Healthy adipose tissue expansion and metabolism during weight gain require coordinated angiogenesis and lymphangiogenesis. These vascular growth processes rely on the vascular endothelial growth factor (VEGF) family of ligands and receptors (VEGFRs). Several studies have shown that controlling vascular growth by regulating VEGF:VEGFR signaling can be beneficial for treating obesity; however, dysregulated angiogenesis and lymphangiogenesis are associated with several chronic tissue inflammation symptoms, including hypoxia, immune cell accumulation, and fibrosis, leading to obesity-related metabolic disorders. An ideal obesity treatment should minimize adipose tissue expansion and the advent of adverse metabolic consequences, which could be achieved by normalizing VEGF:VEGFR signaling. Toward this goal, a systematic investigation of the interdependency of vascular and metabolic systems in obesity and tools to predict personalized treatment ranges are necessary to improve patient outcomes through vascular-targeted therapies. Systems biology can identify the critical VEGF:VEGFR signaling mechanisms that can be targeted to regress adipose tissue expansion and can predict the metabolic consequences of different vascular-targeted approaches. Establishing a predictive, biologically faithful platform requires appropriate computational models and quantitative tissue-specific data. Here, we discuss the involvement of VEGF:VEGFR signaling in angiogenesis, lymphangiogenesis, adipogenesis, and macrophage specification – key mechanisms that regulate adipose tissue expansion and metabolism. We then provide useful computational approaches for simulating these mechanisms, and detail quantitative techniques for acquiring tissue-specific parameters. Systems biology, through computational models and quantitative data, will enable an accurate representation of obese adipose tissue that can be used to direct the development of vascular-targeted therapies for obesity and associated metabolic disorders. The prevalence of obesity has tripled since 1975, affecting over 650 million adults worldwide . ObesityThe dependency of adipose tissue growth and metabolism on adipose vasculature indicates that vascular-targeted therapies could be used to treat obesity and obesity-associated metabolic disorders . Blood aVascular development is primarily regulated by vascular endothelial growth factors (VEGFs) and VEGF receptors . VEGFs wVEGFR1 and VEGFR2 are the primary angiogenic receptors, with different ligand-induced signaling responses  which maIt is also prudent to consider VEGFR heterodimers in adipose tissue angiogenesis, as both VEGFR1 and VEGFR3 can heterodimerize with VEGFR2 on vascular endothelial cells, affecting the angiogenic response. Whether VEGFRs preferentially form homo- or hetero-dimeric complexes is an unresolved matter; however, simulations estimate that 10–50% of VEGFRs are heterodimeric. Furthermore, the VEGFR density is predicted to regulate homo- vs. hetero-dimerization: a ratio of 10:1 VEGFR1:R2 or 10:1 VEGFR2:R1 inhibits homodimerization of the less-abundant receptor  . FunctioAnother key vascular process is lymphangiogenesis, which is primarily driven by VEGFR3 activation on lymphatic endothelial cells. VEGFR3 is necessary for lymphangiogenesis as it activates PROX1, the master transcription factor regulating both differentiation of lymphatic endothelial cells from their progenitors and subsequent maintenance of their lymphatic identity . VEGFR2 165 is the most abundant isoform; other known VEGF-A isoforms include VEGF-A111, VEGF-A121, VEGF-A145, VEGF-A148, VEGF-A183, VEGF-A189, and VEGF-A206 a and VEGF-A(xxx)b which are generated via exon 8 splicing and designated as pro-angiogenic and anti-angiogenic, respectively ((xxx)a/b splice variants are discussed by several reviews (165b properties that are implicated in obesity: (1) VEGFR binding: VEGF-A165b competes with VEGF-A165a for VEGFR binding in pathological conditions (165b:VEGFR2 binding. (2) VEGFR activation: VEGF-A165b induces only weak VEGFR phosphorylation and downstream signaling in ischemia  Sys surgery . Thus, s(xxx)b mRNA in vivo further complicates this story: VEGF-A(xxx)b mRNA was undetectable in RNA-seq data from approximately 7,000 samples from 50 tissues ((xxx)b may be confounded by variants that contain both “a” and “b” C-terminal sequences, termed VEGF-Ax. Such VEGF-Ax variants would be detected by VEGF-A(xxx)b antibody ((xxx)b in obesity . The angiogenic signaling canon describes uni-family interactions: VEGF:VEGFR signaling promoting healthy vascular formation  and PDGFAdipose tissue angiogenesis is driven by either local hypoxia or endocrine signaling . Under hAngiogenesis also supports adipose tissue expansion by providing a niche for preadipocyte recruitment and differentiation . PreadipImpaired lymphangiogenesis is also implicated in the progression of obesity. The peripheral lymphatic vessels are required for lipid transport and immune cell clearance in adipose tissue. Impaired lymphatic vasculature, commonly seen in obesity, can leak lipid-containing lymph into peripheral tissue, inducing adipogenesis, subcutaneous fat deposition, and weight gain in mice . InactivLacteals, the specialized lymphatic vessels adjacent to the small intestines, are important for absorbing digested dietary lipids and conveying the lipids to the circulation . Emerginin vivo obesity models should be used in further studies investigating the molecular and cellular mechanisms that link angiogenesis/lymphangiogenesis, adipogenesis, and related metabolic outcomes. High-fat diet (HFD)-fed mice are widely used models for obesity research because they exhibit (1) increased triglyceride storage via adipocyte hypertrophy, (2) increased overall body weight  y weight . Future Pro-inflammatory macrophage accumulation is considered a major factor that perpetuates chronic inflammation in obese adipose tissue. HFD-induced obesity increased the number of pro-inflammatory M1 macrophages by 65-fold while increasing anti-inflammatory M2 macrophages by only six-fold . M1 macrin vitro study suggests that this increase in M2 macrophages could be a result of M1 macrophages differentiating into M2 macrophages, rather than M2 recruitment. This study found that VEGFR1 mRNA was highly expressed in M1 macrophages but only at low levels in M2 macrophages and that VEGF-A:VEGFR1 signaling stimulates M1 recruitment and subsequent differentiation of M1 macrophages into M2 macrophages  . The evirophages . AltogetBesides the role of M1 and M2 macrophages in inflammation, they also differentially regulate the initiation and continuation of angiogenesis . M2 macrAlthough macrophages are conventionally characterized by M1 and M2 subtypes, other classification schemes based on macrophage functions are useful for identifying the roles of macrophages in specific pathologies . As we hEmerging evidence has shown that adipose vasculature is a promising anti-obesity target , 2016. IAnti-VEGF and anti-VEGFR treatments have also largely been shown to be beneficial in treating obesity in pre-clinical obesity models . HoweverAnti-VEGFR treatments have shown varied effects on weight loss and metabolic enhancement in HFD-fed and chow-fed models. Systemic anti-VEGFR2 treatment significantly slowed weight gain after 6 weeks of HFD, though no weight suppression was shown in the first 6 weeks . In compImproved metabolic function was also observed in two pre-clinical anti-VEGF studies, although there were discrepancies in the weight loss outcomes . One stuVEGFR3-targeted therapeutics for obesity have not yet been significantly explored, although the VEGFR3 activation must be maintained or upregulated to prevent lymphatic vessel leakiness, excess adipocyte hypertrophy, and insulin resistance . A recenWhile vascular-targeted therapies are promising, their efficacy in treating obesity remains unclear in humans. Many factors should be considered in future studies targeting VEGF/VEGFR, including age, treatment duration, as well as the routes of administration (systemic or adipose-targeted). For example, systemic administration of VEGFR2 antibody could undesirably increase lacteal permeability, leading to higher lipid uptake from the small intestines to the lymphatics . Other fSystems biology offers a mechanistic understanding of complex biological networks through mathematical and computational modeling . SystemsSBML.caltech.edu). Reactions and parameters can be modified to test new model implementations in these software. There is also a well-developed community that can critique VEGF:VEGFR model results, which improves the quality of the model and leads to greater credibility in its findings . Therefore, adipose tissue-specific VEGF:VEGFR models would be in line with the field, while offering a platform for predicting the efficacy of VEGF/VEGFR targeted therapies in obese patients.Simulations of VEGF/VEGFR targeted therapy in human patients can be made at the protein level using mass-action kinetic modeling. This approach reconstructs a reaction network using ordinary differential equations (ODEs) , which r121 and VEGF-A165 isoforms to membrane VEGFRs, NRP1, and the ECM  . In addiQuantitative flow cytometry (qFlow) is an emerging quantitative standard for measuring proteins on the plasma membrane . qFlow eFurthermore, cell-by-cell analysis of qFlow data can unmask population heterogeneity when specific subpopulations depart from the “mean” behavior . To idenin vitro cell lines  and literature data, the VEGF:VEGFR kinetics and VEGF concentrations in lean, obese, and treated adipose tissue can be acquired. However, there are minimal proteomic data on VEGFR concentrations on adipose-derived cells, even though adipose tissue is one of the highest VEGFR gene-expressing tissues. qFlow should be used to acquire membrane-bound VEGFR concentrations in adipose tissue; furthermore, cell-by-cell analysis and mixture modeling will be particularly helpful for identifying functionally distinct cell subpopulations within adipose tissue endothelial cells and macrophages.We have reviewed benchmark models that will provide the basis for future efforts to predict the effects of vascular-targeted therapies on obesity and metabolic responses. These models predict VEGF:VEGFR signaling outcomes and metabolic network regulation via mass-action kinetic modeling and CBM, respectively; mass-action kinetic modeling can be used to design effective vascular-targeted therapeutic strategies, and CBM can identify metabolic pathways that are significantly affected by vascular-targeted therapies. Systems biology, through future quantitative data and computational models, will enable a better understanding of obesity-associated vascular dysregulation and advance vascular-targeted therapies for obesity and its associated metabolic disorders.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.YF led the manuscript preparation. TK contributed to the computational modeling review and contributed critical discussion on this work. YF, TK, and PI prepared 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.


Pichia pastoris) recombinant protein comprised of the receptor-binding domain (RBD) of the SARS-CoV formulated on alum and referred to as the CoV RBD219-N1 Vaccine. Potentially, this vaccine could be used as a heterologous vaccine against COVID-19. A second vaccine specific for COVID-19 is also being advanced using the corresponding RBD of SARS-CoV-2. The first antigen has already undergone current Good Manufacturing Practices (cGMP) manufacture and is therefore “shovel ready” for advancing into clinical trials, following vialing and required Good Laboratory Practice (GLP) toxicology testing. Evidence for its potential efficacy to cross-protect against SARS-CoV-2 includes cross-neutralization and binding studies using polyclonal and monoclonal antibodies. Evidence in support of its safety profile include our internal assessments in a mouse challenge model using a lethal mouse-adapted SARS strain, which shows that SARS-CoV RBD219-N1  does not elicit eosinophilic lung pathology. Together, these findings suggest that recombinant protein–based vaccines based on the RBD warrant further development to prevent SARS, COVID-19, or other coronaviruses of pandemic potential.There is an urgent need to advance safe and affordable COVID-19 vaccines for low- and middle-income countries of Asia, Africa, and Latin America. Such vaccines rely on proven technologies such as recombinant protein–based vaccines to facilitate its transfer for emerging market vaccine manufacturers. Our group is developing a two-pronged approach to advance recombinant protein–based vaccines to prevent COVID-19 caused by SARS-CoV-2 and other coronavirus infections. One vaccine is based on a yeast-derived  and caused almost 400,000 deaths . In the At least a dozen COVID-19 candidate vaccines are under development using different technology platforms , with anTowards this goal, we are now also developing the SARS-CoV-2 RBD recombinant protein as a potential vaccine candidate, in parallel with the existing CoV RBD219-N1 candidate vaccine, which was previously developed and manufactured under cGMP in 2016 –10. The P. pastoris. Both approaches reinforce each other, as the processes developed for the CoV RBD219-N1 candidate also apply to the SARS-CoV-2 candidate, and both antigens downstream could be further developed as potentially a bivalent or a universal coronavirus vaccine.Both RBD vaccine candidates have potential as vaccine antigens to prevent SARS-CoV-2 infection and/or COVID-19. Overall, our initial approach relies on advancing the already manufactured CoV RBD219-N1 as a heterologous recombinant subunit vaccine to protect against both SARS and COVID-19 [The SARS-CoV protein known as CoV RBD219-N1 was selected on its ability to elicit high titers of neutralizing antibodies against both SARS-CoV pseudotype virus and live SARS-CoV virus , 8, prioThere are several advantages of the CoV RBD candidate antigens and vaccines for purposes of global health:P. pastoris, a low-cost expression platform, which can be produced and scaled at high yields [High yield and low cost. The antigens are expressed in h yields , 8. By dh yields , 8. A pahttps://www.dcvmn.org/) [P. pastoris–derived recombinant protein is currently produced by several DCVMs, including those in Bangladesh, Brazil, Cuba, India, and Indonesia.Technology transfer. The process is suitable for technology transfer to emerging market vaccine manufacturers  having expertise in fermentation technology (mn.org/) . The P. Shovel ready. The CoV RBD219-N1 antigen was manufactured under cGMP and can be vialed to produce between 20,000 and 200,000 doses, with the possibility of transferring production processes and cell banks to DCVMs for large-scale production sufficient to meet global needs.Beyond low cost and ease of potential technology transfer to DCVMs, an advantage of employing a recombinant protein subunit vaccine is the long-standing safety record of this class of vaccines, and the fact that this technology has been used for the licensure of two other antiviral vaccines—hepatitis B and human papillomavirus, as well as biologics  .In addition to their low cost and suitability for use in public immunization programs in low- and middle-income countries, we pursued RBD recombinant protein–based vaccines as a technology to maximize safety relative to other platforms, such as virus vectors that have previously been found to induce immune enhancement. For instance, immune enhancement in children following a formalin-inactivated respiratory syncytial virus (RSV) vaccine was first reported in the 1960s and later shown to occur in laboratory animals with early prototype SARS-CoV vaccines using virus-vectored platforms or inactivated virus constructs . We haveSome of the earliest SARS-CoV vaccine candidates used vectored-based platforms, and these were associated with immune enhancement or activation. In 2004–2005, scientists at the Public Health Agency of Canada’s National Microbiology Laboratory in Winnipeg, Manitoba (who helped to develop the first successful Ebola vaccine), found that a recombinant modified vaccinia Ankara (rMVA) expressing the S-spike protein resulted in severe liver pathology upon SARS-CoV virus challenge. Similarly, rMVA expressing the S-spike also resulted in lung immunopathology in rhesus macaques, as did other virus-vectored constructs. Lung immunopathology is also linked to whole inactivated viral vaccines. However, it was determined that in many cases eosinophilic pathology is driven by the SARS nucleocapsid (N) protein, although a recent trial in nonhuman primates found that an alum-adjuvanted inactivated SARS-CoV-2 vaccine did not induce immunopathology . Among tGiven the history of virus-vector platforms and inactivated vaccines in eliciting eosinophilic immunopathology, our emphasis has been on the evaluation of inexpensive recombinant proteins produced in microbial systems. These are comprised of the CoV RBD219-N1 antigen, encoding amino acids 319–536 (219 AA) of the SARS-CoV S-spike protein –10, and There is evidence to justify advancing the CoV RBD219-N1 antigen as either a homologous vaccine against SARS –10 or asP. pastoris and formulated on aluminum or other accessible adjuvants represents a highly accessible technology to transfer to low- and middle-income countries. It represents one of several key mechanisms for ensuring that populations across the major affected nations of Africa, Asia, and the Americas will benefit from COVID-19 vaccinations.An international priority is the scale-up and global access of an affordable and safe recombinant vaccine to prevent emerging coronavirus infections, including COVID-19. Our aspirational goal is to protect global populations at risk for this important emerging virus infection. A low-cost recombinant protein antigen expressed in


B-cell maturation antigen (BCMA)-targeting bispecific antibodies and bispecific T-cell engagers (BiTEs) redirect T-cells to BCMA-expressing multiple myeloma (MM) cells. These MM cells are subsequently eliminated via various mechanisms of action including the release of granzymes and perforins. Several phase 1, dose-escalation studies show pronounced activity of BCMA-targeting bispecific antibodies, including teclistamab, AMG420 and CC-93269, in heavily pretreated MM patients. Cytokine release syndrome is the most common adverse event, which can be adequately managed with tocilizumab or steroids. Several clinical trials are currently evaluating combination therapy with a BCMA-specific bispecific antibody, based on preclinical findings showing that immunomodulatory drugs or CD38-targeting antibodies enhance the activity of bispecific antibodies. In addition, bispecific antibodies, targeting other MM cell surface antigens , are also evaluated in early phase clinical trials. Such bispecific antibodies, targeting other antigens, may be given to patients with low baseline BCMA expression, disease with substantial heterogeneity in BCMA expression, following prior BCMA-targeted therapy, or combined with BCMA bispecific antibodies to prevent development of antigen escape. Immunotherapeutic agents are increasingly used for the treatment of both newly diagnosed and relapsed/refractory multiple myeloma (MM) patients –3. This ex vivo response. The efficacy of teclistamab was not associated with cell surface expression of BCMA or cytogenetic risk status. However, samples with a high number of T-cells and thereby a higher T-cell to tumor cell ratio had superior teclistamab-mediated lysis, when compared to samples with low numbers of T-cells or low effector: target ratio. In agreement with our results, other bispecific antibodies or bispecific T-cell engagers (BiTE) targeting BCMA also showed high anti-MM activity in preclinical models [We have recently provided the preclinical rationale for the use of the BCMAxCD3 bispecific antibody teclistamab (JNJ-7957) . Teclistl models –17.Teclistamab is currently evaluated in a first-in-man study in heavily pretreated MM. The first results show high single agent activity and a favorable toxicity profile . TeclistPreliminary data from other clinical trials also shows promising activity of BCMA-targeting bispecific antibodies and BiTEs . One of AMG420 is the first-in-class BCMA-targeting BiTE. AMG420 has a short half-life because of its small size, and therefore needs to be administered via continuous intravenous infusion during 4 weeks of each 6-week cycle [Combination therapy is often used in cancer to prevent the outgrowth of resistant clones. Because of the high single agent activity and good tolerability profile of the BCMA-targeting bispecific antibodies, these agents are ideal components of a highly effective combination therapy. We showed that the direct combination of teclistamab with the CD38-targeting antibody daratumumab enhanced MM cell lysis in an additive fashion . In addiIMiDs, such as lenalidomide and pomalidomide, also have T-cell stimulating effects . Indeed,ex vivo activity of teclistamab was not affected by presence of high-risk cytogenetic abnormalities. In addition, newly diagnosed patients, who do not achieve a deep response with a standard-of care treatment approach, may benefit from early access to a BCMA-targeting bispecific antibody with as ultimate goal the achievement of minimal residual disease-negativity.Because of the high activity and favorable toxicity profile of bispecific antibodies in end-stage MM, these agents may also be beneficial for patients with early relapsed/refractory MM. Furthermore, newly diagnosed MM patients with high-risk cytogenetic abnormalities, who have a poor prognosis, may benefit from the incorporation of a BCMA-targeting bispecific antibody into their upfront treatment strategy. This is also supported by our finding that the Although the BCMA-targeting bispecific antibodies are most advanced in terms of clinical testing, several other T-cell redirecting antibodies are evaluated in early clinical trials. This includes bispecific or trispecific antibodies targeting GPRC5D, CD38, and FcRH5 –33.low clones. Furthermore, although a case report suggests that serial treatment with BCMA-targeting agents is feasible [Bispecific antibodies targeting other MM-associated antigens may be useful in patients with low baseline BCMA expression or heterogeneous BCMA expression. In these patients treatment with a BCMA-targeting bispecific antibody may lead to rapid outgrowth of BCMAfeasible , we needBCMA-targeting bispecific antibodies are highly active in extensively pretreated patients, including those who are refractory to all available drugs. These agents have a favorable safety profile, with CRS as most common adverse event, which can be easily managed with tocilizumab or steroids. A major advantage of bispecific antibodies or BiTEs over CAR T-cells is their direct “off-the-shelf” availability, while production of CAR T-cells takes several weeks. However, treatment with bispecific antibodies is typically continued until progression, while CAR T-cells are generally given as a single infusion and consequently patients will have a treatment-free interval, which is often preferred by patients and care givers. Finally, bispecific antibodies are promising combination partners with other immune stimulating drugs such as IMiDs and CD38-targeting antibodies. Clinical trials evaluating these combinations are ongoing.


SACS gene and without a cure. Attempts to treatments are scarce and limited to symptomatic drugs.Spastic ataxia of Charlevoix-Saguenay is a neurodegenerative condition due to mutations in the SACS underwent oral supplementation (600 mg/die) with docosahexaenoic acid (DHA), a well-tolerated dietary supplement currently used in SCA38 patients. We assessed over a 20 month-period clinical progression using disease-specific rating scales.Two siblings harboring biallelic variants in SACS.DHA was safe over a long period and well-tolerated by the two patients; both showed a stabilization of clinical symptoms, rather than the expected deterioration, warranting additional investigations in patients with mutations in  SACS, which encodes sacsin, a molecular chaperone involved in protein quality control, mitochondrial network dynamics and neurofilament homeostasis. The pathological mechanisms underlying neurodegeneration in sacsin-mutated neurons are not fully understood. A recent study in neuronal models of sacsin depletion suggests that loss of the protein could selectively impair mitophagy, leading to accumulation of damaged mitochondria and subsequent bioenergetic dysfunction, increased levels of oxidative stress products, and peroxidation of membrane phospholipids [Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a relatively common early-onset inherited disorder characterized by the combination of cerebellar ataxia, pyramidal signs, peripheral neuropathy and retinal involvement . ARSACS holipids . The hypDocosahexaenoic acid (DHA) is a well-tolerated dietary supplement able to cross the blood-brain barrier. It is the main component of brain phospholipids and is essential for normal central nervous system development. DHA derives mainly from diet and liver metabolism, while production within the brain itself is limited . DHA haspes cavus and hammertoes. She scored 16/52 and 7/40 on the Spastic Paraplegia Rating Scale (SPRS) [We here report clinical observations in two siblings with ARSACS who underwent DHA oral supplementation, 600 mg per day, for 20 months. Their full clinical and molecular data have been described elsewhere . Brieflye (SPRS)  and the e (SPRS) , respecte (SPRS)  score wae (SPRS)  in 15.8 e (SPRS) . ElectroOver the 20-month period, DHA was safe and well-tolerated by the two patients; both showed an overall stabilization of clinical symptoms, as measured by SARA, SPRS, DSI-ARSACS, 6MWT and 10MWT (Table Description of pharmacological management in rare neurodegenerative conditions is useful to advice patients in case of self-administration and use of over-the-counter drugs, especially when no cures are seen over the horizon . The repDHA supplementation is associated with modifications in lipids profile and some studies suggest that DHA down-regulates expression of LDL receptors and LDL-cholesterol clearance . Therefo


Many patients with peripheral arterial disease (PAD) cannot undergo conventional medical or surgical therapy due to the extent or severity of atherosclerotic disease. Stem cell therapy has shown promising results as an angiogenic therapy in PAD patients. However, the poor survival of transplanted cells due to early immunodestruction and the inability to noninvasively monitor and track the distribution and proliferation of transplanted cells hinders stem cell therapeutic efficacy. We present here a multifunctional mesenchymal stem cell (MSC) microencapsulation and trafficking method utilizing perfluorooctylbromide (PFOB) incorporated alginate-poly-L-lysine-alginate microcapsules (PFOB Caps) for MSC delivery and noninvasive engraftment tracking using clinical X-ray and MR imaging equipment.6cells/ml) were performed by extruding a PFOB-impregnated 2% (w/v) alginate solution from a syringe pump in conjunction with an electrostatic droplet generator, followed by cross linking with poly-L-lysine to form X-ray- and MRI-visible microcapsules. MSCs viability was examined and compared between unlabeled capsules and PFOB Caps. Using 19F MRI and rotational angiograms reconstructed into CT-like images, the minimum detectable concentration was determined in phantoms using standard clinical imaging systems. X-ray delivery and tracking of intramuscular injections of PFOB Caps (~5000 capsules/injection) was assessed in a rabbit PAD model.Microencapsulation of bone marrow-derived rabbit or human MSCs . PFOB Caps containing human MSCs had enhanced cell viability relative to unlabeled capsules . Viability of human MSCs in PFOB Caps was maintained up to 100 days, while it decreased sharply to <10% in unlabeled capsules at 80 days post-encapsulation. in vivo.By adding PFOB, a dual contrast agent and oxygen carrier, to alginate microcapsules, we have demonstrated the enhanced viability of MSCs within PFOB Caps, and the ability to deliver and track engraftment of stem cells using multiple conventional clinical imaging systems


The use of immune checkpoint inhibitors (ICIs) to treat oncologic diseases is progressively increasing. Computed tomography (CT) features of ICI therapy-related pneumonitis may overlap with other diseases, including coronavirus disease 2019 (COVID-19). Thus, oncologic patients undergoing ICI therapy and developing pneumonitis are at risk of being misdiagnosed. Exploring the strengths and weaknesses of artificial intelligence in distinguishing between ICI therapy-related pneumonitis and COVID-19 is of great importance for oncologic patients and for clinicians in order to increase awareness on this topic and stimulate novel strategies aimed to promptly and correctly classify and treat this category of vulnerable patients.n = 30), a COVID-19 group (n = 34), and a group of patients with ICI therapy-related pneumonitis (n = 21). Computed tomography images were analyzed with an artificial intelligence (AI) algorithm based on a deep convolutional neural network structure. Statistical analysis included the Mann–Whitney U test (significance threshold at p < 0.05) and the receiver operating characteristic curve (ROC curve). Results: The algorithm showed low specificity in distinguishing COVID-19 from ICI therapy-related pneumonitis  = 0.62). ICI therapy-related pneumonitis was identified by the AI when compared to pneumonia-free controls . Conclusions: The deep learning algorithm is not able to distinguish between COVID-19 pneumonia and ICI therapy-related pneumonitis. Awareness must be increased among clinicians about imaging similarities between COVID-19 and ICI therapy-related pneumonitis. ICI therapy-related pneumonitis can be applied as a challenge population for cross-validation to test the robustness of AI models used to analyze interstitial pneumonias of variable etiology.Background: Coronavirus disease 2019 (COVID-19) pneumonia and immune checkpoint inhibitor (ICI) therapy-related pneumonitis share common features. The aim of this study was to determine on chest computed tomography (CT) images whether a deep convolutional neural network algorithm is able to solve the challenge of differential diagnosis between COVID-19 pneumonia and ICI therapy-related pneumonitis. Methods: We enrolled three groups: a pneumonia-free group ( SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus that was first identified in Wuhan, China, and that is responsible for a highly contagious respiratory disease named coronavirus disease 2019 (COVID-19) .Coronaviruses are composed of four major structural proteins, including the envelope (E) protein, the membrane (M) protein, the nucleocapsid (N) protein, and the spike (S) protein, the latter being of paramount importance since it mediates viral attachment to the host cell membrane receptor .Real-time reverse transcription polymerase chain reaction (RT-PCR) and next-generation sequencing methods applied to respiratory tract specimens  are considered the reference standard for the diagnosis of SARS-CoV-2 infection .Computed tomography (CT) of the chest can detect lung manifestations, which are often associated with COVID-19 ,7,8. TheRecently, the use of ICI therapy as first- and second-line treatment of different types of malignancies has rapidly grown . ICIs acInterestingly, COVID-19 pneumonia and ICI therapy-related pneumonitis have been suggested to share critical biological mechanisms, including the hyperactivation of immune cells associated with a significant increase in proinflammatory cytokines . Indeed,Distinguishing between COVID-19 pneumonia and ICI therapy-related pneumonitis is a diagnostic challenge.Artificial intelligence (AI), using deep learning technology, is highly promising in the medical imaging field due to its capability of feature extraction and analysis ,21,22. IIn the present study, given the similar biological and clinical characteristics of COVID-19 pneumonia and ICI therapy-related pneumonitis, we tested whether a deep learning algorithm is able to distinguish between COVID-19 pneumonia and ICI therapy-related pneumonitis.We designed a retrospective observational study. This study was performed in accordance with the Declaration of Helsinki. The local Ethical Committee approved the study (the ethic code is: Prot.: 88/20 OSS.NOT ComEt CBM) and waived the written informed consent for the participants.All of the subjects underwent chest CT scan and were consecutively sampled from our electronic database. In this study, three groups of patients were included and classified according to both medical history and CT imaging findings . Inclusion criteria for group selection were as it follows:COVID-19: a group of consecutive symptomatic  patients, with confirmed COVID-19 pneumonia by positive RT-PCR  on nasopharyngeal  specimen with the swab technique and positive chest CT scans acquired between 15 March and 5 April 2020.ICI therapy-related pneumonitis: a group of consecutive oncological patients with a positive history of ICI therapy-related pneumonitis and a positive chest CT scan acquired between 2017 and 2019, before the occurrence of any proven case of COVID-19 in Italy. The diagnosis of ICI therapy-related pneumonitis was clinically established based on the absence of other proven microbiological or pharmacological causes of pneumonia, full recovery after drug discontinuation, and medical therapy (mainly based on corticosteroids), and was confirmed by resolution of the findings at follow-up chest CT scan.Pneumonia-free patients (control group): a group of consecutive symptomatic  patients with a negative chest CT scan acquired between 15 March and 5 April 2020.All chest CT acquisitions were obtained by maintaining the patients in the supine position during end-inspiration, with or without contrast medium injection. Chest CT images were acquired on a Dual Source 384-slice (2 × 192) CT  80–250 mAs; spiral pitch factor: 1.8; collimation width: 0.6 mm)), on a 128-slice CT  80–250 mAs; spiral pitch factor: 1.2; collimation width: 0.6 mm)), and on a 40-slice CT ).A meticulous decontamination of the CT room and passive air exchange was conducted after every scan performed on patients with clinical or imaging suspicion of COVID-19.TM CT Lung (COVID-19) , an AI solution specifically developed for diagnosis and management support of COVID-19 pneumonia. Among its features, the algorithm module includes automated segmentation of the core features of COVID-19 lung lesions and the segmentation of the lung lobes . The output also includes the estimated risk probability for the diagnosis of COVID-19 pneumonia. The core algorithm is based on a deep convolutional neural network structure and uses the U-net network structure as the core segmentation network [n = 2191 adult patients; Wuhan Chinese COVID-19) were mixed, including all stages and clinical presentation of the disease  [3 or percentages based on automatic opacity segmentation [The artificial intelligence analysis was performed by means of InferRead network . The mod severe) . In the  severe) . The quaentation .Descriptive statistics, including means, medians, ranges, and percentiles, were calculated to understand central tendencies of the enrolled cohorts. Data distribution normality was checked by means of Kolmogorov–Smirnov test. The Kruskal–Wallis and the chi-square test were used to compare age and sex distribution among groups, respectively.This study has three objectives: (1) to assess the AI performance in identifying COVID-19 pneumonia in a population of Italian patients; (2) to test whether the AI could differentiate COVID-19 pneumonia from ICI therapy-related pneumonitis; (3) to test whether the AI could potentially be geared towards the identification and quantitative evaluation of ICI therapy-related pneumonitis.p < 0.05. Statistical Package for the Social Sciences (SPSS) software version 26.0  was applied for all the aforementioned statistical computations. Additionally, receiver operating characteristic curve (ROC curve) fitting was performed by using the maximum likelihood fit of a binormal model, and the area under the curve (AUC) was calculated with a 95% confidence interval (95% CI). Sensitivity, specificity, the positive predictive value, and the negative predictive value are presented as point estimates (95% CI).To investigate the three objectives, we performed the following comparisons: COVID-19 pneumonia vs. pneumonia-free groups, COVID-19 pneumonia vs. ICI therapy-related pneumonitis groups, and ICI therapy-related pneumonitis vs. pneumonia-free groups. COVID-19 disease risk as well as affected lobe percentages and volumes were compared between pairs of groups by using the Man– Whitney U test with a significance threshold of n = 30), patients with COVID-19 pneumonia (n = 34), and patients with ICI therapy-related pneumonitis (n = 21), used to test the AI model.p = 0.70). Patients with COVID-19 pneumonia and ICI therapy-related pneumonitis were older than pneumonia-free patients (p = 0.013). The positive SARS-CoV-2 RT-PCR was available for all patients with COVID-19 and a negative RT-PCR test was available for 14/30 (47%) pneumonia-free patients. Within the ICI therapy-related pneumonitis group of patients, non–small-cell lung cancer (NSCLC) was the most prevalent primary tumor . The diagnosis of ICI therapy-related pneumonitis occurred as a complication during immunotherapy with nivolumab , pembrolizumab , and atezolizumab . Three patients  received concomitant systemic therapy, with paclitaxel, lenvatinib or carboplatin–etoposide, respectively. Six patients  were also under long-term prednisolone in doses of 5 mg/day.The number of males was higher than females across all patient groups .The results of the comparison between COVID-19 pneumonia and ICI therapy-related pneumonitis, in terms of total and lobar involvement, are summarized in Representative CT images are shown in In this study, we assessed the performance of a deep learning algorithm, initially trained on a population of Wuhan in China, in solving the challenge of differential diagnosis between COVID-19 pneumonia and ICI therapy-related pneumonitis.The patients with RT-PCR confirmed diagnosis of COVID-19 pneumonia, showing the typical CT findings reported in several studies ,29,30,31ICI therapy-related pneumonitis is an uncommon but important complication of ICI therapy. Since the clinical manifestations of ICI therapy-related pneumonitis are nonspecific, CT plays an important role in the diagnosis of this adverse event. ICI therapy-related pneumonitis might present with several patterns that include organizing pneumonia, nonspecific interstitial pneumonia, and acute interstitial pneumonia–acute respiratory distress syndrome . These pGiven the capability of the COVID-19 algorithm to capture features shared between COVID-19 pneumonia and ICI therapy-related pneumonitis, we were able to show an excellent accuracy of the AI in distinguishing ICI therapy-related pneumonitis from pneumonia-free cases (AUC = 0.97). The result suggests that the algorithm might be geared towards the identification and quantitative evaluation of lungs affected by ICI therapy-related pneumonitis. This interesting hypothesis needs to be further verified with a larger CT dataset of ICI therapy-related pneumonitis, also given the limited sample size of the present study. Additionally, a “transfer learning” strategy, including some ICI patients’ CT data, should be tested and adapted for the partial training of algorithms. This is particularly important, as COVID-19 pneumonia cases are expected to decrease in the future and might become epidemiologically less relevant with the development and distribution of adequate vaccination therapies, whereas the number of ICI therapy-related pneumonitis is predicted to increase according to the increasing usage of ICI in cancer patients .This study has several limitations, including the small size of the samples and the variability of protocols used with the three different scanners. Other viral or bacterial pneumonias were not included in this study; thus, we cannot estimate the accuracy of the AI algorithm for the differential diagnosis with other infectious pneumonias. Moreover, the overlapping chest CT features of several diseases reflect common mechanisms of response of the lungs to different etiologies; therefore, measurements of volume, shape, or density of pulmonary lesions may not be sufficient features to develop powerful deep learning models. Lesion distribution and other radiomics-based data, including clinical information, are promising to further improve the performance of AI algorithms .In conclusion, the AI model, initially developed on the population of Wuhan, classified ICI therapy-related pneumonitis as COVID-19 pneumonias due to shared features between the two conditions, and it distinguished ICI therapy-related pneumonitis from pneumonia-free controls. Awareness must be increased among clinicians about imaging similarities between COVID-19 and ICI therapy-related pneumonitis.ICI therapy-related pneumonitis can be applied as a challenge population for cross-validation to test the robustness of the AI models used to analyze interstitial pneumonias of variable etiology.


Macrocycle-based host–guest chemistry has played an important role in the development of supramolecular chemistry. Burgeoning progress has been continuously made in the development of supramolecular assemblies by using various host molecules, such as cyclodextrins  supramolecular polymer which was constructed from a cyanostilbene based ditopic benzo-21-crown-7 and a ditopic dialkylammonium salt. In another review paper, Gatiatulin et al. summarized the possible alternatives to the classical key-to-lock principle with higher selectivity for molecular recognition. These alternatives are based on cooperativity of phase transitions, which adds up the small differences in molecular structure of different bound guests. In a minireview, Duan et al. (b) summarized broad approaches for the preparation of graphene nanomaterials functionalized with calix[n]arene/pillar[n]arene, and their applications in molecular recognition, fluorescent sensors, electrochemical biosensors, and as catalytic, antibacterial, and adsorption materials.Supramolecular polymers, a concept combining elements of both supramolecular chemistry and polymer science, are promising dynamic functional materials. Moreover, the incorporation of fluorophores into supramolecular polymers could endow them with interesting photophysical properties. In this context, An et al. focused on novel dihomooxacalix[4]arene-based anti-tumor agents. In their work, they reported the synthesis of 19 structurally related dihomooxacalix[4]arene amide derivatives in search of optimal efficacy. Guo et al. synthesized a p-tert-butyldihomooxacalix[4]arene, which could form a soft gel in cyclohexane. Moreover, the xerogel with its highly interconnected and homogeneous porous network, may be used for drug storage and controlled release. Pillararenes are a relatively new macrocyclic host and have been employed to fabricate various supramolecular materials. Macrocyclic amphiphiles have attracted much attention due to their unique properties in the construction of functional nanomaterials. The work of Wang et al. investigates a pillararene-based macrocyclic amphiphile, which is responsive to pH. Interestingly, a pH-induced transition between single-chain macrocyclic amphiphile and bola-type amphiphile and the corresponding self-assembly behavior was investigated. Self-assembled peptides can also be used to fabricate new biomaterials for medical applications. Duan et al. (a) developed cationic pillar[6]arene-modified graphene films on glassy carbon electrodes directly from graphene oxide-cationic pillar[6]arene dispersions using a pulsed electrodeposition technique. Experimental results revealed that the electrochemically reduced graphene oxide-cationic pillar[6]arene films could show a much higher electrochemical response to five purine bases than unmodified reduced graphene oxide films and bare glassy carbon electrodes. In other work, Duan, Wang, Zhang et al. designed new host-guest binding motifs based on a water-soluble pillar[6]arene dodecyl-ammonium chloride with two aromatic sulfonic acids in water. Interestingly, both of these host-guest complexes can be tuned reversibly between their complexed and decomplexed states by sequential addition of a base and an acid . Construction of polypseudorotaxanes in high-polar organic solvents is challenging owing to the weak interactions between macrocycles and axles. Su et al. prepared a novel metal-coordinated poly[2]pseudorotaxane by using pillar[5]arene, 1,4-bis(4-pyridyl pyridinium)butane, and [PdCl2(PhCN)2] in dimethyl sulfoxide.Calixarene derivatives have played an important role in developing anti-tumor agents. The contribution of Chaudhuri et al. developed a method to promote clean and environmentally friendly disinfection of phenolic substrates by employing α-cyclodextrin to affect the product distribution in chlorine dioxide-mediated decomposition of organic pollutants. Chen et al. synthesized and characterized two novel cyclic γ-peptides with hydrophobic inner surfaces, which could self-assemble into stacking nanotubes through intermolecular H-bonds and π-π interactions. Notably, the nanotubes could serve as selective water channels to transport water across the lipid membrane. Metallacycles have obtained great interest in recent years. The contribution of Wu et al. constructed two porphyrin-based organoplatinum(II) metallacycles through coordination-driven self-assembly. Interestingly, these metallacycles could be utilized as catalysts for photo-oxidization with high efficiency. Zhu et al. demonstrated a simple protocol to prepare stimuli-responsive surface-active microcrystalline cellulose particles which are able to reversibly self-assemble at a fluid interface via reversible in situ hydrophobization to stabilize stimuli-responsive Pickering emulsions.In summary, this Research Topic has highlighted how the fields of macrocyclic and supramolecular chemistry are still inextricably linked. The field has matured considerably in recent years and is now beginning to show more and more potential for real-world applications to address important issues across the sciences.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors RE.


Subchondral bone marrow lesions (BMLs) detected on MRI in knee osteoarthritis (OA) are associated with knee pain. The prevalence and progression of subchondral BMLs are increased by mechanical knee load. However, associations of subchondral BML location with weight-bearing knee pain are currently unknown. In this study, we aim to demonstrate associations of subchondral BML location and size with weight-bearing knee pain in knee OA.We analyzed 1412 and 582 varus knees from cross-sectional and longitudinal Osteoarthritis Initiative datasets, respectively. BML scores were semi-quantitatively analyzed with the MRI Osteoarthritis Knee Score for 4 subchondral regions  and subspinous region. Weight-bearing and non-weight-bearing pain scores were derived from WOMAC pain items. Correlation and negative binomial regression models were used for analysis of associations between the BML scores and pain at baseline and changes in the BML scores and changes in pain after 24-month follow-up.B = 0.08, p = 0.02. patellofemoral; B = 0.13, p = 0.01). Subanalysis revealed that greater medial femorotibial BML scores were associated with greater pain on walking and standing . Lateral patellofemoral BML scores were associated with pain on climbing, respectively . Increases or decreases over 24 months in BML score in the medial femorotibial compartment were significantly associated with increases or decreases in weight-bearing pain severity after adjusting for non-weight-bearing pain, age, sex, baseline weight-bearing pain, BMI, and BML at the other 4 joint compartments .Greater BML scores at medial femorotibial and lateral patellofemoral compartments were associated with greater weight-bearing pain scores, and statistical significance was retained after adjusting for BML scores at the other 4 joint compartments and other OA features, as well as for non-weight-bearing pain, age, sex, and body mass index (BMI) (medial femorotibial; Subchondral BML size at the medial femorotibial joint compartment was specifically associated with the severity and the change in weight-bearing pain, independent of non-weight-bearing pain, in knee OA. Specific associations of weight-bearing pain with subchondral BMLs in weight-bearing compartments of the knee indicate that BMLs in subchondral bone contribute to biomechanically induced OA pain.The online version contains supplementary material available at 10.1186/s13075-021-02422-0. Knee pain is the major source of disability and reason for hospital visits in patients with knee osteoarthritis (OA). OA knee pain is characteristically experienced during weight-bearing activity, but might also occur at rest on an unloaded joint. Weight-bearing pain indicates biomechanical mechanisms, whereas chemical or neuroplastic mechanisms might dominate in non-weight-bearing pain. Recent accumulating clinical evidence –3 indicaThe exact pathophysiology of subchondral BMLs is still under debate. Histologically, subchondral BMLs are characterized by bone marrow necrosis, trabecular abnormalities, bone marrow fibroses, edema, cellular infiltration, and vascular proliferation , 5. MicrOA structural change particularly affects weight-bearing components of the joint and mechanical knee load increases the prevalence and progression of subchondral BMLs . Knee OAThe purpose of this study was to assess the association of subchondral BML location and size with weight-bearing knee pain using the Osteoarthritis Initiative (OAI) dataset. We hypothesized that subchondral BML size at the medial femorotibial joint compartment in people with varus angulation is associated with severity of weight-bearing pain, independent of non-weight-bearing pain and the other OA-related MRI features, and that increasing subchondral BML size over time is associated with increased weight-bearing pain severity.https://nda.nih.gov/oai). The OAI provides access to the data and images from the OAI longitudinal cohort study and includes data of MRI and radiographic images and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaires [Subjects were selected from the OAI, a publicly available multi-center, longitudinal, prospective observational study of knee OA  at baseline and 24-month follow-up as knee MRI grading. Intra- or inter-rater reliabilities for subchondral BML score data used in this study were very good to excellent  . For subKnee pain was assessed at baseline and 24-month follow-up using the WOMAC questionnaires. A recent factor analysis of the five WOMAC pain questions suggested that there are two constructs within the five questions . Based op values were used for exploratory analysis; therefore, Bonferroni corrections are not used for the correlations. Negative binomial regression was performed for analysis of associations between weight-bearing or non-weight-bearing pain or total WOMAC score and scores of BML, articular cartilage, osteophytes, Hoffa’s synovitis, effusion, and medial meniscus extrusion. p < 0.05 indicated statistical significance.Statistical analyses were performed with SPSS version 26 software. Spearman’s correlation coefficients were used for associations between BML score and weight-bearing pain. Pearson’s correlation coefficients were used for associations between changes in BML scores and changes in weight-bearing pain. Spearman’s and Pearson’s correlation coefficients and the The participant characteristics in this cross-sectional and longitudinal study are shown in Table Greater BML, cartilage, osteophyte, Hoffa’s synovitis, or effusion-synovitis scores were each associated with greater weight-bearing pain adjusted for non-weight-bearing pain, age, sex, and BMI . Only asGreater BML, cartilage, osteophyte, Hoffa’s synovitis, or effusion-synovitis scores were each also associated with greater non-weight-bearing pain adjusted for weight-bearing pain, age, sex, and BMI Table . HoweverChanges from baseline to 24-month follow-up of variables in the longitudinal study are shown in Table B = 0.15  p = 0.33).No significant longitudinal association between change in medial meniscus extrusion score and change in non-weight-bearing pain was found after adjusting for sex, baseline non-weight-bearing pain, age, BMI and medial meniscus extrusion score, and change in weight-bearing pain , was associated with knee OA pain, not dependent on chondropathy and synovitis .In this study, subchondral BMLs at the lateral patellofemoral joint compartment were also associated with weight-bearing pain, especially pain on climbing. The associations of patellar BMLs with any knee pain and with pain when going up or down stairs have been reported . The patSubchondral BMLs represent regions of subchondral bone characterized as displaying increased bone turnover, and expression of factors, including pro-inflammatory cytokines, that can increase nerve sensitization . We prevOur results suggested that subchondral BMLs might mediate mechanically induced pain such as during weight-bearing. Biomechanical factors may reciprocally contribute to the pathogenesis of subchondral BMLs. Previous studies  reportedBiomechanical unloading by patellofemoral bracing reduced subchondral BML volume at patellofemoral joint compartments in patients with painful patellofemoral OA . HoweverPharmacotherapy also has potential to reduce subchondral BMLs. The bisphosphonate zoledronic acid reduced subchondral BML size and knee pain in people with OA . HoweverSubchondral BMLs are also found in healthy pain-free young-middle aged adults, with a prevalence of 13–17% , 29, 30,In this study, medial meniscus extrusion score was significantly associated with non-weight-bearing pain severity after adjusting for weight-bearing pain and the other OA-related MRI changes. A recent study reported that patients with medial submeniscal flap tear complained of pains during sleep, but not during daytime activities . Pain asNot only subchondral BMLs, but also synovitis , effusioOur study has several potential limitations. We investigated only 2 time points (baseline and 24 months). A previous study using OAI dataset demonstrated that changes in total subchondral BML volume after 24 months were positively associated with changes in knee pain severity . HoweverWe demonstrated that subchondral BML size at the medial femorotibial joint compartment in varus knee OA was associated with weight-bearing pain and that this association was specific for weight-bearing rather than non-weight-bearing pain and was over and above any effects of other OA-related MRI features, age, sex, and BMI. Our findings suggest that specific association of weight-bearing pain with subchondral BMLs in weight-bearing compartments of the knee indicates that subchondral BMLs aggravate biomechanical factors leading to OA pain.Additional file 1.Additional file 2.


Exosomes are extracellular vesicles (EV) of endosomal origin  constitutively released by many different eukaryotic cells by fusion of MVB to the plasma membrane. However, inducible exosome secretion controlled by cell surface receptors is restricted to very few cell types and a limited number of cell surface receptors. Among these, exosome secretion is induced in T lymphocytes and B lymphocytes when stimulated at the immune synapse (IS) via T-cell receptors (TCR) and B-cell receptors (BCR), respectively. IS formation by T and B lymphocytes constitutes a crucial event involved in antigen-specific, cellular, and humoral immune responses. Upon IS formation by T and B lymphocytes with antigen-presenting cells (APC), the convergence of MVB towards the microtubule organization center (MTOC), and MTOC polarization to the IS, are involved in polarized exosome secretion at the synaptic cleft. This specialized mechanism provides the immune system with a finely-tuned strategy to increase the specificity and efficiency of crucial secretory effector functions of B and T lymphocytes. As inducible exosome secretion by antigen-receptors is a critical and unique feature of the immune system this review considers the study of the traffic events leading to polarized exosome secretion at the IS and some of their biological consequences. Pr. Pr+ CTLCTL-target cell IS induces the rapid polarization (from seconds to few minutes) of CTL MTOC and lytic granules (secretory granules or secretory lysosomes-SL-with MVB structure) towards the cSMAC at the IS . Lytic gAnother consequence of degranulation is ILV secretion as nanosize EV at the CTL-target cell synaptic cleft, first described by Peters et al. . Althoug+ and CD8+ lymphocytes) with activation agonists produced non-directional secretion of nanosize EV (quoted as microvesicles) carrying pro-apoptotic FasL and Apo2L [Subsequent publications demonstrated that T lymphocyte stimulation of T lymphoblasts  compartments, involved in antigen processing and antigenic peptide binding to MHC-II molecules [+ compartments [+ exosome secretion [Once BCR recognizes the antigen attached to the surface of specialized APC such as follicular dendritic cells (FDC), DC or macrophages, cell polarity is immediately established upon IS formation that seems to allow B lymphocytes to rapidly focus their antigen-extraction machinery to efficiently capture antigen . B lympholecules   towards the F-actin-poor cSMAC. MHC-II+ compartment and SL degranulation at the cSMAC allows localized secretion of lysosomal hydrolases [+ exosomes attached to the FDC surface. As FDC do not express MHC-II at the cell surface [+ compartments degranulation concomitantly with antigen extraction. Supporting this possibility, purified B lymphocyte-derived exosomes can ex vivo efficiently bind to FDC [+ compartment degranulation at the B lymphocyte-FDC IS) will be necessary to formally demonstrate the B cell origin of these exosomes and to study the potential contribution of the exosomal proteases [Remarkably, upon BCR binding to the antigen at the IS formed with APC such as FDC, B lymphocytes quickly polarize their MTOC, together with MHC-IIdrolases ,90, provdrolases ,90. Subsdrolases ,91  can induce further and maintained stimulation of antigen-specific Th lymphocytes [Non-stimulated B lymphocyte-derived pMHC-II T cells . On the phocytes . The facphocytes . Thus, aphocytes ,107, andphocytes ,108 or mphocytes . FDC canphocytes .+ T lymphocytes, CD8+ CTL, NK cells) and DC in the in vivo CTL immune response to exosomal antigen presented on B-cell derived exosomes have been described [Interestingly, a complex role of the different lymphocyte subsets , it has been described that B-lymphocyte derived exosomes may have immunoregulatory functions which are independent of their ability to present antigen as nucletotidase enzymatic activity present on B lymphocyte-derived exosomes (see below) facilitates CTLs function suppression .+ exosomes/EV able to trigger Fas-induced apoptosis were first described as FasL+ APO2L+ microvesicles secreted upon activation agonist-stimulation of T lymphocytes [+ Jurkat cells [+ T lymphocyte-produced exosomes may have an unsuspected antigen-specific immunoregulatory role in vivo, although this has not been formally demonstrated yet.AICD  is a prophocytes ,12, unvephocytes ,68, to bat cells , suggest+CD4+ lymphocytes, supports an in vivo immunoregulatory role of FasL+ exosomes, although the cellular origin of these plasma exosomes was not established [+ T-lymphocytes from ovalbumin (OVA)-specific, TCR-transgenic OT-I mice produced exosomes bearing OVA-specific TCR and FasL. These exosomes bound to DC via pMHC-I/TCR and CD54/LFA-1 interactions, lead to pMHC-I expression downregulation and ex vivo FasL/Fas interaction-dependent DC apoptosis [+ T-lymphocytes from OVA-specific, TCR-transgenic OT-II mice produced exosomes bearing OVA-specific TCR and FasL, that also bound to DC via pMHC-I/TCR and CD54/LFA-1 interactions, leading to in vivo CD8+ CTL response inhibition, although it was not established whether this inhibition was due to FasL/Fas interaction-mediated apoptosis induction [+ and CD8+ T lymphocyte-derived exosomes were described in TCR transgenic mice. Thus, further experiments are necessary to extend the results to a more physiologic scenario.The presence of exosomes (carrying FasL in one of the reports) in human plasma, capable of ex vivo induce Fas-dependent apoptosis of Fasablished ,117. In poptosis . In addinduction . These i+ and CD8+ T lymphocytes bearing FasL appear to be capable of inducing apoptosis and/or regulate the functions of other immune cells in an antigen-specific manner. Supporting these findings, it has been shown that both ex vivo AICD and CTL-mediated cytotoxicity were decreased in T lymphoblasts from mice lacking PKD2, correlating with TCR-stimulated exosome secretion inhibition [Considering all these results, exosomes produced by CD4hibition .+ lymphocytes without the presence of antigen [+ lymphocytes through activated CTL-derived exosomes [More recently, it has been demonstrated by using OVA-specific, TCR-transgenic OT-I mice that full stimulation of murine CTL with IL-12 induces functionally distinct CTL-derived exosomes, which can thereby activate naïve, bystander CD8 antigen . In addi antigen . Thus, Cexosomes , which mexosomes .T regulatory cells (Treg) are important immunoregulatory T lymphocytes involved in the prevention of inflammatory damage produced after infection, autoimmunity, or allergy , and hav+ and CD8+ T lymphocytes and Treg, can modulate immune responses via exosomes. Thus it was hypothesized that exosomes may be useful for the treatment of certain autoimmune diseases [All these results open the possibility that antigen-specific CD4diseases ,119. In diseases . The clidiseases ,129.Many recent reviews dealing with the potential use of exosomes produced by different cell types as biomarkers of several pathologies and therapies have been published ,131,132,Although the exact content of T lymphocyte-derived exosomes and microvesicles remains to be determined (subheading 1.1), in the initial studies it was hypothesized that some of the molecules present in exosomes may provide these EV with important biological functions. The potential effector functions of both B and T lymphocyte-derived exosomes most probably include antigenic specificity since they display either pMHC-II capable of activating antigen-specific Th lymphocytes (B lymphocyte-produced exosomes) or specific TCR capable of recognizing pMHC (T lymphocyte-derived exosomes). For instance, it was early suggested that TCR/CD3 on T-lymphocyte-derived exosomes ,68 would+ T lymphocytes are a major cell population in the inflammatory infiltrate of rheumatoid arthritis patient joints. CD4+ T lymphocytes co-cultured with these exosomes, develop apoptosis resistance, and blockade of exosomal TNFα may lead to partial restoration of CD4+ T lymphocyte AICD [The presence of biologically active molecules, such as TNFα, in exosomes derived from bystander cells different to T and B lymphocytes, might also affect the immune response, and several TNF family proteins have been identified on exosomes. It is interesting to remark that exosomes produced by synovial fibroblasts from patients developing rheumatoid arthritis  contain membrane-bound TNFα  CD4+ T lyte AICD , amelioryte AICD . Howeveryte AICD . Synoviayte AICD . On the yte AICD , providiIn the same context of autoimmune diseases, it has been shown that circulating exosomes from patients suffering from systemic lupus erythematosus (SLE) induce a pro-inflammatory immune response. SLE exosomes elicit a significant inflammatory response (pro-inflammatory cytokines) on peripheral blood lymphocytes (PBL) in a Toll-like receptor (TLR)-dependent manner . AlthougIn summary, several pieces of evidence indicate that exosomes, not only the ones produced by T and B lymphocytes, play important roles in the immunomodulation and are associated with the immune pathogenesis of autoimmune diseases, including rheumatoid arthritis, Sjogren’s syndrome, systemic lupus erythematosus , and sys+ T lymphocyte responses [Recently it has been described that B lymphocyte-derived exosomes may have immunoregulatory functions that are independent of their ability to present antigen since certain enzymatic activities of B lymphocyte-derived exosomes facilitate the suppression of CTL function . It has esponses ,141. Silesponses ,141. ThuDuring the last 15 years, exosomes have been studied as new biological entities involved in intercellular communication during a multitude of physiological and pathological processes, in particular in the context of the immune system. In addition, cumulative pieces of evidence demonstrate that exosomes constitute promising biomarkers for both diagnosis and prognosis in several relevant pathologies . However, it is necessary to improve the existing experimental approaches, but also the characterization techniques, to study exosomes, to surely establish the role of exosomes in these pathologies and to distinguish exosome functions from other EV functions. In this context, the development of existing, but also new, nanotechnology and imaging tools for the analysis and characterization of exosomes will indeed allow addressing the contribution of exosomes to these pathologies. In addition, a better understanding of the signals involved in MVB maturation and traffic will allow designing strategies to modulate exosome secretion and hence modify their function. Cell-free, exosome-based immunotherapeutic strategies have indeed a promising future.


Rhizoctonia solani is an important phytopathogenic fungus with a wide host range and worldwide distribution. The anastomosis group AG1 IA of R. solani has been identified as the predominant causal agent of rice sheath blight, one of the most devastating diseases of crop plants. As a necrotrophic pathogen, R. solani exhibits many characteristics different from biotrophic and hemi-biotrophic pathogens during co-evolutionary interaction with host plants. Various types of secondary metabolites, carbohydrate-active enzymes, secreted proteins and effectors have been revealed to be essential pathogenicity factors in R. solani. Meanwhile, reactive oxygen species, phytohormone signaling, transcription factors and many other defense-associated genes have been identified to contribute to sheath blight resistance in rice. Here, we summarize the recent advances in studies on molecular interactions between rice and R. solani. Based on knowledge of rice-R. solani interactions and sheath blight resistance QTLs, multiple effective strategies have been developed to generate rice cultivars with enhanced sheath blight resistance. Rhizoctonia solani Kühn is considered as one of the most devastating rice diseases worldwide  caused by the necrotrophic pathogen R. solani, quantitative trait loci (QTL) and genetic regions for RSB resistance, and integrative management methods have been well reviewed recently  with high genetic diversity based on their compatibility for hyphal fusion with known tester isolates  at the early and transient establishment stage of infection, while others induce cell death at the late necrotrophic stage of infection and ultimately contribute to pathogen virulence  proteins deacetylate chitin oligomers into ligand-inactive chitosan, and thereby preventing chitin-triggered immunity and promoting virulence  family of putative chitin-binding proteins similar to Cladosporum fulvum Avr4 and Ecp6, which suppress chitin-triggered immunity through blocking the release of chitin from fungal cell walls by plant chitinases and competing with CEBiP for binding of chitin oligosaccharides, respectively , is the second most abundant polysaccharide on earth and is the major component of fungal cell walls. As a major pathogen-associated molecular pattern (PAMP) in rice, chitin is recognized by the pattern recognition receptor (PRR) CEBiP and coreceptor OsCERK1 on plant cell membrane, and thus initiating immune responses , 3-methylthiopropionic acid (MTPA), phenylacetic acid (PAA) and its derivatives  to cause pH instability and cell wall degradation -one (DIBOA)-glucoside, while inhibit the generation of the jasmonic acid (JA) precursors 3-oxo-2-(2’-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) and 12-oxo-phytodienoic acid (OPDA) -encoding genes involving in the biosynthesis of primary and secondary metabolites including phytotoxins are differentially regulated during R. solani are categorized into seven major families including PG, endo-β- 1,4-glucanase, polymethylgalacturonase, pectin methylesterase, filter paper enzyme, polymethylgalacturonase trans-eliminas and pectin methyltrans-eliminase , particularly including cell wall-degrading enzymes (CWDEs) , glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs) and non-catalytic CBMs are the important CAZymes involving in the degradation of host cell walls and in To counteract the effects from pathogenicity factors in plant pathogens, plants develop multiple layers of defenses against pathogen attacks. During pathogen infection, PAMPs are recognized by plant PRRs, and thus triggering PTI. As the first layer of plant defense, PTI responses include the activation of defense gene expression and mitogen-activated protein kinase (MAPK) cascades, reactive oxygen species (ROS) burst, accumulation of secondary metabolites and defense-related phytohormone signaling pathways -mediated biosynthesis of phenolic compounds including lignin in the shikimate pathway, and thereby improves plant antioxidant capacity and thickens cell walls  is highly susceptible to R. solani AG8, while the transgenic plants overexpressing ethylene response factors (ERFs) or the ethylene biosynthesis enzyme ACS2 exhibit resistance to R. solani  encoding transport inhibitor response 1 (TIR1)-like protein. The siR109944-overexpressing rice plants have a significantly enhanced susceptibility to R. solani, while FBL55-overexpression causes the transgenic plants to be more resistant to RSB  in rice through binding to its promoter  family members differentially regulate the expression of various defense genes and are involved in modulating resistance to 2+ regulates plant physiology and metabolism and plays an important role in plant disease resistance  specifically recognize and bind to fungal PGs, and thereby inhibit the cell wall degradation activity and prevent fungal infection  homologues, RPMK1-1 and RPMK1-2 in R. solani. The transgenic rice plants show an increased resistance to RSB  between hosts and pathogens opens an avenue to develop disease resistant transgenic plants producing sRNAs and double-stranded RNAs, which can silence fungal pathogenicity genes  and simultaneous overexpression of OsOXO4 and the chitinase gene OsCHI11 driven by green tissue-specific promoters both significantly confer enhanced and durable resistance to sheath blight  in rice as a common consequence of many abiotic and biotic stresses. The transgenic rice plants overexpressing glyoxalase for MG detoxification have been demonstrated to have much less accumulation of MG and enhanced resistance towards damage caused by PAA. The finding provides another transgenic technology to develop RSB resistant rice plants  was first identified in Arabidopsis to be a master regulator of systemic acquired resistance, which confers broad-spectrum resistance to various pathogens  and three QTLs  has higher levels of resistance than a single QTL (Wang et al. Multiple QTLs for RSB resistance have been detected in several resistant varieties, including Teqing, Jasmine 85, Zhaiyeqing 8, Xiangzaoxian 19, Tetep and Pecos (Datta et al. 8 recombinant inbred lines of two rice varieties from the United States, which are allelic to the QTLs for plant height and heading date (Goad et al. qRlh11, qSBR11-3, qSBR11-1, qSBR9-1, qShB3-2 and qSB-9 (Hossain et al. Sheath blight resistance QTLs are often mapped to the same region as the QTLs for heading date and plant height (Pinson et al. R. solani and host plants reveals many pathogenicity factors in R. solani and defense-associated genes in rice. Accordingly, the effective strategies to develop RSB resistant germplasms include disarming essential pathogenicity factors in R. solani via host-derived RNAi and transgenic technology, manipulating expression of plant defense-associated genes, and pyramiding the RSB resistance QTLs.Sheath blight has become one of the most important diseases in rice. Genetic resistance has been well considered as the most economic, effective and environmentally friendly strategy to control crop diseases. However, no RSB resistance gene has been isolated and identified. Increasing knowledge on molecular interaction between


Can the favorable properties of an ultrasmall fluorescent core-shell silica nanoparticle aid real-time image-guided detection, localization, and surgical management of sentinel lymph nodes (SLNs) in patients with head and neck melanoma?In this nonrandomized clinical trial of 24 patients, real-time, particle-based fluorescence imaging of SLNs was feasible and safe at the microdosing level and enabled deep-tissue nodal detection. There was high concordance in identifying nodes between preoperative lymphoscintigraphy and particle-based fluorescence-guided biopsy.The findings of this study suggest that ultrabright, optical contrast–conferring particles for SLN identification hold promise for overcoming current probe limitations and improving surgical outcomes. This nonrandomized clinical trial evaluates whether an ultrasmall, molecularly targeted core-shell silica nanoparticle can safely and reliably identify optically avid SLNs in head and neck melanoma during fluorescence-guided biopsy. Sentinel lymph node (SLN) mapping agents approved for current surgical practice lack sufficient brightness and target specificity for high-contrast, sensitive nodal visualization.To evaluate whether an ultrasmall, molecularly targeted core-shell silica nanoparticle (Cornell prime dots) can safely and reliably identify optically avid SLNs in head and neck melanoma during fluorescence-guided biopsy.This nonrandomized clinical trial enrolled patients aged 18 years or older with histologically confirmed melanoma in whom SLN mapping was indicated. Exclusion criteria included known pregnancy, breast-feeding, or medical illness unrelated to the tumor. The trial was conducted between February 2015 and March 2018 at Memorial Sloan Kettering Cancer Center, with postoperative follow-up of 2 years. Data analysis was conducted from February 2015 to March 2018.Patients received standard-of-care technetium Tc 99m sulfur colloid followed by a microdose administration of integrin-targeting, dye-encapsulated nanoparticles, surface modified with polyethylene glycol chains and cyclic arginine-glycine–aspartic acid–tyrosine peptides (cRGDY-PEG-Cy5.5-nanoparticles) intradermally.The primary end points were safety, procedural feasibility, lowest particle dose and volume for maximizing nodal fluorescence signal, and proportion of nodes identified by technetium Tc 99m sulfur colloid that were optically visualized by cRGDY-PEG-Cy5.5-nanoparticles. Secondary end points included proportion of patients in whom the surgical approach or extent of dissection was altered because of nodal visualization.Of 24 consecutive patients enrolled , 18 (75%) were men. In 24 surgical procedures, 40 SLNs were excised. Preoperative localization of SLNs with technetium Tc 99m sulfur colloid was followed by particle dose-escalation studies, yielding optimized doses and volumes of 2 nmol and 0.4 mL, respectively, and maximum SLN signal-to-background ratios of 40. No adverse events were observed. The concordance rate of evaluable SLNs by technetium Tc 99m sulfur colloid and cRGDY-PEG-Cy5.5-nanoparticles was 90% , 5 of which were metastatic. Ultrabright nanoparticle fluorescence enabled high-sensitivity SLN visualization (including difficult-to-access anatomic sites), deep tissue imaging, and, in some instances, detection through intact skin, thereby facilitating intraoperative identification without extensive dissection of adjacent normal tissue or nerves.This study found that nanoparticle-based fluorescence-guided SLN biopsy in head and neck melanoma was feasible and safe. This technology holds promise for improving lymphatic mapping and SLN biopsy procedures, while potentially mitigating procedural risks. This study serves as a first step toward developing new multimodal approaches for perioperative care.NCT02106598ClinicalTrials.gov Identifier:  The few approved SLN mapping agents, such as technetium Tc 99m sulfur colloid, technetium Tc 99m tilmanocept,7 and blue dye, lack a strong optical signal to guide real-time intraoperative identification, and they are limited by nonspecific high macrophage uptake and what is known as shine-through if SLNs are close to the primary lesion.5 Lymphoscintigraphy with technetium Tc 99m is also subject to poor spatial resolution (approximately 10 mm) and, therefore, imprecise SLN localization.8 Collectively, these considerations highlight a critical need to advance the adoption of new optical imaging probes that generate high-contrast, reliably localized SLNs and identify nodal metastases while potentially enabling real-time tailoring of surgical approach and reducing the extent of surgical dissection, duration of anesthesia time, and risk of nerve injury.Since the implementation of SLN biopsy procedures for early-stage melanoma,13 The superior brightness and photostability of fluorescent nanoparticles relative to free dyes is the result of encapsulating near-infrared (NIR) dyes  within its silica core matrix.16 In addition, as αν integrins are known to be highly expressed on the surface of neoangiogenic endothelial and melanoma cells, we functionalized the particle surface with multiple integrin-targeting peptides  via polyethylene glycol chains (PEGs) to create cRGDY-PEG-Cy5.5-nanoparticles.17 Relative to native monovalent peptides, the multivalent nanoparticle platform shows enhanced cellular binding and internalization.18To address these issues, we developed and clinically translated a first-in-kind ultrasmall (sub–8-nm diameter) integrin-targeting, ultrabright, fluorescent core-shell silica nanoparticles (Cornell prime dots).In this study, we present the results of a phase 1/2a particle-driven SLN mapping trial in patients with head and neck melanoma, an orphan disease, using cRGDY-PEG-Cy5.5-nanoparticles and a handheld NIR fluorescence camera system  for accurate intraoperative detection and high-contrast visualization of SLNs, which had been localized preoperatively with standard-of-care technetium Tc 99m sulfur colloid. The particle probe described is, to the best of our knowledge, the first inorganic renally clearable and optically active platform in the United States to have reached clinical trials in patients with melanoma. Primary end points aimed to assess particle safety, procedural feasibility, the particle dose and volume that maximize nodal fluorescence signal and image contrast, and the proportion of nodes identified by technetium Tc 99m sulfur colloid that were optically visualized by cRGDY-PEG-Cy5.5-nanoparticles . Secondary study end points included time from injection to SLN visualization, the proportion of patients in whom the surgical approach or extent of dissection was subjectively altered, risk of reduced nerve injury, and shortened duration of anesthesia because of improved SLN visualization. The distinct optical and structural properties of nanoparticles suggested that key limitations of technetium Tc 99m sulfur colloid could potentially be overcome and that the proportion of excised SLNs identified by technetium Tc 99m sulfur colloid may be concordant with that visualized by nanoparticles.TREND) reporting guideline.This clinical trial was approved by the institutional review board at the Memorial Sloan Kettering Cancer Center (MSKCC) and cleared by the US Food and Drug Administration, with physician-sponsored Investigational New Drug 121544. The trial protocol appears in 19 Participants were clinically node-negative by history, physical examination, and ultrasonographic evaluation.20 Patient exclusion criteria included known pregnancy, breast-feeding, or uncontrolled intercurrent medical illness unrelated to the tumor which, in the opinion of the attending staff, contraindicated administering the study agent. All patients were followed up during a 2-year period.Eligibility criteria included being aged 18 years or older with a histologically confirmed diagnosis of cutaneous melanoma in which SLN mapping was indicated.Given that this was a pilot trial to determine whether the preclinical experience and observations would translate to the operating room, sample size was not chosen based on power calculations but rather on estimating concordance between technetium Tc 99m sulfur colloid and cRGDY-PEG-Cy5.5-nanoparticles. With 31 patients, the expected half-length of a 95% CI, assuming 90% concordance, was 12%.11 They were purified using gel permeation chromatography, sterile processed, and characterized by fluorescence correlation spectroscopy and UV-visible spectroscopy  were produced using a previously described 1-pot synthesis approach  . A primary efficacy end point was the patient concordance rate, ie, the proportion of nodes successfully identified by preoperative technetium Tc 99m sulfur colloid that were optically visualized by cRGDY-PEG-Cy5.5-nanoparticles. The proportion of nodes that were detected only by technetium Tc 99m sulfur colloid alone or by cRGDY-PEG-Cy5.5-nanoparticles alone was also evaluated. Concordance between technetium Tc 99m sulfur colloid and cRGDY-PEG-Cy5.5-nanoparticles was evaluated using 95% exact binomial confidence intervals. Statistical analysis was conducted in R version 3.6 . Statistical significance was set at 19  enrolled from February 2015 to March 2018, 18 (75%) were men . Most paAlthough 40 SLNs underwent histopathological evaluation, 9 nodes were precluded from establishing whether the proportion of lymph nodes identified by technetium Tc 99m sulfur colloid was equivalent to that for cRGDY-PEG-Cy5.5-nanoparticle visualization because of technical factors relating to early dose-optimization efforts (cases 1 and 2) and camera malfunction (cases 11 and 13)  cases of ex vivo SLNs . PatienteTable 3 in Representative cases are shown in 21 Head and neck melanoma is a life-threatening orphan disease with 56.1% survival at 10 years.22 As the presence of metastases in regional lymph nodes is a vital prognostic factor for melanoma, the NCCN recommends wide excision and SLN biopsy for patients with stage IB or II disease.24 However, using standard methods for SLN mapping, false-negative rates range from 0% to 34% , with rates higher than those for non–head and neck melanomas.26Malignant melanoma is among the fastest rising cancers in the United States, with approximately 100 350 new cases diagnosed in 2020, resulting in 6850 deaths.28 Radioactivity guidance alone may be adequate for SLN identification in the groin or axilla where the lymphatic drainage is not as complex as in the head and neck; in the latter case, intraoperative visual guidance is extremely helpful. Importantly, gamma probes used intraoperatively to localize radiotracer accumulation often lack sufficient spatial resolution to discriminate SLNs, particularly deeper-lying nodes and/or those close to the primary tumor.31 Moreover, the very small size of many nodes  makes them difficult to identify exclusively by radio-guided surgery.Current SLN practice for head and neck melanoma presents several unique challenges including (1) complex lymphatic drainage with multiple primary channels and SLN sites; (2) technical difficulties associated with nodal excision, given that short distances between primary lesion and SLNs can make detection and isolation difficult; (3) high risk of facial nerve injury, given that 25% to 30% of SLNs are found within the parotid gland area; and (4) lack of reliable, optical visualization for discriminating SLNs from adjacent fibrofatty tissue intraoperatively.32 and that SLNs are less intensely stained than those in other body regions.33 Other known limitations include needing to dissect overlying soft tissue to directly visualize the blue node36 and risk of anaphylactoid reactions and permanent tattoos. NIR fluorescent dyes, such as indocyanine green, have also been used as an adjunct to radioguidance38 or as a hybrid fluorescent-radioactive tracer39 but have provided only incremental benefit over conventional radio-guided localization.42Some of the limitations of radio-guided SLN localization have been addressed, in part, by traditional use of vital dyes , but these dyes are not as sensitive as the radiotracer technique in the head and neck, with reports indicating inconsistent drainage pathways that create challenges in tracing blue channels to SLNs44 including a phase 1 first-in-human positron-emission tomography (PET) study.43 In an M21 human melanoma xenograft model, particle radiolabeling with zirconium 89 (89Zr)45 and iodine 124 (124I)17 yielded a renally clearable, dual modality  imaging tracer showing target-or-clear detection capabilities and high TB ratios. Relatively uniform particle tracer distribution and retention were also observed at sites of disease.45 In addition, higher sensitivity and specificity were found for radioiodinated nanoparticles compared with the standard-of-care radiopharmaceutical 18F fluorodeoxyglucose (18F-FDG) in a spontaneous melanoma miniswine model.5 In miniswine, PET-avid metastatic SLNs not detected by 18F-FDG were identified following injection of the dual-modality product around the primary lesion.44 More recently, fluorescence-based multiplexing in the same model highlighted the excellent optical imaging capabilities of dual-modality cRGDY-PEG-Cy5.5-nanoparticles for localizing metastatic nodes with varying tumor burden as well as for assessing tumor heterogeneity.48 Its high brightness yielded pronounced tumor-bearing node-to–normal tissue contrast well below the microdosing level . Using a high-resolution  multichannel fluorescence camera system, SLNs bearing micrometastases  could also be detected, underscoring the benefit of combining such a high-resolution, high-sensitivity real-time intraoperative imaging strategy with preoperative PET imaging. Importantly, both PET and optical imaging parameters  were found to be strongly correlated with nodal tumor burden.46To address these limitations, we developed a targeted Cy5.5 dye-encapsulating silica nanoparticle with an approximately 6-mm diameter (cRGDY-PEG-Cy5.5-nanoparticles), which exhibited favorable physicochemical and biological properties for image-guided surgical applications. Translation of this product to the clinic for fluorescence-guided SLN biopsy in head and neck melanoma patients was based on extensive in vivo biological and safety data,The results of our pilot study show that the use of cRGDY-PEG-Cy5.5-nanoparticles, in conjunction with real-time intraoperative optical imaging guidance, is a safe, reliable, and clinically feasible procedure for the accurate and sensitive detection of SLNs in patients with head and neck melanoma. Our findings suggest that this particle-based technology overcomes a number of existing limitations associated with currently approved SLN contrast agents, ie, radiolabeled SC or tilmanocept, blue dye, and indocyanine green, while achieving clinically meaningful end points, including (1) favorable safety profile with high-contrast visualization using very low nanomole dosing regimens; (2) high concordance rate with technetium Tc 99m sulfur colloid, and (3) the ability to tailor surgical approach and/or reduce the extent of planned tissue dissection while (4) minimizing risk of nerve injury within the field of surgical dissection.Based on optimization of the nanoparticle dosing strategy, we were able to obtain very high TB ratios of up to 40 at the low (nanomole) doses used. This level of contrast is not achievable with small-molecule dyes, such as indocyanine green or blue dye, which require substantially higher doses  for nodal detection. Unlike the ultrabright Cy5.5 dye-encapsulating nanoparticles, which can be optically detected as deep as approximately 2 cm below the skin, the indocyanine green signal is weakly intense at any significant depth, with limited penetration of only several millimeters, and has rapid extravasation into surrounding tissues, with marked reductions in tissue contrast.49 although this same trial showed a slightly lower disease-free survival and an increased rate of regional disease control in patients who underwent completion lymphadenectomy. These findings underscore the importance of sensitively identifying and removing metastatic nodes with less invasive and more focused procedures.Importantly, while we did observe lower optical signal in many benign SLNs vs the few metastatic SLNs, larger sample sizes would be needed to establish the statistical significance of this finding and to establish disease-burden thresholds for detectability. Development of tumor-specific probes that can selectively target melanoma is a highly desirable goal given the results of the second Multicenter Selective Lymphadenectomy Trial, in which no difference in melanoma-specific survival was found between the group of patients who had a positive SLN and underwent completion lymphadenectomy and those who had a positive SLN and underwent observation. Completion lymphadenectomy is no longer routinely recommended,It is expected that SLNs should accumulate cRGDY-PEG-Cy5.5-nanoparticles in a time-dependent manner because of mechanical inflow of the tumor-draining lymphatics, acknowledging that specific binding to cancerous cells within the node will depend on a number of factors that include binding affinity, extent of localization, and whether enough time has elapsed for washout of unbound particles from the node. While integrin-targeted nanoparticles may not be entirely tumor-specific , this remains an area of active investigation in our studies as we attempt to determine the association between nodal localization of nanoparticles in patients with head and neck melanoma and nodal tumor burden.99mTc lymphoscintigraphy. High concordance (90%) was found between the proportion of ex vivo SLNs identified by technetium Tc 99m sulfur colloid and that optically visualized by cRGDY-PEG-Cy5.5-nanoparticles following early optimization procedures. Importantly, in 1 case, the SLN could not be detected in situ using technetium Tc 99m sulfur colloid because its location made it difficult to use a gamma probe, but it was able to be visualized optically using the particles. In all patients, nanoparticles led to high conspicuity of the draining tumor lymphatics and SLNs. This visual aid was considered a significant improvement by the surgeon compared with conventional radiocolloid-based gamma probe detection methods that are strictly based on count rate–related audible signals.A primary end point of the study rested on the need for accurate intraoperative detection of SLNs identified on preoperative 99mTc dissection alone. The latter technique also poses significant challenges because the SLN cannot always be easily identified with a gamma probe, leading to an often lengthy surgical procedure. For the head and neck region especially, such extensive dissections can result in an increased risk of significant morbidities for these patients. In some of our other cases, the extent of the initial skin incision and subsequent tissue dissection was reduced given the more superficial location of optically identifiable SLNs that could be visualized through the intact skin prior to incision. For procedures in which the SLNs could be detected and removed, we estimated 30% to 50% reductions in the duration of the surgery using cRGDY-PEG-Cy5.5-nanoparticles as compared with technetium Tc 99m sulfur colloid alone. This range reflected variations in the type of procedure, depth of nodes from the skin surface, and the need for nerve dissection, in addition to other technical factors, such as equipment setup.Visual cues provided by nanoparticles also led to alterations in surgical approach in several cases. With detection of optical signal due to depth penetration beyond 1 cm, optically bright nodes within the parotid gland could be resected based on their location without first identifying and dissecting the main trunk or divisions of the facial nerve. Thus, this nanoparticle-based image-guided surgical procedure was beneficial in terms of reducing the extent of dissection and operative time under anesthesia that otherwise would have been needed using gamma probe–aided Our results set the stage for a larger randomized clinical trial that would overcome the aforementioned limitations in addition to facilitating comparisons between the conventional paradigm of preoperative lymphoscintigraphy with strategies that harness the all-in-one dual-modality imaging capabilities of these nanoparticles. Such a design could potentially aid preoperative treatment planning, intraoperative SLN visualization, and in the future, multiplexed detection of more than 1 marker while limiting extensive normal tissue resection. An important additional end point would include the correlation of the optical signal of ex vivo SLNs with nodal metastatic tumor burden to assess the ability of the technology to discriminate metastatic from nonmetastatic nodes. Importantly, this technology could also be applicable across several surgical fields in which SLN biopsy has been used, such as breast cancer, prostate cancer, gastric cancer, colorectal cancer, and non–small cell lung cancer.This study has limitations. The study design could not quantitatively correlate gamma counts with optical signal as part of the nodal mapping procedure. Gamma counting is semiquantitative, at best, due to coarse spatial resolution, depth-dependent sensitivity, and scattered radiation, thereby making correlative assessments between nonvisualizable radioactive counts and optical signal measurements difficult, if not impossible. By contrast, PET activity is absolutely quantitative and can improve such correlative evaluations in planned near-term future work with a dual-modality  nanoparticle platform. In addition, because these particles are extremely bright, many images were saturated, limiting quantitative analysis. In the future, measures will be introduced to minimize signal saturation and to otherwise standardize acquisition of optical images.This study found that ultrasmall fluorescent core-shell silica nanoparticles can be safely used intraoperatively at nanomole doses for accurate, reliable, and high-contrast visual identification of SLNs in patients with head and neck melanoma. To our knowledge, there is currently no available comparable alternative for fluorescence image–guided SLN biopsy. This novel particle-based technology could significantly change routine surgical practice and lead to safer intraoperative approaches overall, while achieving more consistent clinical results during SLN biopsy among surgeons of variable experience.