Several MRI measures have been developed in the last couple of decades, providing a number of imaging biomarkers that can capture the complexity of the pathological processes occurring in multiple sclerosis (MS) brains. Such measures have provided more specific information on the heterogeneous pathologic substrate of MS-related tissue damage, being able to detect, and quantify the evolution of structural changes both within and outside focal lesions. In clinical practise, MRI is increasingly used in the MS field to help to assess patients during follow-up, guide treatment decisions and, importantly, predict the disease course. Moreover, the process of identifying new effective therapies for MS patients has been supported by the use of serial MRI examinations in order to sensitively detect the sub-clinical effects of disease-modifying treatments at an earlier stage than is possible using measures based on clinical disease activity. However, despite this has been largely demonstrated in the relapsing forms of MS, a poor understanding of the underlying pathologic mechanisms leading to either progression or tissue repair in MS as well as the lack of sensitive outcome measures for the progressive phases of the disease and repair therapies makes the development of effective treatments a big challenge. Finally, the role of MRI biomarkers in the monitoring of disease activity and the assessment of treatment response in other inflammatory demyelinating diseases of the central nervous system, such as neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte antibody disease (MOGAD) is still marginal, and advanced MRI studies have shown conflicting results. Against this background, this review focused on recently developed MRI measures, which were sensitive to pathological changes, and that could best contribute in the future to provide prognostic information and monitor patients with MS and other inflammatory demyelinating diseases, in particular, NMOSD and MOGAD. Magnetic resonance imaging has become a key investigation in different scenarios of multiple sclerosis (MS), including diagnosis, monitoring of disease course, and assessment of treatment response . IdentifThe aim of this review was to describe the prognostic role of conventional and non-conventional advanced MRI measures, with a particular focus on recently developed techniques, in patients with MS and other inflammatory demyelinating diseases of the CNS. Because of the overlapping clinical and MRI findings with MS, special attention would be devoted to neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD).Neuromyelitis optica spectrum disorder is an autoimmune astrocytopathy of the CNS with secondary demyelination, which can be associated with a specific auto-antibody against the antigen aquaporin-4 (AQP4) in 50–90% of cases  whose clIn this review, we gave an overview on the prognostic role of MRI, moving from conventional and established MRI techniques to more recently developed measures, which although not yet considered established prognostic markers in MS, could provide additional information about diseases pathogenesis. Finally, we considered novel MRI developments, including the advent of ultra-high field scanners, and suggested future areas of research. For this purpose, this review included scientific literature of the last 10 years from PubMed using the following search terms: multiple sclerosis, neuromyelitis optica spectrum disorder, myelin antibody glycoprotein associated disease, magnetic resonance imaging, prognosis, and pathogenesis.Several MRI measures obtained using conventional MRI sequences have been suggested as prognostic markers in patients with MS. Their ability to predict disease course was consistently found to be higher than that of clinical measures . EvidencWhite matter lesions count and volume are the most used MRI biomarkers for quantifying the inflammatory activity in MS and represent the primary and secondary efficacy outcomes in many MS clinical trials . In patiRecent evidence suggests that lesion topography and gadolinium (Gd)-enhancing lesions are independent predictors of long-term outcomes in patients with CIS. In a cohort of patients with relapse-onset MS, spinal cord lesions, combined with inflammatory activity on MRI at baseline, were found to predict long-term disease outcomes. The presence of spinal cord lesions and at least two Gd-enhancing lesions in CIS patients was associated with a 45% risk of developing secondary progressive (SP) MS after 15 years and with a worse EDSS . HoweverIn MS, especially in the progressive phases, some pre-existing T2 lesions may show chronic activity, which can be associated with clinical progression . HistopaRecent MRI studies using deformation-based techniques , allowed us to identify in MS those WMLs with a constant and concentric volume increase and defined them as slowly expanding lesions (SELs). SELs represents a promising marker for chronic active lesions, as they can be detected on routinely acquired MRI scans, and may predict clinical progression . ChronicIn patients with NMOSD, several MRI features, including spinal cord T1-hypointensity, cavitation, or atrophy, were associated with a higher risk of poor recovery, refractory pain, and permanent disability, and lesions in the upper cervical region extending to the brainstem carried a risk of respiratory failure . In a reThe persistence of Gd-enhancing lesions, due to the breakdown of the blood-brain barrier, turned out to be an important predictor of poor prognosis after an NMOSD attack, a finding which can guide treatment options and suggests an adequate short-term follow-up .In a large French cohort of adult patients with MOGAD, those presenting with an abnormal brain MRI had a higher disability at onset than those with a normal brain MRI . In addiIn addition to lesions visible on conventional MRI sequences, inflammatory demyelinating diseases may lead to neurodegeneration and consequent brain and spinal cord atrophy, which are more related to disability progression. MRI is also sensitive to the detection of brain tissue loss, and this may be qualitatively assessed in the form of macroscopic brain volume contraction, ventricular enlargement, and widening of the cerebral sulci.MRI techniques able to assess the whole brain and grey matter (GM) atrophy are now widely accessible using automated analysis methods, and recommendations to improve their measurement and interpretation have been suggested –27. FurtBrain atrophy is an important feature of MS pathology, mainly reflecting neuroaxonal loss within lesions and normal-appearing tissue . It has In addition to baseline measures, the rate of brain volume loss at both whole and regional levels have been correlated with subsequent disability in MS , althougThe so-called “brain-predicted age” (brain-PAD) paradigm has been proposed as a potential prognostic biomarker in MS, to capture the earliest progressive processes from MRI data (T1-weighted images) using machine learning analysis. Indeed, brain-PAD was independently associated with higher disability, younger age at diagnosis, and longer disease duration, irrespective of disease phenotype .2 was found to be able to differentiate patients with progressive MS (below the threshold) from those with relapsing MS (above the threshold)  MS and higher atrophy rates than those detected in the brain . In PPMSreshold) . The increshold) .Differences in atrophy patterns seem to exist between MS and NMOSD. Indeed, while MS patients showed more whole brain and thalamic atrophy than NMOSD and healthy controls, NMOSD patients showed more spinal cord atrophy and milder brain atrophy, especially in the WM, than healthy controls, thus supporting the presence of different underlying pathogenic mechanisms . TherefoDifferent patterns of brainstem atrophy were also observed in the two diseases, with the midbrain being most severely affected followed by pons in MS whereas only the medulla oblongata was affected in NMOSD . GM atroRecently, distinct structural brain alterations were identified in MOGAD. Indeed, they included atrophy in the fronto-orbital cortex, temporal gyrus, and deep GM, with hippocampal atrophy correlating with clinical and cognitive disability . A signiin vivo, even when their prognostic role was not yet demonstrated  MRI  detected at “clinical” field strengths using specific MRI sequences  is an early and frequent phenomenon in MS and correlate with a disability, cognitive impairment, and a higher risk of transition to the secondary progressive phase . PatientUHF) MRI . An impoUHF) MRI , while aUHF) MRI . Then, aUHF) MRI .Despite the exact underlying mechanisms are not completely understood, studies on the post-mortem MS brain tissue support the idea that meningeal inflammation and the consequent cortical microglia activation lead to progressive cortical demyelination and neurodegeneration , 64. RecA growing body of work has investigated the role of iron in MS pathogenesis and evolution. The paramagnetic rim is due to the presence of iron-laden activated microglia at the lesion edge and can be easily seen on 7T phase images. Using high-resolution susceptibility-based imaging, nearly all 7T paramagnetic rims can also be seen at 3T, suggesting the possibility of implementing them as an outcome measure in MS MRI-based clinical trials .in vivo. Using quantitative susceptibility mapping (QSM) deep grey matter iron was found to be associated with the secondary progressive course in (MS), independently of tissue atrophy  and correlated with clinical disability in patients with spinal cord lesions .New diffusion imaging models have been developed in the last years and showed interesting results in MS. An increase over time in the RD of the cervical cord of early PPMS was detected using Q-space imaging (QSI), an advanced model-free diffusion imaging technique with higher sensitivity for neuroaxonal alterations than standard DTI. In the same study, higher RD of the cervical cord predicted worse disability at 3 years, thus suggesting ongoing neurodegeneration in the cord independently of lesions . NeuriteMyelin-sensitive MRI measures are crucial to investigate MS pathology. In a combined MRI-pathology study, magnetisation transfer ratio (MTR) showed a strong association with myelin content, especially in WM and cortical GM lesions . In MS aMTR values in NAWM and GM of MS showed a gradient of abnormalities depending on the distance from the surface of the brain . These gRecently, the ratio between conventional T1-weighted (T1w) and T2-weighted (T2w) sequences  has been proposed as a reliable measure to evaluate myelin integrity as well as dendrite density , which wIn the last decade, approaches of network-based connectomics MRI have been developed to explore the relationships between changes in functional or structural networks and clinical measures . For exaThe application of advanced imaging in NMOSD and MOGAD is emerging, but with many controversies and lack of studies assessing changes in these new measures over time, therefore limiting clinical application in the near future.Several studies have investigated the presence of diffuse and “occult” damage within the NAWM of patients with NMOSD by applying different MRI techniques. Proton MR spectroscopic imaging (MRSI) showed normal N-acetylaspartic acid (NAA), creatine and choline levels within the NAWM, arguing against occult neuroaxonal damage, inflammation, and gliosis . By contDTI, which is sensitive to microstructural alterations, has been performed in NMOSD and showed contrasting data. On the one hand, no DTI abnormalities were reported in different brain regions, except for the visual and motor WM pathways where a selective trans-synaptic axonal degeneration may occur secondary to destructive lesions in the optic nerves and spinal cord, respectively , 100. OnSome imaging and histopathological studies in NMOSD showed abnormalities and neuronal loss in cortical GM . Less isIn a MOGAD patient, longitudinal clinical and MRI follow-up showed progressive neurologic deterioration without any relapse associated with progressive WM changes, thus suggesting a possible tissue loss over time. Using diffusion imaging, WM disruption in the optic radiation and corona radiata was identified in both MOGAD and AQP4-Ab positive NMOSD, although milder in the former condition .Results from studies applying advanced MRI techniques to the spinal cord in NMOSD are also controversial. When using DTI analysis for the assessment of cervical spinal cord damage in NMOSD, abnormal DTI-derived metrics  in the cervical cord of patients with NMOSD (even in the absence of hyperintensities on T2-weighted imaging) were found . MoreovePatients with AQP4-antibody disease showed a significant reduction in the cervical cord MTR, FA, and increased mean diffusivity, and the damage was localised to areas of the cord involved in the acute attack. By contrast, MOGAD patients did not show significant differences compared with healthy subjects in any MRI modality .Functional MRI is a powerful tool for studying cortical functional reorganisation and brain plasticity. Studies have assessed functional connectivity (FC) abnormalities within the various brain networks in patients with MS, with the aim of identifying trajectories of changes over the disease course and thus increasing the understanding of MS pathology . RestingCognitive impairment has been demonstrated in NMOSD patients, as a result of functional alterations within specific neuronal circuits . PrelimiSevere functional impairment in the visual areas and increased FC in the temporal gyrus were also detected in MOGAD , which iNeurodegenerative changes along the visual pathway are common in both MS  and NMOSCross-sectional studies of OCT have shown that peripapillary global retinal nerve fibre layer (RNFL) and inner retinal layer thicknesses are reduced in MS and correlate with clinical disability and MRI-derived brain volume measures . These fOCT has been extensively used in MS and NMOSD to detect levels of axonal damage after an episode of ON as well as to help to differentiate the two diseases . Retinalin-vivo detection of the molecular processes involved in neuroinflammation and neurodegeneration  have the potential to improve our understanding of the mechanisms of progression in MS, thus providing important prognostic information. PET is a non-invasive molecular imaging technique enabling the neration . In a 4-neration . Using aneration . HoweverMore recently, UHF MRI  has gained more interest in clinical imaging. Indeed, a number of studies have shown the benefits from the application of this powerful tool not only for research purposes but also in clinical settings to facilitate a correct diagnosis, prognosis and improve patient management. For example, using UHF MRI, it has been demonstrated that the rate of CL accumulation was higher in patients with RRMS who developed SPMS than in those who remained RRMS , independently from the accumulation of WMLs . In MS, *-weighted images, arguing against a widespread hypometabolism in NMOSD  and ophthalmological imaging . Serum and CSF NFL light- and heavy-chain proteins have recently been proposed as reliable biomarkers of neuroaxonal damage in MS due to the correlation with MRI biomarkers of disability progression, such as brain and spinal cord atrophy . Nonethein vivo, and a protocol to detect sodium concentration in the cervical cord of MS patients has been recently developed, showing correlation with diffusion metrics (Second, although quantitative MRI techniques are technically challenging at the level of the spinal cord, they are able to provide valuable information on microstructural tissue damage. Multi-parametric imaging models are very important for the future, in order to look at the underlying driver of structural abnormalities. Indeed, sodium-MRI is a promising new metabolic imaging technique able to provide information on energy failure  metrics . It woulThird, alongside neurodegeneration, remyelination is a possible mechanism of tissue repair as it may contribute to the shrinking of lesions in MS. Future longitudinal studies may assess whether a reduction in lesion size or lesion disappearance can be seen in MS and can be used as an outcome measure for repair treatments. Lesions may evolve differently in the two Ab-mediated disease diseases, and a complete resolution of T2 lesions occurs more frequently in MOGAD than AQP4-NMOSD and MS . StudyinMoreover, when considering NMOSD, some previous studies did not separately analyse results by antibody type, therefore possibly including MOGAD patients which can also fulfil criteria for seronegative NMOSD. The distinction between AQP4 seropositive and seronegative NMOSD is further emphasised by recent prospective randomised clinical trials which included both groups but had an effect only on seropositive patients , 137. ThFinally, the role of new MRI biomarkers in the clinical management of inflammatory demyelinating diseases still needs to be established and the high cost of UHF MRI scanners makes it unlikely this type of imaging to be routinely and widely used in clinical practise in the near future.Conventional and non-conventional advanced MRI measures play an important prognostic role in inflammatory demyelinating diseases. The growing application of advanced imaging in MS, NMOSD, and MOGAD could improve the identification and validation of new pathological hallmarks in large clinical studies, leading to the development of novel diagnostic and therapeutic strategies.RC designed the project and wrote the manuscript. AG designed the project and revised the manuscript. GS revised the manuscript. ND designed the project, supervised the project, and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A key hurdle to making adeno-associated virus (AAV) capsid mediated gene therapy broadly beneficial to all patients is overcoming pre-existing and therapy-induced immune responses to these vectors. Recent advances in high-throughput DNA synthesis, multiplexing and sequencing technologies have accelerated engineering of improved capsid properties such as production yield, packaging efficiency, biodistribution and transduction efficiency. Here we outline how machine learning, advances in viral immunology, and high-throughput measurements can enable engineering of a new generation of de-immunized capsids beyond the antigenic landscape of natural AAVs, towards expanding the therapeutic reach of gene therapy. Recently approved AAV-based therapeutics and numerous therapeutic candidates in advanced clinical development  have demin vivo transduction efficiency in many target tissues, particularly following intravenous administration. Improving in vivo transduction of target cells and organs would enable gene therapies to more effectively treat diseases, to perdure, and to address new therapeutic applications. Importantly, pre-existing humoral and cellular immunity against natural AAV capsids limits patient eligibility for therapies as well as their therapeutic efficacy  of the kind we are intending to predict. However, it is also possible to train models solely based on a set of good examples without additional measurements. For instance, training models on the rapidly growing set of publicly available protein sequences to learn relationships among them has shown promise in protein structure and function prediction , in which a linear transformation of input data allows for the identification of data elements that contribute most to the variance in the data set. PCA and other more complex non-linear dimensionality reduction methods transform high-dimensional raw input data to a lower-dimensional representation (a latent space) that is easier to interpret, visualize, and optimize  delivered .in silico screening of variants  responses against cells presenting capsid antigens, for which immunosuppression is the primary clinically viable remedy.In an ideal therapeutic scenario, a single dose of GT would provide a durable, curative effect throughout a recipient’s lifetime. In practice, this goal has been difficult to realize as therapeutic transgene expression from current vectors decays over time . Waning + T cells recognize presented peptides via their highly specific T cell receptors, which in turn determines cell stimulation, proliferation and cytotoxic activity. CTL activation results in killing of transduced cells as well as generation of immunologic memory that poses a barrier for vector redosing. Unlike B cells, which interact with surface exposed capsid epitopes, T cells can in theory sample the full peptidome of an AAV capsid, including buried capsid sequences that drive assembly or disassembly, and which may be more difficult to alter by conventional engineering approaches. Extensive mapping of CD8+ T cell epitopes within AAV capsid proteins and evaluation of their propensity to activate T cell responses would identify the key sequences which must be modified to de-immunize AAV capsids. The large diversity of HLA alleles among people and distinct patterns of peptide presentation and recognition determined by them makes this challenging. While it is currently not possible to exhaustively assess peptide presentation by all variants of MHC class I found in humans, emerging ML methods in peptide presentation and immunogenicity prediction  class I molecules , 34. CD8ediction , 37 willediction , 39 will+ T cell epitopes and learn general properties that influence which peptides tend to be presented in particular genetic backgrounds  capsids. Models can serve as esponses . For insesponses  or genomesponses ] to predMany gaps remain in our understanding of how anti-capsid antibodies can be evaded. Serology studies with naturally occurring AAVs have been useful in defining population-level prevalence of anti-AAV immunity but such bulk-level measurements have had limited value for engineering antibody-evading capsids. Some monoclonal antibodies isolated from mice have been characterized in detail , 61 provuniversal capsids capable of treating all patients.One such promising approach would be to measure the activity of serum antibodies against highly diverse libraries of capsid variants using immune human serum samples. Such data would enable ML models to learn the quantitative relationship between AAV capsid sequences and their abilities to evade pre-existing antibodies, and to learn commonalities in anti-capsid antibody responses among people. Similarly, intravenous immunoglobulin (IVIg) preparations containing antibodies from thousands of donors may be useful in such screens for identifying the predominant patterns in human antibody responses. Recent work characterizing B cell and antibody responses to a number of important human pathogens , 63–65 rML-powered capsid design and engineering will transform the landscape of GT delivery modalities, however non-capsid improvements are also relevant from an immunological perspective and can also increase therapeutic effectiveness. Reducing the activation of innate immunity by engineering the vector genome , 67, co-As we have outlined, ML approaches to engineer improved AAV capsids have multiple applications: enabling gene therapies that are effective in a lower dose regimen, removing capsid peptides which elicit cytotoxic T cell responses thereby leading to longer lasting gene expression, and resurfacing capsid exteriors allowing potentially universal treatment of all patients. While these goals are ambitious and each individually worthy of study, combining all such properties in a single capsid would be transformative for the field. ML approaches will facilitate this goal by incorporating information from diverse experimental systems and improving the efficiency of multi-trait capsid optimization. We are optimistic that safe, efficient, target-specific, non-immunogenic and universal capsids will one day enable gene therapy to reach its full potential by delivering therapeutic DNA to cure, treat and prevent disease and even to improve overall health for all patients. Interdisciplinary collaborations focused on combining HT measurements with ML-powered sequence design algorithms will dramatically accelerate progress towards achieving these goals.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.AW, KL, JK, SS, JG and EK conceptualized, wrote and edited the manuscript. AW and SS prepared figures. All authors contributed to the article and approved the submitted version.AW, KL, JK, SS, JG and EK are employees and shareholders in Dyno Therapeutics Inc.


Early goal-directed treatment is an evidence-based approach to guide hemostatic therapy during major periprocedural bleeding. If viscoelastic coagulation tests are not available, an algorithm, termed the pyramid of hemostatic interventions, can help manage severe bleeding. Pregnant women accumulate huge reserves of prothrombotic and antifibrinolytic hemostatic elements to avoid peripartum blood loss. We provide comparison of therapeutic hemostatic approaches and natural gestational process and identified remarkable analogy between early goal-directed management of bleeding and hemostatic adaptation of pregnant woman. Therefore, gestational hemostasis serves as a natural model for goal-directed hemostasis resuscitation and can foster understanding of hemostatic management of periprocedural bleeding. Patients with major perioperative bleeding can be managed by the pyramid of hemostatic intervention  can form a circulating reversible complex with plasminogen, preventing it from binding to the clot. The plasma half-life of TXA is 1.5 h; therefore, a loading dose should be followed by infusion  with anticoagulant elements  in a balanced formula (4PCC). The main indication for 4PCC is to suspend coumarin effect or to replace vitamin-K-dependent factors in patients with liver disease who need an emergency surgical intervention    may be up to three times higher Bremme . Plasma 2-antiplasmin and locally by thrombin activated fibrinolysis inhibitor (TAFI) (Hellgren Expression of the fibrinolytic tissue plasminogen activator (t-PA) level can be decreased or slightly increased during pregnancy Hellgren . The plaHellgren . Thus, tThus, the global gestational hemostasis is well balanced with a huge prothrombotic reserve to prevent maternal bleeding at delivery. This strong prothrombotic potential is controlled by the high levels of the first cascade-elements reacting to the trigger of the aggregation, coagulation, and fibrinolytic processes  and the final volume-substrates of these cascades and .most of the steps of the therapeutic recommendations can be matched with the “successful natural approach.” These analogies between the therapeutic and the gestational hemostatic approaches .If bleeding persists despite the best clinical practice, off-label rFVIIa administration can be considered. It is noteworthy that the increase in FVII concentration is the highest among the plasma coagulation factors in pregnancy at term, which allows for a fast and efficient response to the high amount of TF following placental abruption, resulting in rapid local clot formation.Administration of FXIII is rarely recommended by the algorithms. Similarly, at term, FXIII decreases by ~ 50%, facilitating postpartum clot elimination. High amount of FXIII would impede clot dissolving leading to complications and pain.The prothrombotic standby state of gestational hemostasis at term underlines the fact that a multicomponent biological system can only be effective if all the elements are available simultaneously. This “philosophy of the physiology” has a clear message to the clinician. Consequently, all components judged necessary by traditional or POC tests should be administered within ~ 30 min, not by portions, which would preserve the shortages in a dangerous and costly manner.not only treat but to prevent massive bleeding and can be considered as secondary prevention of further severe periprocedural bleeding. This is an important difference in morbidity and mortality for patients, and it is of great value for health care providers. Furthermore, the analogy for prothrombotic reserves is still valid in term of in vitro reserves. Pharmacological hemostatic products must be stored and be readily available at bedside.Periprocedural bleeding cannot be predicted; thus, the mechanism of gestational accumulation of prothrombotic reserves is not adaptable for the perioperative care of massive bleeding per se. Nevertheless, the time of severe hemorrhage can be often anticipated, such as in complex cardiac, vascular, or hepatic surgery. In these cases, supplementing the estimated losses of prothrombotic components may start immediately after correcting surgical bleeds. This management enables ultimum refugium application of rFVIIa emerges particularly during life-threatening bleeding in young patients . As a third level, a high capacity, rebalanced hemostatic system with huge prothrombotic reserves is developed by pregnant women. These categories of the protecting power should be considered perioperatively as well. Massive bleeding requires if enough procoagulant components are given with the right timing. Therefore, European guidelines propose early goal-directed individualized factor-based hemostasis resuscitation, leaving a high RBC:FFP ratio to be applied in a context-sensitive way, i.e., depending on the volume status of the severely bleeding patient (Rossaint et al. Gestational hemostasis can physiologically break the massive bleeding-massive transfusion coupling. The application of the principles of this “natural approach” may help prevent massive bleeding. It highlights that management of massive bleeding might be possible without a transfusion of huge quantities of allogenic blood products less invasive mechanisms of clinical management for perioperative massive bleeding applying less homologous blood, and thus may guide future practice.Studying gestational hemostasis facilitates natural based, There are remarkable analogies between the early goal-directed clinical management of severe bleeding and the mechanisms of global gestational hemostasis. Therefore, hemostatic changes in pregnancy may serve as a natural model for periprocedural hemostasis resuscitation. With the application of the “philosophy of physiology,” the massive tissue injury-massive bleeding-massive transfusion coupling can be broken, making hemostasis resuscitation a natural-based, less invasive procedure saving more blood and lives.


Primary nodular chest wall amyloidoma, in which a solitary mass of amyloid is deposited in and around the lungs with no evidence of systemic amyloidosis, is extremely rare, most often asymptomatic, and may resemble primary bronchogenic carcinoma. As a result, there are fewer than 100 cases published in the literature and no controlled clinical trials. Primary nodular chest wall amyloidoma is typically diagnosed either as an incidental radiological finding or after very serious and destructive mass growth at which point late-stage respiratory and pain symptoms finally develop, most often in elderly patients. We present imaging studies of a 61-year-old male patient with an unusually massive and destructive chest wall mass, originating in the chest wall, diagnosed as chest wall amyloidoma by histopathology analysis. Our CT, MRI, and PET scan findings are consistent with and contribute to the developing pattern of imaging characteristics seen in other case studies, which can be used to identify amyloidoma before it becomes destructive using non-invasive imaging analyses. Amyloidosis is a rare disease, characterized by the deposit of an abnormal insoluble protein, amyloid, in extracellular tissues and organs, resulting in serious dysfunction and potentially death, if untreated Primary nodular pulmonary amyloidoma is typically diagnosed either as an incidental finding or after very serious and destructive mass growth at which point late-stage respiratory and pain symptoms finally develop, most often in elderly patients A 61-year-old male was transferred from an outside facility for worsening shortness of breath while performing yard work over the prior three weeks. The patient had a past medical history of hypertension, congestive heart failure, severe chronic obstructive pulmonary disease (COPD), smoking (until age 46), and a right apical lung mass confirmed to be a plasmacytoma (at age 50). Upon arrival at our facility, the patient was afebrile with heart rate of 76 and blood pressure of 156/92. Routine labs including complete blood count with differential as well as basic metabolic and liver function panels were unremarkable. His venous blood gas revealed a mild respiratory alkalosis. COVID-19 test was negative. The patient was admitted for further evaluation.Radiographs demonstrated a large right apical lung mass with foci of intramural calcifications . ComputeMagnetic resonance imaging (MRI) of the right brachial plexus , Fig. 6 Differential diagnoses of the chest wall mass, based upon imaging studies, included plasma cell neoplasm , plasmacytoma, or amyloidoma. An image-guided biopsy was performed to clarify the diagnosis. Multiple 16-gauge core biopsies of the soft tissue component of the right lung mass were obtained . PatholoImage-guided cardiothoracic and neurosurgical resections were performed to reduce the impact of the mass on thoracic structures including right-sided T1-T3 hemilaminectomies and facetectomies. Pathology confirmed the diagnosis of amyloidoma. The surgeries were successful and the patient was discharged to outpatient follow-up care. Our last medical contact with the patient was two months after surgery, when he tested positive for COVID-19. Follow-up two months later determined that he recovered from COVID-19 and is doing well.The term amyloid was first used by Rudolf Virchow in 1854 Diagnosis of localized pulmonary amyloidoma is extremely rare, in part due to the unusual nature of the condition, but also because the patients are often asymptomatic and the mass is very slow-growing. While respiratory symptoms, such as cough, dyspnea, and hemoptysis are seen in certain forms of localized pulmonary amyloidosis, such as tracheobronchial, which facilitates detection Only one case with a clinical course similar to our patient has been reported recently by Desai et al There have only been a few published case reports of patients with pulmonary amyloidoma and plasmacytoma Therefore, regular imaging studies are suggested, especially for patients with presenting respiratory symptoms, such as shortness of breath, cough, dyspnea, and hemoptysis, as well as known pulmonary conditions including COPD, plasmacytoma, and amyloidosis, in order to identify the development of expansile and destructive pulmonary nodes. Primary nodular pulmonary amyloidoma mimics a variety of serious lung diseases including malignant tumors as well as interstitial, granulomatous or inflammatory respiratory diseases Case reports in the literature suggest that preliminary evidence of primary nodular pulmonary amyloidoma may be revealed as incidental findings on chest radiographs, though differential diagnosis is limited In the most recent and detailed analysis of CT chest scans in 41 patients with nodular pulmonary amyloidoma, Brandelik et al. found that all patients presented predominately with a nodular parenchymal pattern, with 39 of them exhibiting solitary or multiple pulmonary nodules Only a few studies have described the MRI findings in patients with primary pulmonary amyloidoma Several cases have been published with PET/CT scan findings for solitary nodular pulmonary amyloidoma. One of the first case reports showed increased metabolic activity of the calcified mass with numerous osseous lytic lesions (1)Unusually large solitary destructive mass in and around the lungs that is not detected until the patient is elderly, in part, due to slow asymptomatic growth over the years, most often initially identified as an incidental finding on a radiograph.(2)CT scans have generally shown lobulated margins with calcifications in a heterogeneously enhancing mass and unusual cystic radiological features.(3)MRI analyses generally demonstrate heterogeneously enhancing mass with highly variable signal intensities; specifically, the lung mass may appear isointense relative to skeletal muscle on T1-weighted axial MRI, but relatively low intensity on T2-weighted MRI.(4)PET/CT scan may show pathological utilization of the glucose analog and hypermetabolic activity with unusually high SUV, along with lytic lesions.In summary, imaging features that may suggest primary nodular amyloidoma include the following characteristics, which may be useful in differentiating it from bronchogenic carcinomas, other tumors, and infectious or inflammatory diseases:While the diagnosis of slow-growing asymptomatic pulmonary nodular amyloidoma can be challenging, we have provided evidence that routine imaging studies, using our expanding knowledge of specific imaging patterns, may be useful to identify growing masses before they become destructive as well as avoid unnecessary invasive procedures. Once imaging studies are suggestive of amyloidoma, pathohistological diagnostic confirmation is still required by a pulmonary biopsy and demonstration of apple-green birefringence with congo red stain under polarized light.Excision of the pulmonary mass is often recommended in the management of these patients, especially for large amyloidoma lesions, in order to reduce the destructive impact on adjoining organs, tissue, and bones as well as to improve respiratory functioning. Radiotherapy can be a viable option for the treatment of solitary pulmonary amyloidoma Primary pulmonary nodular amyloidoma is an extremely rare diagnosis. Our case report of an unusually massive chest wall amyloidoma, with a history of plasmacytoma, contributes to the accumulation of specific imaging features regarding diagnosis of rare pulmonary diseases as differentiated from more commonly seen disorders, which is critical in developing appropriate treatment plans and prognostic guidance. Early imaging detection is critical in order to avoid serious displacement and destruction of adjoining organs, tissues, and bones.Written informed consent was obtained for this Case Report including publication of imaging studies.


Blood vessels are essential for the formation and maintenance of almost all functional tissues. They play fundamental roles in the supply of oxygen and nutrition, as well as development and morphogenesis. Vascular endothelial cells are the main factor in blood vessel formation. Recently, research findings showed heterogeneity in vascular endothelial cells in different tissue/organs. Endothelial cells alter their gene expressions depending on their cell fate or angiogenic states of vascular development in normal and pathological processes. Studies on gene regulation in endothelial cells demonstrated that the activator protein 1 (AP-1) transcription factors are implicated in angiogenesis and vascular development. In particular, it has been revealed that JunB (a member of the AP-1 transcription factor family) is transiently induced in endothelial cells at the angiogenic frontier and controls them on tip cells specification during vascular development. Moreover, JunB plays a role in tissue-specific vascular maturation processes during neurovascular interaction in mouse embryonic skin and retina vasculatures. Thus, JunB appears to be a new angiogenic factor that induces endothelial cell migration and sprouting particularly in neurovascular interaction during vascular development. In this review, we discuss the recently identified role of JunB in endothelial cells and blood vessel formation. They display heterogeneity and different characteristics depending on the state of angiogenesis and tissue type. The differences noted between vascular endothelial cells include the basic properties of arteries, veins, capillaries, tip cells, and stalk cells. In addition, they include a wide variety of tissue-specific endothelial cells, such as the blood-brain barrier structure bearing cerebral blood vessels, and liver sinusoidal vascular endothelial cells that have a loose basement membrane structure. It has been shown that these differences are related to differences in gene expression. For example, blood-brain barrier transporters major facilitator superfamily domain containing 2A (Mfsd2a) and solute carrier family 2 member 1 (Slc2a1) are specifically expressed in endothelial cells of the central nervous system ,4,5. Thery 2021) . The trary 2021) . They alry 2021) .The process of vascular development, termed vasculogenesis, is initiated at the early developmental stages by the formation of a primitive vascular plexus, a beehive-like structure of endothelial progenitor cells. In the process of angiogenesis, the primordial vascular plexus invades the vascular-free area in response to angiogenic cues, such as hypoxia and vascular-inducing factors. Budding, branching, and fusion occur repeatedly to create more complex capillary networks. The vascular endothelial growth factor (VEGF) is a primary regulator of angiogenesis and blood vessel formation that controls endothelial cell proliferation, survival, and migration to form blood vessels. VEGFA controls angiogenic sprouting by guiding filopodia extension from tip cells at the vascular-sprouting frontier . At the The AP-1transcription factor family consists of Jun , Fos , and ATF . These factors form homodimers or heterodimers at the N-terminal leucine zipper common motif, which bind to DNA through the DNA binding motif, thereby regulating the transcription of target genes ,30,31. APreviously, it was found that JunB is involved in the differentiation of erythroid cells  and T ceExpression of JunB in human tissues/organs during different developmental, normal, and pathological conditions has been described. Moreover, human placental JunB expression in JunB/cyclin-D1 imbalance in placental mesenchymal stromal cells derived from pre-eclamptic pregnancies with fetal placental complications has been described . AmplifiJunB has been implicated in angiogenesis, and its expression is induced by hypoxia and VEGF. In endothelial cells, JunB regulates endothelial cell functions as a downstream factor of VEGF signaling . MoreoveJunB is expressed in endothelial cells and regulates their morphogenesis by regulating the core-binding factor beta subunit (CBFβ), which controls MMP13 expression, cell migration, and tube formation . In thisIn the sprouting region of the primordial vascular plexus, vascular endothelial cells located at the tip of the growing vessel extend numerous filopodia toward the vessel-free region. The endothelial cells that extend these filopodia are termed tip cells, and the endothelial cells that proliferate behind the tip cells and support vascular growth are called stalk cells . The morJunB is reportedly upregulated in tip cells at the angiogenic frontier and contributes to vascular development in mouse embryonic skin  and retiThe in vivo functional study of JunB using conventional JunB knockout mice was the first to describe the loss of JunB resulting in embryonic lethality through placental malformation and failure of cardiac vasculogenesis . More spRetinal vasculature has been widely used as an analysis system for angiogenic sprouting and vascular development due to its postnatal development and ease of tissue accessibility. VEGF plays a central role in retinal vascular development , and it Another study on retinal angiogenesis demonstrated that JunB is involved in the process of retina-specific vascular network development . RetinalIt is established that nerves and blood vessels align parallel to each other. However, until recently, the molecular mechanism involved in this process was unclear. Recent reports described that both neuronal signaling to the blood vessels  and vascDuring lymphatic vessel development in vertebrates, Prospero homeobox protein 1 (PROX1) expressing endothelial cells in cardinal veins eventually differentiate into lymphatic endothelial cells during lymphangiogenesis, which is considered the master regulator of lymphatic endothelial cell fate specification ,83. ForkThe main original articles that describe the functions of JunB in angiogenesis and vascular development, discussed in this review, are summarized in The mechanism of vascular “directional” control appears to be maintained mainly through the signaling balances between attractive and repulsive effectors from various tissues during vascular formation. For example, arterial and venous juxtapositional alignment is controlled by the balance of two types of action: Repulsive effects between arterial EphrinB2 and venous EphB4 ,91 and aIn the process of retinal vascular development, JunB is specifically upregulated in tip cells at the angiogenic frontier in the upper layer of retinal nerve tissue and migrates radially on the upper layers of the retina. Expression of JunB is also observed specifically in tip cells vertically protruding into the deep retinal plexus layer, leading to the formation of longitudinally oriented retinal blood vessels . In the


Herpes zoster involving all three divisions of the trigeminal nerve is extremely rare and may pose a diagnostic challenge, especially in young and immunocompetent patients. We present a unique case of herpes zoster involving all three divisions of the trigeminal nerve and illustrate that this uncommon eruption can be a presenting sign of varicella zoster aseptic meningitis. This case emphasizes the importance of fundamental morphology recognition, particularly its ability to aid in clinical diagnosis and its potential to decrease patient morbidity and mortality by expediting the initiation of appropriate treatment. Herpes zoster is a dermatomal, painful, vesicular rash caused by the reactivation of the varicella zoster virus (VZV) . It is mHerein, we describe the first known presentation of multi-dermatomal herpes zoster involving all three branches of the trigeminal nerve with unilateral oral vesicles and aseptic viral meningitis in a young, immunocompetent adult. This case highlights the importance of analyzing the morphology and distribution of a dermatologic process to not miss an atypical aggressive case of VZV in an otherwise healthy individual and prevent the possible morbidity and mortality associated with delayed diagnosis.A 33-year-old previously healthy male presented with oral pain and a painful left-sided facial rash of two days duration. The patient reported a left-sided headache with subjective fevers, body aches, night sweats, and odynophagia. He had an uncomplicated left upper molar extraction one month prior. Upon presentation, skin examination revealed erythematous, edematous, well-demarcated plaques to the left jawline, cheek, eyebrow, and nose Figure .Oral examination showed multiple coalescing vesicles to the left buccal mucosa and soft and hard palate, all strictly obeying the midline Figure . A lumbaGiven its rarity, a multi-dermatomal vesicular rash may pose a diagnostic challenge, especially in young and immunocompetent patients. The patient in this case report was found to have multi-dermatomal herpes zoster of the trigeminal nerve with disseminated disease to the central nervous system, which has not previously been reported in an immunocompetent individual. However, aseptic meningitis with an associated abdominal herpes zoster eruption has been observed, highlighting the ability of herpes zoster to present with concomitant central nervous system invasion . The iniThere have been only a few published cases of herpes zoster affecting all three branches of the trigeminal nerve, although none were associated with disseminated central nervous system infections ,5,6. SimThis case reemphasizes the importance of analyzing distribution, morphology, and arrangement in addition to conducting a thorough examination, including mucous membranes, when evaluating a patient with a new eruption. Clinicians should maintain high clinical suspicion of central nervous system dissemination in all patients with systemic symptoms, especially in the setting of multi-dermatomal eruptions, as such aggressive presentations will require prompt treatment with intravenous acyclovir. Viral PCR from either cutaneous lesions or CSF can take several days to result. Clinical diagnosis made on the basis of morphology, even in rare or atypical cases, has the ability to decrease the morbidity associated with delayed diagnosis of VZV.


Pirfenidone is an orally bioavailable drug with antifibrotic and anti-inflammatory properties already approved for the treatment of idiopathic pulmonary fibrosis. Pirfenidone has been recently tested in animal models of myocardial fibrosis with promising results. Here we will review the rationale underlying the potential therapeutic effect of Pirfenidone in HFpEF.Heart failure with preserved ejection fraction (HFpEF) is a major public health problem with growing prevalence and poor outcomes, mainly due to the lack of an effective treatment. HFpEF pathophysiology is heterogeneous and complex. Recently a “new paradigm” has been proposed, suggesting that cardiovascular and non-cardiovascular coexisting comorbidities lead to a systemic inflammatory state, perturbing the physiology of the endothelium and the perivascular environment and engaging molecular pathways that ultimately converge to myocardial fibrosis. If inflammation and fibrosis are the “ Myocardial fibrosis precedes the clinical diagnosis of HFpEF and is strongly associated with disease severity and adverse outcomes (Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome characterized by typical symptoms and signs of heart failure (HF) with normal or near-normal left ventricular ejection fraction LV EF ≥ 50%), echocardiographic features of diastolic dysfunction and/or structural heart disease and elevation of natriuretic peptides . The pre0%, echocoutcomes .Based on these evidences, a mechanistic overlap between HFpEF and other fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) seems likely . PirfeniHFpEF is a systemic syndrome, driven by accumulated risk factors and comorbidities, which, in vulnerable subjects, trigger pathways leading to increased ventricular stiffness, diastolic dysfunction and abnormal ventricular-arterial coupling , 9. The The classic risk factors for developing HFpEF include age, female gender, hypertension, diabetes, overweight/obesity, renal dysfunction, metabolic syndrome (present in the 85% of patients) and physical inactivity , 13. AccIn acute HFpEF, changes in levels of inflammatory markers such as pentraxin-3, TNF- receptor1a, myeloperoxidase, and lymphotoxin β receptor are correlated to outcome . The “neThe role of CMD at the basis of HFpEF pathogenesis and evolution has gained growing consent over years . CMD is both diastolic dysfunction and impaired CFR demonstrated a > 5-fold increased risk of HFpEF hospitalization, providing evidence that CMD, alongside myocardial stiffness, may play an important role in the pathophysiology of HFpEF.In the multi-national PRevalence Of MIcrovascular dySfunction in Heart Failure with Preserved Ejection Fraction (PROMIS-HFpEF) study , CMD hasMyocardial fibrosis is an endogenous response to different cardiac insults that may become maladaptive over time and contribute to the onset and progression of HF. ECM expansion, secondary to excess collagen accumulation, is considered a key pathophysiological mechanism of HFpEF, a common pathway that exists regardless of aetiology. There is strong evidence demonstrating both the primary aetiological role of myocardial fibrosis in HFpEF, but also the adverse impact that ECM expansion has on myocardial mechanical, electrical and microvascular function, confirming that the myocardial fibrotic burden is strongly and independently associated with adverse outcome –33. HoweInterestingly, myocardial and pulmonary fibrosis share some characteristics and molecular mediators, with TGF-β and Ang II being the major regulating factors. The activation of the AT1 receptor in fibroblasts by Ang II leads to the secretion of TGF-β, which stimulates fibroblast proliferation and ECM synthesis  in an autocrine manner and induces cardiomyocytes hypertrophy in a paracrine manner . TGF-β tTo date, unlike HFrEF, HFpEF is still orphan of treatments proven to significantly reduce major CV events. Beta-blockers, angiotensin-converting enzyme inhibitors, Ang-II receptor blockers, mineralocorticoid receptor antagonists and angiotensin receptor-neprilysin inhibitors have all failed to reach the pre-specified primary endpoints in trials testing their effects on CV outcomes  49–71),–71,49–71Over the last years, the new insights on HFpEF pathophysiology have increased the interest in testing new drugs specifically targeting the molecular mediators involved in HFpEF. Given the role of the oxidative stress and NO metabolism perturbation, it seemed reasonable to propose organic NO donors as potentially useful therapeutic tools. Unfortunately, results from the early studies are at best inconclusive and in some patients, paradoxically, a tendency to reduce the total physical activity was observed , 63. HowDrugs specifically targeting inflammation have been tested in HFpEF: in animal models chemokine antagonists  and immuno-modulatory cytokines  showed promising results . Anakinrin vivo and direct effects on dapagliflozin on the activity of this exchanger in endothelial cells demonstrated in vitro (The sodium–glucose cotransporter 2 (SGLT2) inhibitors  were found to reduce hospitalizations as well as mortality in HF , 74, butin vitro . These dDue to the key pathophysiological role of myocardial fibrosis in the development and progression of HFpEF, there is a growing interest about the potential beneficial effects of anti-fibrotic drugs, commonly used in other fibrotic disease such as IPF, also in HFpEF such as Pirfenidone.Pirfenidone is an orally bioavailable small synthetic molecule, with proven anti-inflammatory and anti-fibrotic properties and it is authorized by the European Commission for the treatment of adults with IPF .Pirfenidone is rapidly absorbed in the gastrointestinal tract and its half-life is about 3 h , 79. It The precise mechanism of action of this drug remains still unclear . PirfeniThe available data on cardio-protective effects of Pirfenidone are still in a preclinical phase, but there are several evidences pointing to that direction .cardiac fibroblasts migratory ability, inhibits their proliferation and the process of myofibroblast differentiation (by inhibition of α-SMA expression) as well as the myocardial fibroblast synthesis and secretion of TGF-β1. Pirfenidone also regularizes ratios of myocardial MMPs and tissue inhibitors of metalloproteinases, enhancing myocardial renin-angiotensin system imbalance and cardiac fibroblast synthesis and secretion of IL-10, an anti-fibrotic cytokine  trial . This isIn humans, only two retrospective studies have investigated the effect of Pirfenidone on LV structure and function in patients with IPF treated with this drug.In the first one, Alansari et al.  hypothesIf cardiac fibrosis and microvascular inflammation are the common pathophysiological substrates in this conundrum, Pirfenidone could have a powerful role in the treatment of all the different HFpEF subtypes, targeting the “core mechanisms” they all share. However, differences in timing of fibrogenesis and in fibrotic burden in each HFpEF-related comorbidity are still poorly understood, being influenced by many co-factors. Nowadays circulating biomarkers are considered a powerful tool to depict the patients pro-inflammatory and pro-fibrotic profile. Moreover, CMR offers the unique possibility to non-invasively estimate the amount of myocardial fibrosis. We can speculate that a “risk stratification” strategy, using inflammation and fibrogenesis biomarkers and CMR, might identify patients who will benefit most from Pirfenidone: those showing an intense inflammatory and fibrogenetic activation before the evolution toward an advanced, irreversible, stage of diffuse myocardial fibrosis. Trials specifically testing Pirfenidone effects on patients with this profile might help to optimize treatment in HFpEF.fil rouge in the HFpEF conundrum, thus they can be considered reasonable targets treatment. Pirfenidone is a well-established drug for the treatment of IPF and in animal studies it showed its anti-inflammatory properties and its ability to reverse cardiac fibrosis. Taken together these data suggest that Pirfenidone could have a role in the treatment of HFpEF by targeting inflammation and myocardial fibrosis, however, at present, clinical trials are lacking. Large, specifically-designed studies with hard end-point in this setting are needed.Up to now, HFpEF treatment has been borrowed from the HFrEF experience, with poor results. The complex and not completely understood HFpEF pathophysiology is probably the key to develop a tailored effective treatment. The new HFpEF paradigm states that the coronary microvascular endothelial inflammation is the main driving factor, activating complex molecular pathways that eventually converge to myocardial fibrosis. Coronary microvascular inflammation and myocardial fibrosis can be considered the FG and RL wrote sections of the manuscript. FC contributed to manuscript revision. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Laryngeal cancer is a prevalent head and neck malignancy, with poor prognosis and low survival rates for patients with advanced disease. The recurrence rate for advanced laryngeal cancer is between 25 and 50%. In order to improve surgical resection of laryngeal cancer and reduce local recurrence rates, various intraoperative optical imaging techniques have been investigated. In this systematic review we identify these technologies, evaluating the current state and future directions of optical imaging for this indication. Evidently, the investigated imaging modalities are generally unsuitable for deep margin assessment, and, therefore, inadequate to guide resection in advanced laryngeal disease. We discuss two optical imaging techniques that can overcome these limitations and suggest how they can be used to achieve adequate margins in laryngeal cancer at all stages.Laryngeal cancer is a prevalent head and neck malignancy, with poor prognosis and low survival rates for patients with advanced disease. Treatment consists of unimodal therapy through surgery or radiotherapy in early staged tumors, while advanced stage tumors are generally treated with multimodal chemoradiotherapy or  laryngectomy followed by radiotherapy. Still, the recurrence rate for advanced laryngeal cancer is between 25 and 50%. In order to improve surgical resection of laryngeal cancer and reduce local recurrence rates, various intraoperative optical imaging techniques have been investigated. In this systematic review, we identify these technologies, evaluating the current state and future directions of optical imaging for this indication. Narrow-band imaging (NBI) and autofluorescence (AF) are established tools for early detection of laryngeal cancer. Nonetheless, their intraoperative utility is limited by an intrinsic inability to image beyond the (sub-)mucosa. Likewise, contact endoscopy (CE) and optical coherence tomography (OCT) are technically cumbersome and only useful for mucosal margin assessment. Research on fluorescence imaging (FLI) for this application is sparse, dealing solely with nonspecific fluorescent agents. Evidently, the imaging modalities that have been investigated thus far are generally unsuitable for deep margin assessment. We discuss two optical imaging techniques that can overcome these limitations and suggest how they can be used to achieve adequate margins in laryngeal cancer at all stages. With almost 40,000 cases in Europe in 2018 . More adIn head and neck cancer, margin status remains one of the most important factors for local recurrence and overall survival, demonstrating the need to improve surgical resection. In patients undergoing  laryng(-opharyng)ectomy, inadequate margins are clearly associated with poor survival ,6. In paImportantly, it was clearly demonstrated that tumor-positive additional wound bed biopsies, taken after transoral CO2 laser microsurgery (TLM) of laryngeal cancer, significantly impacted local control . This fiIn addition, margin adequacy cannot be determined from sampling the wound bed. When a total laryng(-opharyng)ectomy is performed in advanced stage laryngeal cancer, a specimen-driven analysis of the surgical margins will likely be better, as has been shown in similar cases of oral cancer surgery ,14. IdeaThe surgeon’s ability to distinguish between malignant and healthy tissue during laryngeal surgery remains suboptimal, elucidating the need for objective intraoperative tools for margin assessment that facilitate both specimen and wound bed evaluation. Optical imaging has long been investigated and implemented for the early diagnosis of laryngeal cancer. Over the years, a number of these imaging modalities have been suggested as intraoperative tools to facilitate real-time enhanced visualization of resection margins ,16. ThisThis systematic review is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses , T-stage, surgical procedure, negative margin definition, negative margin rates, extra resection rate, patient-related outcomes , and diagnostic outcomes .The ‘methodological index for non-randomized studies’ (MINORS) was applied to all studies in the final inclusion . The MINThrough the search strategies, 4128 articles were identified. A total of seven additional articles were found through snowballing. After removing duplicates, 2644 articles were screened on title and abstract. Then, 68 articles were screened on full text and 19 articles were included for qualitative assessment in the systematic review. After careful consideration, the contributions from articles with a MINORS score of 4 and lower were deemed of insufficient quality, and were excluded from the systematic review. This additional screening resulted in 18 studies for final inclusion. Narrow-band imaging (NBI) is an endoscopic technique that uses selective light wavelengths to visualize the abnormal vascular patterns of the mucosa associated with premalignant or neoplastic lesions. NBI uses optical filters to select blue and green light . Narrow-band blue light, because of its low penetration depth, highlights mainly the mucosal surface, making subjacent capillaries appear brown. At the same time, the narrow-band green light with its higher penetration depth, allows it to enhance visibility of vessels in the submucosa, making them appear dark green A,B.The value of NBI during the diagnostic phase for early detection of laryngeal cancer and its precursor lesions is undisputed . Variousp = 0.02) . It. It42].  = 0.02) . NonetheOverall, NBI was found to modify intraoperative decision-making in several ways: better visualization of tumor borders led to more secure resection margins, detection of additional lesions prompted additional biopsies, and NBI-led upstaging resulted in changes to the surgical plan . With itAutofluorescence (AF) is the natural fluorescence emission of tissue when exposed to light of a suitable wavelength . AF is bThe fundamental principle of AF is related to changes in tissue morphology, optical properties, and the concentration of endogenous fluorophores in tissue as a result of neoplasia. When exposed to blue light, healthy laryngeal mucosa emits green fluorescence, whereas neoplastic mucosa fluoresces red-violet C,D. The In total, three clinical studies evaluate the intraoperative benefit of AF for margin assessment. Succo et al. reported high sensitivity and specificity (96.8% and 98.5%), stating an improvement in diagnostic accuracy over WL alone in 12 out of 73 cases (16.4%) . PaczonaThese studies suggest that direct AF can have a positive impact on disease-free survival and local control. However, more substantive studies are warranted to examine the added benefit of AF for more discrete identification of tumor presence, or positive margin reduction. AF is limited to examination of surface tissues. The illuminating light does not penetrate diseased epithelium, so that neoplastic changes within the basal mucosal layer may be hidden by epithelial hyperkeratosis. The presence of necrosis, scar tissue, bleeding, and inflammation can alter mucosal fluorescence in an unpredictable manner .Fluorescence Imaging (FLI) is an emerging optical modality that facilitates intraoperative guidance aimed at complete resection of tumor tissue . After sThe application of FLI for intraoperative margin detection in laryngeal cancers is thus far underexplored. A 2004 trial reported a sensitivity of 96% for 5-aminolevulinic acid-induced protoporphyrin-IX (5-ALA Pp-IX) fluorescence in the control of pharyngo-laryngeal cancer . In thisThe attenuating effects of light scattering, non-specific autofluorescence and absorption are relatively low for fluorescence in the near-infrared (NIR) range . In a stContact endoscopy (CE) is a technique that allows for in vivo visualization in cellular detail. After staining superficial cells of the mucosa with methylene blue, a microscopic endoscope is placed in direct contact with the surface, providing images of the suspected area at ×60 or ×150 magnification . ContactCE has been studied for the detection of microscopic margins during surgery in 10 patients with early glottic SCC, yielding histopathologically confirmed complete excision (negative margins defined as ≥2 mm) by Dedivitis et al., without local recurrence after a minimum follow-up period of 5 years . This suOptical coherence tomography (OCT) is the optical counterpart of ultrasound, and is capable of imaging cross-sectional anatomy at high resolutions in living tissue . OCT is OCT has only been studied for intraoperative margin assessment during TLM by Shakhov et al. in 26 patients with early laryngeal carcinoma . While tThere is a paucity of trials that analyze intraoperative imaging as a surgical adjunct during procedures for laryngeal cancer. The diagnostic value of NBI and AF in early laryngeal cancer is known. Their intraoperative application as an aid in defining surgical margins has not yet been studied as extensively, with most data available on NBI in this setting. As NBI visualization is limited to microvascular changes of the (sub)mucosa, evaluation of deep margin infiltration is impossible. Nevertheless, some recent studies report encouraging results of intraoperative NBI in patients with moderate-advanced laryngeal tumors. Distinct from intraoperative margin assessment, an interesting application for NBI is the follow-up of patients with early to moderate stage glottic cancer after curative treatment. Witkiewicz et al. recently reported that NBI after TLM is a good predictor of margin positivity, supporting clinical decision-making regarding the need for second-look surgery . NBI durBoth OCT and CE are associated with various technical impracticalities. CE requires topical staining and is only able to clearly image the superficial layers of the epithelium . PreviouAlthough only 2 studies employing FLI for margin assessment were identified, laryngeal cancer surgery might be amenable to the more prevailing developments in FLI. An important advantage of FLI is that it can be used to scan large surfaces. In addition to the possibility to image fluorescence at considerable depths and tissues other than the mucosa, FLI is thus ideally suited for wound bed inspection and detection of clinically occult lesions. An interesting area to explore for FLI would be to identify suitable tumor-specific tracers for intraoperative imaging of early to advanced laryngeal cancer . PreviouAs per the confines of this systematic review, there currently is no real-time application for Raman spectroscopy (RS) in margin assessment of laryngeal cancer. However, its demonstrated potential for both in and ex vivo tissue discrimination is worth noting. RS is a technique based on spectral differences between normal and malignant tissue, and is capable of near-instant, accurate, and non-invasive analysis of a tissue’s molecular composition . In oralThe demonstrated effectiveness of NBI in the early diagnosis of laryngeal cancer has led to reasonable evidence for its application in intraoperative margin detection, although inclusion of patients with T3–T4 laryngeal cancer is rare. An important disadvantage remains the technique’s intrinsic inability to image structures other than the (sub-)mucosa. Similar limitations hold for AF, OCT, and CE. Finally, FLI has scarcely been studied for intraoperative margin assessment in laryngeal cancer. Indeed, one might need to consider optical imaging advancements in adjacent anatomical regions and similar applications to gain insight into which modalities warrant further exploration in achieving adequate margins. NIR FLI with tumor-specific fluorescent agents and RS are emerging technologies that overcome many of the limitations associated with NBI, AF, CE, and OCT. With evidence supporting their value in assessing tumor depth infiltration, we believe it is worthwhile to pursue their clinical development for margin assessment in laryngeal cancer.


Herein, we summarize existing evidence on the impact of homogenization on HM, with a particular focus on changes to the macromolecular structure of the milk fat globule and the subsequent impact on digestion kinetics. We use these published data to create a conceptual framework for the potential implications of homogenized HM-based nutritional products on preterm infant health. Importantly, we underscore that the safety and efficacy of homogenized HM-based products warrant investigation.An exclusive human milk diet (EHMD) has been shown to reduce health complications of prematurity in infants born weighing ≤1250 g compared with cow milk–based diets. Accordingly, the number of available human milk (HM)-based nutritional products continues to increase. Newly available products, and those reportedly soon to enter the market, include homogenized donor HM and homogenized HM–based fortifiers. Existing literature demonstrating the benefits of an EHMD, however, is limited to  Human milk (HM)-based nutritional products are not created equally. Homogenization alters the evolutionarily evolved structure of HM, yet safety and efficacy research are lacking for homogenized HM-based nutritional products. The best source of nutrition for premature infants born weighing <1500 g is their mothers’ own milk (MOM) fortified appropriately with protein, minerals, and vitamins; when MOM is unavailable or contraindicated, donor human milk (DHM) should be substituted .The benefits derived from an EHMD diet are 2-fold: delivering the nutrition required to offset missed nutrient accretion that occurs during the third trimester and reducing the complications associated with enteral feeding , 11. BefAs the use of HM-based nutritional products grows, so do the number of products available in the market. Currently available products include DHM, HM-based multinutrient fortifiers, and HM-based caloric fortifiers. These products are used in combination with MOM and/or DHM under the direction of health care professionals to meet the individual nutritional needs of the most medically fragile infants , 11, 12.Newly available products, and those reportedly soon to enter the market, include homogenized DHM and homogenized HM-based fortifiers , 18. HerHomogenization is a physical process that evenly disperses 2 mutually nonsoluble liquids to create a single uniform mixture. Milk is homogenized to disperse fat droplets and prevent the cream from rising to the top. In the dairy industry, homogenization is used to improve cow milk's taste, consistency, and appearance as well as to extend its shelf-life .Some newly available and upcoming HM-based nutritional products are homogenized, although the rationale for homogenization is unclear. The strongest rationale is based on the hypothesis that homogenization may improve weight gain of very-low-birth-weight premature infants. This hypothesis gained traction after evidence showed that fecal excretion of fat is lower in infants fed homogenized HM than in those fed nonhomogenized HM . These dAnother rationale for homogenizing HM-based nutritional products may be to reduce fat loss during enteral feeding. Continuous enteral feeding can lead to fat loss of HM because the fat sticks to plastic tubing . HomogenMore likely, the rationale for homogenizing HM-based nutritional products may be to counter the effects of high-temperature processing, such as retort sterilization or ultra-high temperature (UHT) processing. Retort sterilization is a heat treatment common in canned food manufacturing during which milk is exposed to high temperatures and pressure to eliminate viral and bacterial pathogens. UHT processing is a higher heat treatment for shorter duration than retort sterilization. New and upcoming homogenized HM-based nutritional products also undergo either retort sterilization or UHT processing. Unfortunately, high-temperature processing methods can lead to loss of fat content in HM because they cause the fat to adhere to the container. Sterilization of HM resulted in loss of >10% of fat content, whereas pasteurization of HM had no effect on its fat content . It is pUltimately, the rationale for homogenizing HM-based nutritional products is based on the weak scientific premise that it is advantageous for infant weight gain, although it is more likely performed to counter the negative effects of harsh heat treatments on HM fat.Homogenization affects the properties of milk in a variety of ways. The most serious of these relate to changes in the macromolecular structure of the milk fat globule. Specifically, homogenization removes a large proportion of the outer membrane, resulting in smaller milk fat droplets to which milk serum proteins adsorb . This chThe milk fat globule is the primary vesicle that delivers fat and fat-soluble nutrients to the infant . The natHomogenization disrupts the MFGM, exposing the hydrophobic lipid core to the surrounding aqueous environment , 36. WheThe MFGM and its various components have been implicated in favorable neurodevelopmental outcomes and low risk of infections and feeding intolerance , and areIn vivo studies of formula-fed infants support the hypothesis that the MFGM may contribute to the benefits of HM-feeding. For instance, the addition of cow or whey-sourced MFGM to infant formula has been shown in clinical trials to result in fewer infections and improved neurodevelopment outcomes compared with standard, MFGM-free formulas . NeverthThe effects of a disrupted MFGM on the neurocognitive benefits of HM for preterm infants is a critically important area of research in need of investigation . PrematuThe neurocognitive benefits of HM are attributed, in part, to the numerous HM components important for brain development, including sialic acid, gangliosides, sphingomyelin, choline, cholesterol, and PUFAs . These cIn addition to causing premature digestion of HM lipids, homogenization can alter the accumulation patterns of free fatty acids. Fatty acids are a normal part of the inner triglyceride core of the milk fat globule. Three fatty acids connected to a glycerol molecule make up a single triglyceride. Typically, long-chain fatty acids are released before SCFAs so they can be absorbed in the small intestine . HoweverLong-chain fatty acids, especially long-chain PUFAs (LCPUFAs), are important for brain development. LCPUFAs, including DHA (22:6n–3), are primarily accumulated in the fetal brain during the third trimester .Achieving optimal accretion of LCPUFAs in the brain of infants born prematurely remains a serious clinical challenge, although recent evidence suggests feeding an EHMD using nonhomogenized HM-based fortifiers may be beneficial. Among premature infants fed fortified HM, those fed HM with a nonhomogenized HM-based fortifier maintained normal blood concentrations of DHA in the first 3 wk of life compared with a 30% decline in infants fed HM fortified with a homogenized cow milk–based fortifier .These results are intriguing because all infants in this study were fed HM (>70% MOM), but 1 group received a nonhomogenized HM-based fortifier with intact MFGM and the other group received a homogenized cow milk–based fortifier, lacking MFGM. Further investigation of the effect of a disrupted MFGM structure on blood DHA concentrations should be undertaken before homogenization is deemed appropriate for HM-based fortifiers.New and upcoming homogenized HM-based nutritional products also undergo high heat processing, such as UHT or retort sterilization—a combination that plausibly could have implications for infant epithelial barrier function and risk of infection and NEC . An MS-bFeeding tolerance and growth are critically important clinical outcomes for preterm infants, and the role of the microbiome in these outcomes has gained attention . A recenBifidobacterium spp., and lactic acid bacteria (It has been suggested that the macromolecular structure of the milk fat globule enables advantageous absorption (which may not necessarily be rapid or complete) that optimizes lipid tissue distribution , 58. Spebacteria . These ibacteria . This mabacteria . The posbacteria .In summary, homogenization changes the macromolecular structure and composition of the milk fat globule in ways that influence protein and fat bioavailability and bioactivity. Although preliminary studies indicate that this may increase fat absorption , 62, 63,HM-based nutritional products as part of an EHMD have been shown in clinical studies to improve health and reduce the complications of prematurity in very-low-birth-weight infants. Accordingly, the use of these products and EHMD protocols are increasing globally as are the number of available products. Some new and upcoming products are homogenized which alters MFGM structure, a complex mixture of proteins, lipids, and carbohydrates shown to be important in infection, neurodevelopment, and growth. Changes to the macromolecular structure of the milk fat globule have unknown consequences for the health of premature infants. Thus, investigation of homogenization-induced changes to the macromolecular structure and function of the milk fat globule is critical to establish the safety and efficacy of homogenized HM-based nutritional products, especially those that also undergo high heat treatments including retort sterilization and UHT processing. Importantly, the published clinical benefits of HM-based nutritional products only apply to currently available nonhomogenized products. The safety and efficacy of homogenized HM-based nutritional products have not been established.Box 1Traditionally used in the dairy industry, homogenization is used to improve cow milk's taste, consistency, and appearance as well as to extend its shelf-life.Homogenization disrupts the milk fat globule, which may lead to premature digestion of bioactive components linked to favorable neurological outcomes, including sphingolipids and long-chain PUFAs. The implications of these changes for infant health are unknown.The described clinical benefits of human milk (HM)-based nutritional products only apply to currently available nonhomogenized products. The safety and efficacy of homogenized HM-based nutritional products have not been established.


Prevention of neurodegeneration during aging, and support of optimal brain function throughout the lifespan, requires protection of membrane structure and function. We review the synergistic action of different classes of dietary micronutrients, as well as further synergistic contributions from exercise and stress reduction, in supporting membrane structure and function. We address membrane-associated inflammation involving reactive oxygen species (ROS) that produce immune regulators from polyunsaturated fatty acids (PUFAs) of membrane phospholipids. The potential of dietary micronutrients to maintain membrane fluidity and prevent chronic inflammation is examined with a focus on synergistically acting membrane-soluble components  and water-soluble components (vitamin C and various phenolics). These different classes of micronutrients apparently operate in a series of intertwined oxidation-reduction cycles to protect membrane function and prevent chronic inflammation. At this time, it appears that combinations of a balanced diet with regular moderate exercise and stress-reduction practices are particularly beneficial. Effective whole-food-based diets include the Mediterranean and the MIND diet . Humans need micronutrients to assist with, and protect, vital functions. Essential micronutrients—which cannot be synthesized in the human body and must be consumed with the diet—include not only vitamins but also a host of other compounds of plant origin termed phytochemicals . Phytochemicals can be classified by solubility into hydrophobic, or membrane-soluble, compounds and hydrophilic compounds soluble in aqueous cell environments. Plants produce hundreds of largely water-soluble compounds , as wellMembranes are the site of neuronal function and are thus abundant in the mammalian brain. For both neuronal and other functions, membranes need to have a specific fluidity. Membrane fluidity is influenced by the type of fatty acids in the phospholipids that make up membranes and can be further modulated by metabolites that are dissolved within these membranes. A well-known example of such a metabolite is cholesterol that acts as a stabilizer of membrane fluidity. The dietary xanthophylls, zeaxanthin and lutein, also act as membrane stabilizers in human membranes  or terminate the immune response  [Furthermore, membranes are a key site in the initiation of an immune response. When a series of events produce oxidants, polyunsaturated fatty acids (PUFAs) of membrane phospholipids become oxidized to hormone-like gene regulators that either 3 PUFAs) . Convers3 PUFAs) . A varieSeveral classes of phytochemicals must work in tandem with each other to prevent excessive oxidation of the fatty acids of membrane phospholipids (see details on this topic below). A direct interaction of membrane-soluble and water-soluble antioxidants was demonstrated by in vitro studies with isolated membrane vesicles. More research is needed to clarify if, and where, in the human body dietary phytochemicals co-occur and interact directly in the same sites in vivo. Vitamin E, zeaxanthin, and lutein are readily taken up from the gut and cross the blood–brain barrier . It wasLike the xanthophylls, vitamin E also has both hydrophobic and hydrophilic portions  and is iZeaxanthin and vitamin E are both capable of reducing, and thereby detoxifying, oxidants (such as ROS) directly as well as reducing lipid peroxyl radicals, which slows the chain reaction of lipid oxidation induced by ROS . While zMore research is needed to clarify how these interactions occur in vivo with respect to the relative contribution of the different cycles shown in Water-soluble dietary phytochemicals with antioxidant capacity include vitamin C and the large, diverse group of phenolics . PhenolEvidence for direct interaction between membrane-bound vitamin E or carotenoids on the one hand and water-soluble antioxidants on the other hand mainly comes from in vitro studies that showed direct interaction between membrane-soluble and water-soluble phytochemicals in preventing oxidation of membrane phospholipids. For example, the ability to re-reduce vitamin E and zeaxanthin radicals was documented for ascorbic acid  as well as phenolics such as anthocyanidins (that can interact with zeaxanthin ), catechFuture research should elucidate how the many phenolic phytochemicals of a plant-rich diet interact synergistically with membrane-bound phytochemicals in vivo, as there are several possibilities of how this may occur. While many polyphenols are absorbed poorly beyond the gut in their original forms , some poAdditional indirect interaction is possible between membrane-soluble antioxidants (located in membranes throughout the body) and water-soluble polyphenols that remain in the intestinal tract. For phenolic phytochemicals that do not move beyond the intestinal tract, an alternative mechanism exists that allows them to exert synergistic effects with membrane-bound phytochemicals throughout the body d. This aThe available evidence is consistent with an ability of zeaxanthin and/or lutein to attenuate neuroinflammation ,46. IrreWhile there is substantial evidence for inverse correlations between zeaxanthin and/or lutein levels in blood or tissues and the risk for various diseases, the levels of these two xanthophylls often correlate closely with those of other carotenoids, including provitamin A (β-carotene) and lycopene . Caroten-3 fatty acids specifically and significantly increased visual processing speed [There is a growing number of supplementation studies with zeaxanthin and/or lutein. Supplementation with zeaxanthin and lutein enhanced cognitive performance in both young and older adults  as well as the need for interaction between membrane-bound and water-soluble antioxidants, clinical trials are needed that examine the efficacy of combinations of multi-component supplements. A supplementation study on a combination of lutein and the omega-3 PUFA DHA (docosahexaenoic acid) showed that this combination synergistically enhanced memory as well as the rate and efficiency of learning ,58. PosiIn addition to synergy among diverse dietary components, synergistic interactions take place between diet and other lifestyle factors, such as physical activity and psychological stress. promoting gene regulators during an acute infection, as well as formation of inflammation-terminating gene regulators after an acute infection is brought under control and to prevent detrimental chronic inflammation . This dAs stated above, synthesis of endogenous antioxidant enzymes is triggered by ROS formed in working muscles and suppressed by high-dose antioxidant supplements  and results in continuous low-grade activation of the immune system, i.e., chronic inflammation . This faSince diet, physical activity level, and psychological stress level interact to modulate ROS levels and the production of immune-system regulators , a combiOn the other hand, profound changes in just a single lifestyle factor are unlikely to be equally effective. Interventions that inherently combine different components are indeed emerging as particularly effective. Therapies/activities that effectively combat chronic inflammation tend to be the ones that naturally combine several lifestyle factors. An example is yoga, which combines physical activity with stress reduction (see above). Another example is horticultural therapy (gardening) that combines physical activity with stress reduction, nature exposure, and even social interaction. Such horticultural therapy has been shown to reduce markers of stress and chronic inflammation in older adults ,106. MorAny additional environmental factors that trigger ROS production can contribute to chronic inflammation. Such factors include a wide variety of environmental toxins, including pollution of air, water, and food with organic toxins, heavy metals, and other agents Essential dietary micronutrients are required to support membrane function and prevent chronic inflammation, especially in the human brain. Membrane-soluble micronutrients  interact synergistically among each other and with water-soluble antioxidant metabolites (vitamin C and various phenolics) and antioxidant enzymes. Several of these dietary micronutrients have multiple essential functions as antioxidants, anti-inflammatories, and/or membrane stabilizers. The synergy among different types of micronutrient antioxidants consists of intertwined oxidation-reduction cycles that protect membrane function and allow a controlled, well-orchestrated inflammation response that does not escalate to chronic inflammation. More research is needed to assess localization and interaction among different classes of micronutrients in vivo, and to test for causal relationships via clinical trials using supplementation with combinations of micronutrients. Additional lifestyle factors  interact with dietary micronutrients in affecting the internal balance (redox homeostasis) of ROS and antioxidants. More research is needed to test interventions that combine diet, physical activity, and stress reduction. Overall, multi-component interventions should be a target of future research as well as of recommendations for lifestyle changes. At this time, it appears safe to recommend a combination of a balanced diet (modeled after whole food diets like the Mediterranean and/or MIND diet) with regular moderate exercise and stress-reduction practice. On the other hand, recommendations for any specific supplements, especially in higher doses ,66, requ