . ResveraHerbs have been considered a source of powerful antioxidants and polyphenols, with potential anti-inflammatory actions in the periodontium. The daily consumption of green tea had a positive adjunctive therapeutic benefit with nonsurgical periodontal therapy in patients with chronic periodontitis ,63, attrN = 1152), a small but significant decrease in the number of teeth with periodontal bone loss was linked to greater coffee consumption [Caffeine has been found to modulate both innate and adaptive immune responses and reduce inflammation . In a lasumption . The orasumption . A 6-monsumption . Turmerisumption . Howeversumption .Periodontitis is a major source of oral and systemic inflammation and a significant cause for tooth loss in adults. The role of a dysregulated hyperactivated host immune response in periodontitis is clear. Differences between individuals in the response to bacterial plaque may be a result of variation in host susceptibility and immune response, with some individuals being very sensitive and developing aggressive forms of periodontitis at a relatively young age, whilst others are resistant and never develop periodontitis ,72. LiviModifying our diet to a Mediterranean diet one that involves a high intake of vegetables, fruits, olive oil, herbs, and fatty sea fish seems to decrease gingival inflammation and bleeding on probing. A high consumption of fruit seems to counteract inflammation and favors the return to periodontal health. Although the role of vitamin D in regulating bone homeostasis has been recognized, the vitamin D supplementation had a modest effect as an adjunct to periodontal therapy, with limited clinical relevance. Limited evidence on the adjunctive effect of supplementation with other vitamins and micronutrients, such as collagen peptides, on periodontal therapy is currently available, and more clinical trials are needed. Despite the positive therapeutic effect of omega-3 PUFAs, with or without aspirin, in patients with periodontitis, more studies are needed to elucidate the most effective doses of administration of omega-3 PUFAs. Plant-derived compounds including polyphenols, herbs, and phytonutrient supplements are promising therapeutic agents in the management of periodontal inflammation; however, more clinical investigations are required to draw a decisive conclusion. In summary, several clinical studies on food ingredients and types of diet have revealed their positive impact on periodontal therapy due to their anti-inflammatory effects. The limitations of the current available clinical studies are the small sample sizes and the short follow-up periods. More human studies with larger sizes and longer follow-up periods are needed to verify these positive therapeutic benefits of food ingredients and supplements in periodontal treatment.The nutritional assessment of our patients with questionnaires has not received adequate attention in clinical practice and needs to be more frequently integrated. It is very important that clinicians identify patients who might receive inadequate nutrients with anti-inflammatory properties in their diet and consider recommending them to modify their diet and enrich it with food compounds and supplements that may ultimately enhance the outcomes of periodontal treatment and reduce their risk of the progression of periodontal diseases. It is also critical to investigate further nutrients that would promote the maintenance of homeostasis in the periodontium, where inflammation is controlled and eubiosis (symbiosis) in the oral cavity is retained. Therefore, more clinical studies on several other food ingredients such as cocoa, honey, mastic gum, red wine, and coconut oil are needed, to investigate their effect on modifying the inflammatory response in the periodontium and improving the periodontal status and wound-healing process. Unique formulations of different nutrients in oral health products  need also to be explored and developed further as possible therapeutic agents for periodontal inflammation.