Nanoparticle-antibody conjugates have emerged as promising systems to confer increased sensitivity and accuracy for in vitro diagnostics . Meanwhile, in vivo applications have benefited from the targeting ability of nanoparticle-antibody conjugates, as well as payload flexibility and tailored biodistribution. This review provides an encompassing overview of nanoparticle-antibody conjugates, from chemistry to applications in medical immunoassays and tumor imaging, highlighting the underlying principles and unique features of relevant preclinical applications employing commonly used imaging modalities .Modern diagnostic technologies rely on both  Notably, various nanoparticle systems have been studied as a means of targeting and increasing the accumulation of their cargo in tumor tissues, taking advantage of the tumors’ leaky vasculatures, which allow nanoparticles to extravasate out of the vasculature and be retained in tumor tissues. This tumor physiology-based phenomenon is known as the enhanced permeation and retention (EPR) effect. Nanomedicines may offer increased safety profiles, as healthy tissues are less exposed to the particle’s payload due to their limited biodistribution in vivo, but not necessarily greater dose accumulation at the tissue of interest. It has been recognized that the nanoparticle delivery efficiency to tumors is minimal, which can significantly impair product performance as the absolute amounts of nanoparticles extravasating into tumors may not be enough to achieve their purpose. Recent surveys of preclinical data from xenograft tumor models have demonstrated that less than 2.25% (mean) of the injected nanoparticle-based dose accumulates in solid tumor tissues  or be secreted to bind and neutralize their target antigens . CurrentIn IgG antibodies, each of four polypeptide chains has a variable (V) region responsible for binding antigen and a constant (C) region. They can be divided into two fragments based on their functions, which are the antigen-binding fragment (Fab) and the fragment crystallizable (Fc) region . DiffereEmploying recombinant antibody technologies, single-chain variable fragment (scFv) can be achieved, which includes the variable heavy and variable light domains. With two antigen-binding domains linked by a flexible peptide, scFv can provide excellent affinity and alteration of specificity to specific targets , while dAntibody functionalization on nanoparticle surface includes mainly two conjugation methods: adsorption and covalent binding ,12. The Adsorption, including physical and ionic, is one of the simplest conjugation methods. The antibodies attach to the material surface owing to intrinsic surface interactions, such as Van der Waal forces, electrostatic forces, hydrophobic interactions, and hydrogen bonds. Using this simple and gentle method, fragile antibodies can be immobilized onto surfaces with limited damage . On the Accompanied with high stability and excellent reproducibility, covalent binding is preferred because covalent bonds are less susceptible to disassembly , leadingN,N′-dicyclohexylcarbodiimide (DCC), to crosslink carboxylic acids to primary amines (N-hydroxysuccinimide (NHS) or N-hydro-xysulfosuccinimide (sulfo-NHS) are often used because they can increase the EDC-mediated coupling efficiency. The existence of NHS or sulfo-NHS produces a more stable intermediate that helps to prevent intra and intermolecular crosslinking of the antibody  carbodiimide (EDC) and y amines ,21. The y amines . During antibody –25. Baseantibody ,26.via an oxidative process (N-succinimidyl S-acetylthioacetate (SATA), can modify the amine group of lysine residues with thiol groups (A site-selective conjugation approach that involves binding through the sulfhydryl groups (-SH), also called thiol group, of the antibody. Sulfhydryls exist in proteins on the side chain of cysteine amino acids. Pairs of cysteine sulfhydryl groups are coupled by disulfide (-S–S-) bonds  process . For IgG process ,29. Howel groups . HoweverThis refers to a group of simple chemical reactions with stereospecificity and high efficiency ,32. Its in vitro assays conducted with biological samples to investigate analytes of biochemical interest. Due to its reliance on antibody binding mechanisms, immunoassays have been acclaimed for their relative sensitivity and specificity, but improvements are still warranted. Detection and quantification of small amounts of biomolecules can be optimized by the application of nanoparticles with adjustable surface chemistries, tunable optical properties and biocompatibility  nanoparticles, with their outstanding plasmon resonance properties, can be employed as signal enhancement tools for SPR . PlasmonAu substrate and antibodies can be used to fabricate a biosurface, which functions as the bottom of the sandwich providing a stable layer for amplification of the SPR signal. A typical binding structure of a sandwich immunoassay leveraging SPR is shown in Fig. In conclusion, the signal amplification produced by the nanoparticle-antibody conjugates enables the reduction of the limit of the detection (LOD), thereby leading to a significant increase in the sensitivity.LSPR is generated by the interaction of light and noble metals capable of producing a collective oscillation of conduction band electrons when the size of the surface is much smaller than the photon wavelength. This phenomenon fosters non-propagating excitations, denominated localized surface plasmons, where the plasmon oscillation distributes throughout the entire particle volume, creating a mass-spring harmonic oscillator effect . CompareAntibodies, acting as bioreceptors, can be carried by metal nanoparticles to improve the limits of detection for various analytes and increase the LSPR shift because of the changes in the local refractive index around the metal nanoparticle . A schemSilver nanoparticles can be conjugated with antibodies to provide a peak shifting in LSPR. As Valdez reported, polyclonal antibodies for respiratory syncytial virus, which is a paramyxovirus that leads to mild, cold-like symptoms, can be functionalized on silver nanoparticles using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry. The functionalized silver nanoparticles can interact with the respiratory syncytial virus and have a specific peak shifting at 60 min .Overall, both silver and gold are excellent materials for nanoparticle manufacturing and have demonstrated suitable optical properties for analytical purposes. In general, plasmon resonance is considered insensitive to metal composition as the bulk plasma frequencies of noble metals are similar . Among tConsidering the complexity involved in antibody-nanoparticle conjugates, the conjugation stability may play a significant role in the analytical performance of this methodology and therefore must be addressed. For stability purposes, the most crucial factor is pH. The conjugate’s electrostatic attraction can be affected by pH-dependent flocculation if the antibodies are conjugated to the metal nanoparticles non-covalently . To protin vivo applications, the vast majority of the research conducted with nanoparticles for diagnostic purposes focuses on tumor imaging. A variety of colloids have been widely used in imaging preclinically and more recently with limited clinical applications. Many types of nanoparticles, including sulfur colloid, albumin colloidal nanoparticle, and iron oxide nanoparticle, received approval from the U.S. Food and Drug Administration (FDA) for their application in radionuclide imaging and MRI (Enhance permeation and retention (EPR) effect: tumor vasculature, especially concentrated at the tumor-host interface and within the stroma, does not mature properly and faulty vasculature allows easier extravasation of macromolecular structures into the tumor stroma. Poor lymphatic drainage kinetically entraps nanoparticles in tumor tissue, increasing their residence time at the site of interestLow volume of distribution as the vast majority of the nanoparticle-loaded dose is retained within the blood vasculature Prolonged half-life in circulation increases the likelihood of nanoparticle extravasation due to the probabilityCombination of EPR effect with long circulation can universally enhance tumor accumulationFor  and MRI . As one  and MRI . Upon sy and MRI ,62:EnhanIn reality, the assumptions mentioned above have been established over many years of preclinical studies using well-controlled tumor-bearing mouse models. Nowadays, an overall understanding is that the EPR effect is highly variable and preclinical tumor models usually fail to translate these challenges (as observed in human patients). This is shedding light onto the challenges of nanoparticle-based delivery to tumors of both imaging agents and therapeutic molecules. In general, nanoparticle-antibody conjugates have been portraited as a viable approach to increase the targeting-ability of nanoparticles and their retention at the tissue of interest. However, unless a specific vascular-targeting ligand is used, nanoparticle extravasation into tumor tissues through the EPR effect is still required for antibody-ligand interactions to occur. Therefore, most “active-targeting” nanoparticle systems will still be affected by inter and intra-individual variations in the EPR effect and will perform mainly by increasing nanoparticle-cell interactions and retention in the tumor tissue.In vivo tumor imaging can be performed using a variety of modalities, whereas the most commonly used in the clinic include positron emission tomography (PET), magnetic resonance imaging (MRI), and X-ray computed tomography (CT). Nanoparticle-antibody conjugates can be used as active-targeting nanoparticle-based contrast agents for multimodality imaging and they may provide significant insights regarding tumor extravasation and “leakiness”. The vasculature leakiness is due to the abnormal and rapid growth of tumor vasculatures and is highly variable depending on the type and growth of tumors. Considering the intra- and inter-individual heterogeneity observed for the EPR effect in the clinic, it is genuinely considered a significant bottleneck hindering product performance in vivo . In fact, such an approach has been explored at the clinical level. For example, Merrimack Pharmaceuticals (USA) has administered 64Cu-labeled HER2-targeted PEGylated liposomal doxorubicin to screen metastatic breast cancer patients as part of their MM-302 clinical trial  or spin-spin (T2) relaxation times of the protons on nearby water molecules and therefore increase sensitivity and resolution ,80. MagnManganese oxide nanoparticles, which shortened T1 relaxation times, are the most commonly used manganese-based nanoparticles in MRI imaging in preclinical studies. Manganese oxide nanoparticles can be synthesized and conjugated with anti-CD105 antibody TRC105, which can target CD105, an antigen exclusively expressed on proliferating endothelial cells . In addiSuperparamagnetic iron oxide nanoparticles (IONPs) are also widely used as nanoparticle-based MRI contrast agents; and it is commercially available for clinical use for gastrointestinal imaging  . Based oFrom a preclinical perspective, IONPs have been widely studied as possible systemic contrast agents for parenteral administration. When combined with the advantages of high sensitivity from IONPs and selectivity from antibodies, antibody-conjugated IONPs became promising contrast agents for early-stage cancer detection . Human eAntibody-nanoparticle conjugates can be employed to enhance nanoparticle-cellular interactions and potentially increase tumor retention for enhanced imaging capabilities. Considering tumor accumulation is a kinetic phenomenon resulting from a balance between nanoparticle extravasation from blood vasculature into tumor tissue and lymphatic drainage, increasing cellular interactions and internalization of nanoparticles can assist in reducing drainage post extravasation. Nanoparticle-antibody conjugates are more likely to be internalized by tumor cells, increasing their residence time in the tissue. As a consequence of increased residence time in the tumor site, visualization of tumors is facilitated when employing multimodal imaging methods, even as nanoparticles are removed from the blood circulation. This is a feature that cannot be achieved with small-molecule contrast agents. This allows for easier logistics between contrast administration and imaging, improvement of imaging signal, and reduction of the need for multiple contrast agent dosing.In vivo functionality  and led to a two-fold increase in KPL-4 xenograft tumor contrast when compared with images obtained with an iodinated small-molecule contrast agent in X-ray CT, although the majority of the accumulated dose was retained within neovascular regions of the tumor tissues Fig.  91). In. In91). In general, increased gold conjugate retention in tumor tissues has been attributed to both the longer circulation time of gold nanoparticles, small particle size which enables improved tumor penetration and distribution, and increased retention in the tumor tissue due to antibody interactions with targeted cells. However, the contribution of the former appears to be less relevant when compared with nanoparticle size for the majority of preclinical studies. Conflicting results are found in the literature, indicating the inconsistency of product success when nanoparticle retention is solely attributed to the use of targeting antibodies. Considering the complexity of systemic nanoparticle delivery to tumors, it appears a variety of factors can significantly influence the efficacy of antibody targeting for retention purposes, mainly nanoparticle size, antibody density on the surface of the particle, antibody configuration, as well as antigen expression levels on the targeted cell; and these parameters must be considered concurrently while designing nanoparticle-antibody conjugates.in vitro, these systems usually have failed to translate such increase in vivo to the same magnitude. Although there is an incomplete understanding of particle extravasation, diffusion, and internalization within tumors, it is generally understood that smaller nanoparticles may exhibit increased tissue diffusion . Nanoparticles can be directly tagged with high specific activity radiotracers to provide real-time imaging in vivo at much lower concentrations when compared with other imaging modalities. Photons emitted by a radionuclide such as 64Cu, 89Zr, 13N, 18F, 72As, or 68Ga can be detected by a scanning device, typically scintillator crystals coupled to a photomultiplier  desired for improved intratumoral distribution. Furthermore, the small particle size ensures the bulk number of injected nanoparticles is removed from the circulation via renal filtration instead of mainly accumulating in the liver and spleen.A single-chain variable fragment (scFv) format of the HER2-targeting antibody Trastuzumab, which is the first FDA-approved monoclonal antibody for the treatment of metastatic breast cancer, can be used to functionalize ultrasmall silica nanoparticles (5–10 nm), namely C’ dots . The antex vivo immunohistochemical staining, providing an optimal platform for molecular imaging and antigen detection in real time. In contrast, for HER-2 low expression tumors, the signal was diminished and predominantly localized along the tumor periphery and seen within stromal tissue , has also been studied as a targeting tool to functionalize small gold nanoparticles. Because of the over-expression of EGFR in many epithelial solid tumors, EGFR-functionalized gold nanoparticles labeled by ng group .Overall, the use of ultrasmall nanoparticles enables enhanced intratumoral distribution beyond perivascular regions and may provide an optimal platform to investigate the presence of a wide variety of antigens within the tumor tissue. Considering their small size and easier removal from the tissue, nanoparticle retention as a function of antibody functionalization may be used to estimate the presence and density of antigens within the tissue without need for invasive procedures  and immunohistochemistry.in vitro diagnostics regarding the increase in sensitivity and reliability of immuno-based assays  and/or passive targeting . However, only a small fraction of the injected nanoparticle-based dose is expected to accumulate in solid tumor tissues, whereas the vast majority of the dose exhibits non-specific accumulation and elimination ,2. This in vivo kinetics of particulates in blood circulation instead of seeing it as a limitation for applications for which this approach is not ideal. For instance, nanoparticle-based imaging agents could be employed to investigate the likelihood of nanoparticle accumulation in certain tissues in the clinic, offering imaging data that could enable imaging-guided therapies . This approach does not focus on imaging the tumor tissue as a whole, but rather focuses on imaging the extravasation of nanoparticles into the tissue regardless of the extent of accumulation, and the information obtained will be valuable for treatment strategy determination.Considering the difficulties associated with tailoring the pharmacokinetics of nanoparticles, the best path forward is to take advantage of these limitations to invent a new generation of imaging agents with specific functionalities, leveraging the natural


The next-generation sequencing (NGS) analysis of human antibody repertoires has enabled a heightened appreciation and comprehensive characterization of the B-cell receptor (BCR) landscape at an unprecedented resolution –4. This Hong et al. used cord blood samples from 10 newborn babies and peripheral blood from 33 healthy adults to perform an in-depth analysis of human neonatal and adult IgM heavy chain repertoires. Their comparative study revealed unexpectedly high levels of similarity between the neonatal and adult repertoires although antibody repertoire of healthy adults was more diverse than that of neonates. These results are helpful in understanding the antibody development and diversity in newborn babies and adults. Kirik et al. used NGS to analyze human bone marrow B cells to elucidate how different mutational paths are traversed by antibody lineages stemming from different germline gene origins both in terms of amino acid substitutions, insertions, and deletions. Specifically, they identified germline gene-specific mutational patterns as found in selected and non-selected repertoires. These findings provide a framework for understanding patterns of evolution of antibodies arising from specific, defined germline genes. D'Angelo et al. showed that many different heavy-chain complementarity determining regions 3 (CDR-H3s) could be identified within a target-specific antibody population after in vitro selection by using a data set of 32,138 CDR-H3 sequences derived previously from the yeast display sorting and analysis of CDK2-specific antibodies. One of the remarkable observations demonstrated numerous rearranged heavy chains, derived from 19 different germline IGHV genes, were found to contain the same CDR-H3. Their main finding concludes that the same CDR-H3 can be generated by many different rearrangements, but that specific target binding is achieved by only certain unique V-D-J rearrangements and VL pairing. Jeliazkov et al. determined the structural flexibility of the CDR-H3 loops, using previously published algorithms, for thousands of homology models of antibodies derived from the NGS data to find if affinity maturation reduces their conformational flexibility or not. They also used a total of 922 antibody crystal structures from the Protein Data Bank  covering full length scFvs following in vivo panning in an animal model of atherosclerosis. They tested its performance by tracking and analysis of known, identical and related scFv-phage clone P3. Rosenfeld et al. and Vergani et al. present on a topic of bulk B-cells which provides a way for computationally assessing B-cell clone sizes and a library preparation method for NGS to capture an exhaustive full-length repertoire for nearly every sampled B-cell to be sequenced respectively. Rosenfeld et al. used three different measures of B cell clone size: copy numbers, instances and unique sequences, and then showed how these measures can be used to rank clones, analyze their diversity, and study their distribution within and between individuals. Overall, this method showed how different clone size measures can be used to study the clonal landscape in bulk B cell immune repertoire profiling data. On the other hand, the methodology as adopted by Vergani et al. serves as a useful protocol for Ig-seq where every IGHV-D-J rearrangement in the starting B-cell populations can be detected. Finally, advancements in NGS and error corrections have enabled antibody repertoire sequencing with single mutation precision but still compromising with sequencing accuracy. This opens the possibility for undocumented novel germline alleles. To address on this important issue, Wendel et al. present a method that can be quickly and easily applied to any antibody repertoire data set to mitigate the effects of germline mismatches on SHM patterns.Technical landscape for NGS analysis of human antibodies has changed tremendously and will continue toward the improvement of methods, immunoinformatics and data analysis tools. In this respect, we have four exciting articles devoted to methods/protocols. Chaudhary and Wesemann, which provides a sound introduction to practical steps involved in the process of immune repertoire profiling including sample preparation, platforms available for NGS, sequencing data processing and annotations, and fundamental measurable features of the immune repertoire such as V/D/J gene-segment frequencies, CDR-H3 diversity and physicochemical properties, and immunoglobulin somatic hypermutation (SHM). They also highlight additional analyses using the NGS-derived repertoire data: isotype analysis, which offers insights into the effector biology mediated by heavy chain constant regions, such as complement fixation or binding to Fc receptors; clonal lineage analysis, which is used to trace clonal evolution of HIV-1 broadly neutralizing antibodies; and B-cell network analysis that can link mature antibody sequences to their germline precursor sequences. Extrapolation of these procedures for analyzing paired VH:VL repertoires was also discussed. The readers attracted to this review article will likely appreciate the detailed description of statistical tools and their features that can be used for analysis and interpretation of NGS big data sets, along with a comprehensive list of software tools available for sequence error correction, annotation, and evaluation of B cell repertoires. This is followed by a review in which Miho et al. discuss four computational strategies: (i) measuring immune repertoire diversity, (ii) clustering and network approaches to resolve the sequence similarity architecture, (iii) phylogenetic methods to retrace antigen-driven evolution, and (iv) machine learning methods to dissect naïve and antigen-driven repertoire convergence. Furthermore, they summarize outstanding questions in computational immunology and propose new directions for systems immunology by possibly linking NGS-based potential metrics with computational discovery of immunotherapeutics, vaccines, and immunodiagnostics. These two reviews are followed by a mini-review article by Rouet et al., which specifically addresses the strategies for NGS of phage- and other antibody-display libraries, and list NGS platforms and analysis tools. This review also touches briefly on bioinformatic tools and applications to design validation with analyses of naïve antibody libraries, affinity maturation and epitope mapping with specific examples from literature. After these three reviews, our Research Topic addresses a challenging question of how B-cell receptor repertoire sequencing can potentially be enriched when coupled with structural antibody data, as described in the review by Kovaltsuk et al.. This review covers the basic principles about structural architecture of IgG, repertoire sequencing technologies and antibody structural properties. Further, they highlight on computational approaches and tools that leverage antibody structure information and provide a generalized workflow of antibody modeling. Overall, the authors illustrate how these two data types—NGS DNA sequences  and atomic structures, that can enrich one another and yield potential for advancing our knowledge of the immune system and improving antibody engineering and developability. Along this line of work, Mishra and Mariuzza review the structural basis of antibody affinity maturation from NGS data. Interestingly, they looked at the studies of antibody affinity maturation prior to and after NGS. They further emphasized how important the NGS is for the reconstruction of antibody clonal lineages in immune responses to viral pathogens, such as HIV-1. They discussed in detail about various mechanisms of paratope preorganization, rigidification, reorientation, and indels as described for many antibodies. Overall, this review provides a more holistic perspective to structural basis of antibody affinity maturation from the point of next-generation sequencing.Next, we provide five excellent reviews in the Research Topic, starting with a review by Breden et al.), which provides an overview of the founding principles and presents the progress it has made to develop and promote standards and recommendations for best practices and data-sharing protocols. In conclusion, NGS combined with innovative single-B-cell technologies has the potential to yield millions of native human antibody sequences and some of them that could match with therapeutic antibodies  community (tibodies , 14. Thitibodies –17.PP wrote the manuscript. All authors contributed to this work and approved the final version of the manuscript.PP is an employee of Sanofi Genzyme. JG is an employee and CEO of Distributed Bio. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors GI.


Neuroimmunological and neurodegenerative disorders are major healthcare issues, associated with high individual and socioeconomic burden. For many neurological disorders an early diagnosis is key for efficient management and therapy. However, neural tissues from brain, spinal cord, or peripheral nerves are difficult to sample, and their extraction is often associated with functional deficits. Therefore, in most cases a combination of biomarkers such as radiologic or biochemical findings are used to define a diagnosis in the clinical practice. The development of pathologically-sensitive and easy-to-measure disease biomarkers is a key factor for investigating new medications in phase II trials aiming to quickly screen their efficacy, and in phase III trials where they are coupled to clinical measures. Moreover, biomarkers are used to predict treatment efficacy and adverse event risk, paving the way for personalized medicine with individual risk assessment and prevention strategies.In the last few decades the enormous technological progress, especially omics technologies, expanded the definition of biomarkers from biochemical and clinical, to genetic, proteomic, metabolic, or microbial markers. Interestingly, some of the biomarker studies additionally provided important insights into the pathophysiology of neurological disorders. In multiple sclerosis TNF-blocking drugs can promote onset or exacerbation of MS and GWAS (genome-wide association studies) data informed about the underlying mechanisms instructing clinical practice . In ParkThis Research Topic reviews current knowledge and provides new insights into pathomechanisms of a wide variety of neuroimmunological and neurodegenerative disorders from studies on (novel) risk factors and outcome predicting biomarkers.Pawlitzki et al. shows the value of CSF-based biomarker approaches indicating ongoing chronic CNS inflammation even before first clinical sign of symptoms in patients with radiologically isolated syndrome, while Schwenkenbecher et al. demonstrate the importance of oligoclonal bands in the diagnostic algorithm of multiple sclerosis assessing the impact of the newest McDonald criteria. Until now, we have only a limited understanding of inflammatory and neurodegenerative processes within the CNS compartment in multiple sclerosis. Here, Birkner et al. assessed the role of ICAM-5, a CNS-restricted adhesion molecule, in mediating T cell-induced neuronal damage in the animal model of experimental autoimmune encephalomyelitis (EAE), Huhn et al. investigated the use of sodium MRI as a translational approach in MS patients. Another part of this Research Topic section focuses on the value of biomarker approaches in different real-world therapeutic settings, namely blood-based biomarkers in patients treated with fingolimod , dimethylfumarate , and in a longitudinal setting in patients treated with natalizumab . The study by Vohl et al. investigates a cohort of patients treated with intrathecal corticosteroid therapy, a long-established, but sometimes under-appreciated therapeutic approach, in numerous centers specialized on the treatment of MS patients, that so far has suffered from the lack of large studies. In a cohort of 38 patients with autoimmune encephalitis (AE), Körtvelyessy et al. showed that CSF Progranulin levels were elevated during the acute phase, suggesting ongoing neuro-axonal damage, and that total-Tau (t-tau) and neurofilament light chain (NfL) levels were associated with the risk of clinical, laboratory, and radiological progression. Future studies on AE could benefit from the work of Amedonu et al., with the use of a novel assay to study molecular mechanisms of autoantibody-induced receptor internalization and to screen new molecular-based therapies.There is still an unmet need for novel, innovative biomarker approaches in different stages of central nervous system inflammation ranging from first diagnosis to evaluation of inflammatory and neurodegenerative processes and therapy responses. In this Research Topic, Goltermann et al. showed that apolipoprotein E (APOE) ε4 homozygosity, a well-known risk factor for Alzheimer's disease (AD), was associated with worse visuospatial working memory and global brain structural alterations in 62 non-neurological subjects from the FOR2107 Marburg-Münster Affective Cohort Study.Neurodegenerative diseases have been conventionally studied in relation to the genetic risk of clinical/cognitive dysfunction and neuronal damage. In line with this, Hu et al. demonstrated an inflammatory component in the normal aging and in AD, by showing an accentuated shift from T-helper (Th)-1 to non-Th1 inflammatory pathways in the cerebrospinal fluid (CSF) (similar to what happens in multiple sclerosis). Hotter et al. evaluated 91 stroke patients not suffering from stroke-associated infections (derived from the PREDICT and 1000 Plus studies), and found that IL-6 was associated with several MRI measures of stroke severity , suggesting that inflammation is related to vascular parenchymal damage. Also, neuroinflammation during the fetal period could be related to neurodevelopmental disorders . Cao et al. studied the role of α7nAChR on fetal sheep astrocytes, a classic model of fetal neurobiology, and further unraveled the complex interactions between inflammation and neurodegeneration at neurodevelopmental stages.More recently, an interplay between inflammation and neurodegeneration has been recognized. Looking at conditions that are conventionally unrelated to inflammation, Johnen and Bertoux suggested that the early clinical diagnosis of the behavioral variant frontotemporal dementia (bvFTD) requires an in-depth assessment of clinical signs, behavioral and psychological symptoms as well as cognitive performance. Authors strongly encouraged the design of novel neuropsychological tests covering understudied aspects of dementia , and further expanding the number of clinical and neuropsychological biomarkers for the neurology clinical practice (Johnen and Bertoux).However, our Research Topic did not aim to underestimate clinical examination, which remains of utmost importance in the neurology clinical practice. In particular, in their review, Doppler et al. were now able to show that dermal phospho-alpha-synuclein (p-syn) in PD patients with glucocerebrosidase gene (GBA1) mutations seems to offer a similar distribution and frequency as observed in PD patients without a known mutation. Therefore, skin biopsy may be suitable to study p-syn deposition in these patients or even to identify premotor patients with GBA1 mutations . However, a panel of serum immune markers  as analyzed using principal component analysis (PCA) was not found to allow for reliably predicting clinical PD subtypes . Varghese et al. introduced a technology-based system called Smart Device System (SDS) that was tested over 2 years to train an Artificial Intelligence System; this system will be further implemented for multi-modal high-resolution acceleration measurement of patients with PD or essential tremor (ET). Furthermore, Warnecke et al. used flexible endoscopic evaluation of swallowing (FEES) to detect specific pharyngolaryngeal movement disorders, such as arytenoid tremor that may help to differentiate multiple system atrophy (MSA) from PD.Over the last decade increasing scientific efforts have been made to establish biomarkers for early diagnosis of Parkinson's disease (PD) and other movement disorders. These potentially helpful biomarkers include findings from neuroimaging techniques, blood and cerebrospinal fluid (CSF) immune markers, technology-based objective measures (TOMs) provided by wearable devices, results from skin biopsies, and several others. Prell et al. provide an overview of recent developments of bio-fluid and imaging biomarkers for dementia, depression, and fatigue in PD. They conclude that more research needs to be undertaken to find reliable combinations of predictors of these neuropsychological symptoms in PD on an individual level, and that standardization and harmonization of protocols in particular in CSF handling and neuroimaging has to be further implemented . Richter et al. assessed the utility of brainstem raphe (BR) alterations evaluated by transcranial sonography (TCS) as a potential biomarker for apathy and depression in PD patients. These BR alterations correlated with depression and apathy scales indicating that a serotonergic signal disturbance might exist for both symptoms in PD . Beside these neuropsychological symptoms, significant attention has being paid to gastrointestinal dysfunction and particularly swallowing disorders. Clinical research has shown that dysphagia is an often overlooked, but complex syndrome in many patients with neurodegenerative and neuromuscular disorders mainly contributing to disease burden in the majority of cases. Schröder et al. were able to show that reduced saliva substance P (SP) concentrations may predict early pharyngeal swallowing dysfunction in PD patients and may help to identify PD patients with a higher risk of developing clinically relevant dysphagia. Labeit et al. reported the first case of isolated dysphagia due to Jo-1 antibodies associated myositis. They suggest that myositis-focused diagnostics including an autoantibody panel should be done in patients with unclear dysphagia that show typical sings for myositis in the instrumental dysphagia assessment .In addition, biomarkers for detection of specific symptoms related to PD and other movement disorders have been studied throughout the last years. In their narrative review Schlenstedt et al. presented pilot data indirectly supporting the hypothesis that exercises specifically designed to target freezing of gait (FOG) might be more beneficial than non-FOG-specific interventions in PD patients. However, future studies should include larger samples and high frequency interventions to investigate the role of training balance performance to reduce the severity of FOG and to analyze which subgroup of PD patients shows the largest effect size . Finally, Hatz et al. were able to demonstrate that deep brain stimulation of the subthalamic nucleus (STN) used to treat motor complications in advanced PD does not alter verbal fluency performance in a systematic way at group level. Nevertheless, when STN stimulation produces an alteration of verbal fluency performance in an individual PD patient, it was inversely correlated with left temporal delta power as measured by quantitative EEG .In the future biomarkers should not only be available for early diagnosis, but also for indicating responses to specific therapeutic interventions that help to guide treatment decisions in PD and other movements disorders. Stuhlmüller et al. review the role of myositis-specific and -associated antibodies in idiopathic inflammatory myopathies (IIM). They highlight their value in the diagnostic process and prognosis , and elaborate on detection methods and associated clinical phenotypes . De Paepe et al. demonstrate an upregulation of osmolyte accumulators and identify associated inflammatory signaling pathways in skeletal muscle cells upon pro-inflammatory stimuli in vitro and in skeletal muscle biopsies of IIM patients. The authors characterize specific osmolyte accumulators as potential biomarkers for regenerating muscle fibers or autoreactive immune cells .The peripheral nervous system (PNS) consists of nervous structures  and their associated receptors and organs. The diagnosis of PNS disorders is a multi-step process and often complicated by unspecific findings. Instructive cellular and non-cellular biomarkers for diagnosis, prognosis and/or treatment management are largely missing. However, recent years including studies of this Research Topic support the high relevance of biomarkers for an adequate disease management. Correspondingly, Roos et al. identified key molecules involved in the pathogenesis of sporadic inclusion body myositis (sIBM), which were further characterized by qPCR, immunostaining, immunofluorescence in situ and in silico studies. By these means, they were able to demonstrate a key role of specific macrophage subtypes and molecules  involved in their activation and type I interferon and interferon gamma associated pathways in sIBM . With a similar proteomic approach Kölbel et al. identified candidate protein biomarkers in skeletal muscle of laminin-211 deficient type 1 A (MDC1A) congenital muscular dystrophy (CMD), one of the most devastating CMDs characterized by severe muscle weakness, brain abnormalities, and delayed motor milestones. The expression of 86 proteins associated with fibrosis and altered synaptic transmission was specifically linked to MDC1A skeletal muscle. Moreover, the observed alterations in metabolic pathways and mitochondrial function might identify new therapeutic targets .However, biomarker research might not only unravel associations with clinical parameters but also reveal important insights into pathophysiological disease mechanisms. With an unbiased proteomic profiling Gatz et al. introduce an in vitro system identifying the pathogenic consequences of different mutations on a cellular level. In the future, these findings can instruct genetic testing of patients with suspected MSS.Changes in protein function, folding or metabolism is a common feature of several degenerative disorders of the PNS. In Marinesco-Sjögren syndrome (MSS) characterized by a clinical triad of bilateral cataracts, ataxia, and myopathy, mutations in the SIL1 gene lead to protein misfolding. However, read-out systems to identify pathogenic variants of SIL1 mutations are missing. Therefore, Seeliger et al. provide a cross-sectional study of 144 patients with polyneuropathy of whom 44 were diagnosed with Sjögren's syndrome. They describe clinical findings and discuss potential tools to identify Neuro-Sjögren . Other systemic inflammatory disorders such as vasculitides can also affect structures of the nervous system including the PNS resulting in a variety of potential symptoms. Strunk et al. review potential biomarkers in the context of nervous system vasculitides with a focus on body fluid derived markers and discuss their diagnostic and prognostic value.Besides disorders primarily affecting structures of the PNS, systemic autoimmune diseases such as Sjögren's syndrome might involve the PNS as well. In this Research Topic, Overall, the current Research Topic provides a deeper understanding of the pathomechanisms underlying neurological disorders and identifies new biomarkers and their pathophysiologic background. Thereby, the Research Topic might initiate hypothesis-driven refinement of current and novel biomarkers.SB, MM, TW, and TR edited the Research Topic and drafted and revised the editorial manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective assessments of concussion recovery are crucial for facilitating effective clinical management. However, predictive tools for determining adolescent concussion outcomes are currently limited. Research suggests that heart rate variability (HRV) represents an indirect and objective marker of central and peripheral nervous system integration. Therefore, it may effectively identify underlying deficits and reliably predict the symptomology following concussion. Thus, the present study sought to evaluate the relationship between HRV and adolescent concussion outcomes. Furthermore, we sought to examine its predictive value for assessing outcomes. Fifty-five concussed adolescents (12–17 years old) recruited from a local sports medicine clinic were assessed during the initial subacute evaluation (within 15 days postinjury) and instructed to follow up for a post-acute evaluation. Self-reported clinical and depressive symptoms, neurobehavioral function, and cognitive performance were collected at each timepoint. Short-term HRV metrics via photoplethysmography were obtained under resting conditions and physiological stress. Regression analyses demonstrated significant associations between HRV metrics, clinical symptoms, neurobehavioral function, and cognitive performance at the subacute evaluation. Importantly, the analyses illustrated that subacute HRV metrics significantly predicted diminished post-acute neurobehavioral function and cognitive performance. These findings indicate that subacute HRV metrics may serve as a viable predictive biomarker for identifying underlying neurological dysfunction following concussion and predict late cognitive outcomes. The clinical management of adolescent concussion has generally relied upon subjective symptom reporting and neuropsychological assessments for determining the recovery status ,2,3. DesHeart rate variability (HRV) is a widely used and reliable index of cardio-autonomic function , which hRecent evidence has shown that HRV metrics are associated with symptom severity and postinjury deficits in cognitive function following a concussion ,35. AlthThis study is a retrospective analysis of data extracted from part of a larger study on the clinical evaluation of concussions. Four-hundred and twelve youths suspected of having recently sustained a concussion were evaluated at a local pediatric sports medicine clinic. During the initial evaluation, the concussion diagnoses were confirmed by the attending physician affiliated with this study (JPH) using guidelines established by the Consensus Statement on Concussion in Sport  and the Of the youth screened for concussion, 74 (~18%) adolescent patients that initially received a diagnosis of concussion during subacute evaluation (3–15 days) and returned for a follow-up post-acute evaluation within 60 days of injury were identified for further analysis. Exclusion criteria included medical histories known to further alter the outcome following concussion and cardio-autonomic function. Fifty-five (13.3%) patients were eligible and included in the final analyses per the inclusion/exclusion criteria .Comprehensive demographic information ; injury characteristics; and medical and sport histories were collected from parents/legal guardians that accompanied the patients. Information was used to screen for possible inclusion/exclusion criteria and potential moderating variables following adolescent concussion, such as a prior diagnosis of concussion, body mass index (BMI), time since injury, and athletic status.HRV metrics were collected through an EmWave Pro Plus infrared pulse plethysmograph ear sensor . Participants maintained a respiratory rate of 0.13 Hz  in a seated position for the duration of a 5-min recording. HRV measurements occurred in a temperature and light-controlled environment at approximately the same time of day across subjects. Raw HRV data were processed using Kubios HRV Standard version 3.0.2 . HRV data were inspected and corrected for artifacts. Once artifacts were removed, a 10% Hanning window was applied to the corrected data. Finally, time domain and nonlinear HRV metrics were calculated. Data collection and analysis were performed in accordance with the recommendations by the Task for Force of the European Society of Cardiology and North American Society of Pacing and Electrophysiology .Time domain parameters were calculated from the intervals between adjacent QRS complexes resulting from sinus node depolarization, also known as RR intervals . NormaliNonlinear indices have also been shown to be affected following concussion . Thus, sAdditionally, a 1-min HRV recording during physical exertion  was conducted to serve as a quantifiable index of one’s ability to adapt to environmental stress. Nonlinear indices have not been validated for ultra-short-term recordings (<5 min); thus, only time domain metrics were calculated .Clinical symptoms were measured using the Rivermead Post-Concussion Symptoms Questionnaire (RPQ), a 16-item self-report questionnaire used to examine the severity of post-concussion symptoms compared to premorbid levels . A reliaDepressive symptoms were evaluated using the Beck Youth Inventory Second Edition–Depression Scale (BYI-2), a 20-item self-report questionnaire . BYI-2 qNeurobehavioral function was assessed using the Behavior Rating Inventory of Executive Function (BRIEF-P), an 86-item parent-reported inventory of their child’s everyday executive function in the home and school environments . This scCognitive performance was tested using a modified CogState Brain Injury Testing Battery  consisting of three computerized task conditions. The three selected tasks included Groton Maze Learning (GML), One-back (ONB), and Groton Maze Delayed-Recall (GMR) tasks. Correct moves per second and total errors were used to assess cognitive efficiency and working memory during the Groton maze tasks. Accuracy (proportion correct), mean reaction time (msec), and reaction time variability (standard deviation of performance speed) were used to assess working memory and attention during the ONB task. Cognitive tasks incorporated into this modified battery have shown acceptable validity and good reliability across various age-groups and clinical populations ,47,48. It-tests were used to examine differences in outcome measures across the subacute and post-acute evaluations. Multivariate linear regression was used to analyze (1) the association between HRV metrics and concussion outcomes at the subacute evaluation and (2) the predictive value of subacute HRV metrics for determining post-acute outcomes. Covariates known to influence HRV and post-concussion outcomes were entered into regression models, including age, sex, history of concussion, body mass index (BMI), time since injury, and athletic status (athlete/nonathlete). All models met the assumptions of linearity, constant error variance, and the absence of significant outliers and multicollinearity. A priori level of statistical significance was set to p < 0.05.All statistical analyses were conducted using SPSS software version 27.0 . To account for skewed distributions of dependent variables, the RPQ, BYI-2, and BRIEF-P scores were transformed using a natural-logarithm transformation. Additionally, ONB accuracy was transformed using an arcsine square root of accuracy, and ONB mean reaction time was transformed using a logarithmic transformation of the mean reaction time in order to enhance the inference of models. Paired-sample The participant demographic information and injury characteristics are summarized in t-tests found that the clinical and depressive symptoms significantly decreased from the subacute to post-acute evaluation (p < 0.001). Measures of the neurobehavioral functions did not significantly change over time, with the exception of the Organization of Materials scale . Measures of the cognitive performances significantly improved from the subacute to post-acute evaluation (p < 0.05), excluding the GMR total errors and ONB reaction time (RT) variability.2 = 0.082) and RMSSD , whereby concussed adolescents with greater HRV at rest reported lesser somatic symptom severity at the subacute evaluation. HR dispersion at rest was found to have a significant positive association with emotional  and cognitive  symptoms at the subacute evaluation. Subacute HRV metrics were not found to significantly predict the self-reported symptom severity on the RPQ at the post-acute evaluation  and RMSSD  at the subacute evaluation. In contrast, a significant negative association was found between the SampEn at rest and Behavioral Regulation Index scores at the subacute evaluation , whereby a lesser SampEn was associated with poorer Behavioral Regulation scores of the BRIEF-P. No subacute HRV metrics were significantly associated with the subacute Metacognition Index scores of the BRIEF-P  and RMSSD  at rest, whereby the lesser SDNN and RMSSD values were associated with poorer Organization of Materials scores at the post-acute evaluation  and RMSSD  at rest during the subacute evaluation (2 = 0.084), GMR total errors , and ONB accuracy  and greater reaction variability  on the ONB task  at the post-acute evaluation  and during IHGC  significantly predicted ONB accuracy at the post-acute evaluation (2 = 0.070) and RMSSD  during IHGC significantly predicted the ONB reaction time at the post-acute evaluation  and cardiovascular system decouple on multiple levels . This foAccordingly, we observed that diminished SDNN and RMSSD were significantly associated with more severe somatic symptomology at the subacute evaluation. Our findings are consistent with observations of attenuated resting-state HRV during the acute and subacute recovery phases, often characterized by the presence of concussion symptoms ,30. It iIn contrast, we did not observe a relationship between the metrics of HRV and depression at either timepoint. This finding was contrary to our hypotheses and prior evidence suggesting that HRV metrics may predict a later onset of depressive symptoms in adult females with a mTBI (mild traumatic brain injury) . HoweverIn addition, we observed that higher indices of vagal activity were associated with poorer behavioral regulation scores (SDNN and RMSSD) and slower cognitive performance as indexed by correct moves per second on GML (SDNN and RMSSD) at the subacute evaluation. Furthermore, a greater HRV during physical exertion was associated with slower cognitive performance as indexed by the reaction time and reaction time variability on the ONB task at the subacute evaluation (RMSSD). These findings corroborate prior research that observed associations between higher HRV and poorer emotional and cognitive symptoms in concussed adolescents . Recent Beyond traditional HRV variables, we observed consistent associations between HR dispersion and concussion outcomes across the timepoints. We observed that those with greater HR dispersion reported more severe emotional and cognitive symptoms, as well as performed worse on cognitive tasks at the subacute evaluation. Moreover, a greater HR dispersion at rest and during stress predicted a poorer cognitive performance on the GMR and ONB tasks at the post-acute evaluation. These findings corroborate those of Brandt and colleagues, who identified RSA indices as potential predictors of adverse mTBI outcomes .The preliminary research also suggests that HRV complexity  may be attenuated during the acute, subacute, and post-acute phases of concussion recovery ,65. AccoTogether, our results help advance the scientific understanding of the relations between HRV and concussion outcomes. Our results are the first to suggest that HRV may predict neurobehavioral and cognitive outcomes beyond the subacute phase of injury. This is an important point, as prior subacute associations between HRV across domains of functioning have led many researchers and clinicians to suggest that HRV is just a general marker of dysfunction bereft of specificity and predictive power. However, our results point to a specific predictive value of HRV to post-acute neurobehavioral functions and cognitive functions independent of symptom questionnaires. Thus, HRV may be a simple and effective way for clinicians to identify patients who are likely to experience persisting alterations in neurobehavioral functioning and cognition. This would provide clinicians the opportunity to preemptively change management strategies to maximize the positive outcomes.Although the current results serve to provide a specific relation between HRV and concussion outcomes, the present study is not without limitations. First, although age was adjusted for in our regression analyses, our results may not fully account for individual differences in physiological maturity among this sample of adolescents. The current findings also cannot inform us regarding the differences in female populations across estrous cycles or across birth control types. Although both sexes were represented in the current study, menarche, estrous phase, and hormonal contraceptives can influence HRV. Furthermore, as this study was conducted in a clinic and not a laboratory, we cannot determine the central mechanisms  that mediate the relation between HRV metrics and functional outcomes. Additionally, as the preinjury baseline measures were not captured, the current study cannot tell us how these relations may change as a function of sport and sub-concussive impacts, which affect the neuronal integrity centrally and peripherally. Furthermore, while we controlled for the BMI and physical activity level, we did not have objective measures of cardiorespiratory fitness, which may influence the resting HR and HRV. Thus, additional research is still needed from a scientific and clinical perspective to determine the predictive value of HRV across different populations and demographic factors.The present study is the first to suggest that acute HRV at rest and during a physical exertion task may be useful for predicting neurobehavioral and cognitive outcomes in the post-acute phase of injury. Importantly, our results fit with the prevailing model of cardio-autonomic regulation, which posits a strong relation between HRV, learning, memory, and attention. Thus, our findings provide both a theoretical and data-driven impetus for using HRV in the clinical assessment and management of concussions. HRV is feasible for use in clinical settings, as it is noninvasive; cost-effective; and can be used under various conditions . Furthermore, measuring the HRV does not require the level of training or space of other psychophysiological measures and can be collected from simple ear clips or three-lead ECGs (Electrocardiogram). That being said, further research is needed to understand the relation of HRV to concussion outcomes across a wide variety of populations and demographic factors in order to provide clinicians with a more complete understanding of the benefits and limitations of HRV to their clinical practice.


Adult OCD patients showed significant FA reduction in the sagittal stratum  and posterior thalamic radiation . In the sagittal stratum, lower FA was associated with a younger age of onset , longer duration of illness , and a higher percentage of medicated patients in the cohorts studied . No significant association with symptom severity was found. Pediatric OCD patients did not show any detectable microstructural abnormalities compared to controls. Our findings of microstructural alterations in projection and association fibers to posterior brain regions in OCD are consistent with models emphasizing deficits in connectivity as an important feature of this disorder.Microstructural alterations in cortico-subcortical connections are thought to be present in obsessive–compulsive disorder (OCD). However, prior studies have yielded inconsistent findings, perhaps because small sample sizes provided insufficient power to detect subtle abnormalities. Here we investigated microstructural white matter alterations and their relation to clinical features in the largest dataset of adult and pediatric OCD to date. We analyzed diffusion tensor imaging metrics from 700 adult patients and 645 adult controls, as well as 174 pediatric patients and 144 pediatric controls across 19 sites participating in the ENIGMA OCD Working Group, in a cross-sectional case-control magnetic resonance study. We extracted measures of fractional anisotropy (FA) as main outcome, and mean diffusivity, radial diffusivity, and axial diffusivity as secondary outcomes for 25 white matter regions. We meta-analyzed patient-control group differences (Cohen’s  Diffusion tensor imaging (DTI) allows the study of WM at the microstructural level through the analysis of intrinsic, three-dimensional diffusion properties of water within brain tissues2. Prior DTI studies in OCD5 suggest that microstructural alterations are present in a number of WM areas. However, results across studies are inconsistent, with contrasting or conflicting effects of OCD on DTI metrics6. Sources of heterogeneity may include methodological factors , clinical characteristics, and variations in demographic or socioeconomic factors. More importantly, sample size variations may impact reported findings, as small studies may have insufficient power to detect subtle alterations7.Abnormalities in cerebral white matter (WM) are relevant to models of anomalous brain circuitry that posit deficits in connectivity in obsessive–compulsive disorder (OCD). OCD has a childhood onset in over 50% of all cases, and most childhood-onset OCD cases persist into adulthood8, is one such collaboration. Previous findings from the working group focused on subcortical and cortical brain gray matter abnormalities, using subcortical volumes, cortical thickness, and surface area quantification algorithms. An initial analysis of data from 3589 individuals showed distinct subcortical volume abnormalities in adults  and unmedicated children  with OCD9. The second study focused on cortical gray matter differences and showed a lower surface area for the transverse temporal cortex and a thinner inferior parietal cortex in adult patients. In pediatric OCD patients compared to healthy controls, significantly thinner inferior and superior parietal cortices were found10. Medication status was associated with structural differences in both pediatric and adult OCD.Brain imaging consortia offer new opportunities, pooling data and findings from around the world to achieve an appropriate sample size. The OCD working group of the Enhancing Neuro-Imaging Genetics through Meta-Analysis (ENIGMA) consortium11 or lower11 fractional anisotropy (FA) in anterior midline tracts), with more homogenous findings for fronto-temporal and fronto-parietal intra-hemispheric bundles, we expected to find microstructural alterations (as reflected by lower FA11) in the long tracts connecting frontal regions to posterior temporal, parietal and occipital association cortices.Here we aimed to investigate WM microstructural alterations in adult and pediatric OCD using data from the ENIGMA OCD working group, in the subset of participants that had collected diffusion magnetic resonance imaging (MRI). DTI metrics in 700 adult patients were compared to those of 645 adult controls, and separately, 174 pediatric patients were compared to 144 pediatric controls. Analyses also aimed to investigate associations between WM microstructure and demographic and clinical variables. As prior meta-analytic findings in frontal and callosal regions have been inconsistent (with either higher10) showed different patterns of effects in pediatric and adult cohorts; thus, we performed separate meta-analyses for adult and pediatric data. Globally, we analyzed data from 1345 adults  and 318 children (including 174 OCD patients and 144 controls). The diagnosis of psychiatric disorders including OCD and other comorbid conditions (if any) was made using a structured or semi-structured interview; the Structured Clinical Interview for DSM-IV [ n = 9 datasets], the Mini-International Neuropsychiatric Interview [ n = 3 datasets], or the schedule for affective disorders and schizophrenia for school-aged children: Present and Lifetime Version . Patients were administered the Yale-Brown Obsessive–Compulsive Scale (YBOCS)15 and the Child YBOCS16 to assess symptom severity. These tools are clinician-rated, 10-item scales, with each item rated from 0 (no symptoms) to 4 , with separate subtotals for the severity of obsessions and compulsions.The ENIGMA OCD Working Group includes 19 international research institutes. Previous literature (including studies from the present Working GroupCommon exclusion criteria across sites included: (1) history of psychoactive substance dependence or abuse during lifetime, (2) history of neurologic illness or brain injury, (3) presence of any brain pathology as instantiated by standard magnetic resonance imaging (MRI) exams , (4) dementia diagnosis according to DSM-IV-TR criteria.Tables All local IRBs approved the use of measures extracted from completely anonymized data.http://enigma.ini.usc.edu/protocols/dti-protocols/) and already employed to pool harmonized DTI analyses from around the world19.Harmonized preprocessing, including brain extraction, eddy current correction, movement correction, echo-planar imaging-induced distortion correction and tensor fitting, was carried out at each site, using protocols and quality control pipelines provided by the ENIGMA-DTI working group , derived from the Johns Hopkins University21 WM parcellation atlas, were used to extract the mean FA across the full skeleton and mean FA values for 25 ROIs.Once tensors were estimated, each site conducted a harmonized image analysis for FA quantification using the ENIGMA-DTI protocol, consisting of the tract-based spatial statistics (TBSS)Diffusivity measures , axial diffusivity (AD), and radial diffusivity (RD)) were also derived for secondary analysis . In the main analyses, we combined left and right ROI across hemispheres, as we had no a priori hypotheses regarding lateralized effects on FA.d effect sizes were calculated for differences in FA between patients and healthy controls. Age, sex, age-by-sex interaction, and quadratic covariates of age2 and age2-by-sex interaction were included in the model, as linear and nonlinear age and sex interactions have been reported for FA17. Subsequently, a random effects meta-analysis was run at the coordinating site using Comprehensive Meta-Analysis  to combine individual site effect sizes. Heterogeneity scores  were also computed for each test.At each site, Cohen’s d values for case/control effects and z-scores, and were considered significant if p < 0.05. The stability of the overall effect size estimate was tested using a ‘leave one out’ sensitivity analysis. This analysis shows how the overall effect size changes if one dataset at a time is removed, assessing whether potential results are site-dependent with between-sites variations potentially deriving from variability in study population characteristics (sampling error). Furthermore, to ascertain whether the estimated effect size varied as a function of clinical characteristics, mixed-effects meta-regressions were performed on FA, using age of onset, duration of illness, symptom severity, and percentage of medicated patients in the patients’ dataset as regressors. The influence of medication status was also explored through a mixed-effects sub-group analysis, comparing effect sizes in medicated (n = 8) and unmedicated (n = 3) patient cohorts. These analyses were primarily run in those WM areas where effect sizes were significant and stable according to the leave-one-out analyses. Ancillary analyses explored the effect of clinical variables on FA also in WM areas where only partly stable results were observed Effect sizes are reported as overall Cohen’s Demographics and clinical characteristics of the participants in each site are shown in Tables d = −0.17, z = −2, p = 0.045), the posterior corona radiata , the posterior thalamic radiation , the sagittal stratum  and the uncinate fasciculus   only, whereas PCR , PTR , SS , and UNC  showed non-significant variance between sites.Heterogeneity scores revealed a significant high variance for GCC  and higher RD in PTR and SS . No significant results were found for AD.As secondary analyses, we also investigated diffusivity measures  in those WM regions where FA was significantly reduced in OCD. Results revealed that patients diagnosed with OCD showed higher MD in the SS , longer duration of illness  and a higher percentage of medicated patients  = 5.27, p = 0.022) between the effect sizes in medicated  and unmedicated  patients. No relationship was found between FA values of the PTR and clinical measures. No relationship was found between FA and YBOCS scores.As stated, meta-regressions were primarily run in regions where effect sizes were significant and stable . In the SS of adults diagnosed with OCD, lower FA was significantly associated with younger age of onset  and higher percentage of medicated patients  in the PCR, and with higher percentage of medicated patients  in the UNC.Ancillary analyses in those WM areas where results were unstable according to the leave-one-out analyses , revealed that lower FA was associated with longer duration of illness  in the pathophysiology of many psychiatric disorders has been supported by growing research evidence26. In our study, higher MD and RD (and the absence of changes in AD) in the same bundles supports the hypothesis that lower FA reflects a disruption of myelin sheaths28, given that RD is a putative myelin marker26.Although FA is a general measure of microstructure—including variation in regional myelination levels, such as axon demyelination or loss, myelin loss or increased extracellular space—it does not provide a physiologically specific explanation of WM abnormalities30. Moreover, symptoms indicative of obsessive–compulsive traits are related to individual myelination over time, even in otherwise healthy samples. This suggests that the mechanisms underlying compulsivity have long-lasting effects on brain development, possibly affecting myelination trajectories during adolescence, with enduring effects into adulthood23. Alternatively, the paucity of extrinsic factors regulating the development of myelinating glia31 could have driven the altered myelination and its association with prolonged illness. Indeed, impoverished environment is both the cause and the consequence of mental illness in general, and of compulsivity in particular32.The association between myelin degradation in the SS and longer illness duration together with the absence of a detectable alteration in pediatric patients, suggest that neuroplastic changes may reflect prolonged symptomatology, since compulsively engaging in a particular behavior or cognitive process has been suggested to alter brain structure33 and long-term treatment exposure may negatively influence the proliferation of oligodendrocytes and their myelination of axons36. Our findings are consistent with a previous large multimodal meta-analysis3 where increased WM volume and decreased FA were especially pronounced in OCD samples with a high proportion of medicated patients. That said, given the cross-sectional nature of the present study, our interpretation here is a tentative one, and requires confirmation with a longitudinal design.Lower FA in the SS was related to medication status , and present only in the cohort of medicated patient. Moreover, the effect remained significant across combinations of datasets when only medicated patients were considered. Therefore, we cannot rule out the possibility that medication impacts WM microstructure. Indeed, drug-induced reductions in the FA of several WM tracts may be seen in OCD37. Both bundles project to posterior parietal, temporal and occipital cortices, and include many major association fibers (including the UNC) where altered microstructure may be related to clinical phenomenology39. Our findings are consistent with a range of work indicating altered connectivity outside the fronto-striatal circuit in OCD. For example, results of a multimodal structural imaging study suggested that patients with OCD show significant alterations of the interrelated gray and WM networks over occipital and parietal cortices, frontal interhemispheric connections, and cerebellum40. Also, decreased functional connectivity in the occipital cortex, temporal cortex, and cerebellum has been shown in OCD41. Finally, there is evidence in OCD of associations between structural and functional alterations in a complex network including, beyond orbitofrontal and cingulate areas, temporal and occipital cortices42.PTR and the SS  convey projection fibers to the posterior part of the brain. Thus, our results strengthen the hypothesis that OCD involves abnormalities affecting an extensive network of regions3 and the more recent multiple brain system approach43. The present findings are based on the TBSS technique, which by reducing WM tracts into a skeleton, confines statistical testing to a selective group of voxels through a constrained local search for maximal FA. Such an algorithm may therefore produce more consistent results, but also more conservative ones, thus explaining the lack of case/control group differences in more anterior WM structures found in previous meta-analysis where whole-brain WM volumetric and FA studies were combined3.We only partially replicate previous large meta-analytic findings demonstrating the validity of both the classic fronto-striatal model of the disorder44 and clinical45 correlates.Notably, in our study WM microstructural alterations in OCD were associated with age at scanning. Specifically, WM alterations were observed in the adult cohort only, and were associated with longer illness duration. These findings, which were unrelated to OCD symptom severity, complement previous evidence of differences between adult and pediatric OCD patients in brain morphological47 underpins myelin vulnerability along a continuum from early to late stages of development and disease48. Thus, it has been suggested that pediatric OCD could be a neurodevelopmental disorder with potentially differing patterns of myelination occurring throughout life49. Indeed, evidence in healthy subjects indicates that the psychiatric trait of compulsivity is linked to reduced myelin growth that emerges only during adolescence (being present only to a minor extent in childhood) as a result of aberrant developmental processes23. Alternatively, pediatric OCD might be a developmentally moderated expression of etiologic processes that are shared with the adult clinical phenotype.There is evidence that the human brain’s protracted myelination37, the technique has some limitations. Indeed, by reducing WM tracts into a skeleton, delineating the center of the tracts and projecting onto it only the highest FA value along the projection, some information might be lost50, and potential artifacts, resulting from misregistration, might be produced51. Nevertheless, several test–retest and reliability analyses were conducted by the ENIGMA-DTI working group to ensure reproducibility of measures and effects using this TBSS approach52. Future investigations combining various imaging modalities in the same meta-analysis and both pre-analyzed and raw data53 will potentially offer insights that are not apparent from the TBSS approach. Moreover, a word of caution is needed regarding the interpretation of the neurobiological basis of DTI measures since although FA reflects the myelination, orientational coherence, and microtubular axonal structure of fibers, other in vivo markers not explored in the present study have been shown to be a more direct reflection of myelination status55. Another potential limitation of the present study may lie in the differences in clinical characteristics between the studied patients, particularly in the average age of onset (which ranged from 4 to 49). Since the latter is often calculated retrospectively, a reliable and unanimous method for establishing this important effect moderator is warranted. Also, we were not able to calculate specific dosages of different medication types and analyze medication effects in terms of drug dosages or total time of treatment and, as such, potential detrimental/normalizing effects of different medications could not be tested. Furthermore, while disorder severity was assessed cross-sectionally at the time of the scan, WM integrity reflects a process that occurred longitudinally over time and this could explain the absence of disorder severity effects. Lastly, it is worth mentioning that while the adult cohort analysis had sufficient power to detect the observed effect size, as the sample size was adequate to detect microstructural differences as small as d = 0.15, the null result in the pediatric cohort may be a consequence of the relatively small sample size since the power for potentially detecting even very small differences was low (0.32). Nevertheless, this is the largest pediatric dataset investigated in a DTI study of OCD.A number of limitations of the data analyzed here deserve emphasis. First, although TBSS is a widely used method for voxel-based analysis of WM, addressing issues associated with smoothing and misalignment in DTI group analysisIn summary, our results clearly indicate a key role in adult OCD for microstructural alterations in projection and association fibers to posterior brain regions. Our meta-regression results related to duration suggest that microstructural alterations may persist during the course of the illness, although longitudinal data are needed to confirm such trajectories. Future studies to investigate the co-occurrence of abnormal WM microstructure, GM volume and metabolic differences in OCD may shed light on the interactions and trajectories of structural and functional alterations in this condition. In particular, longitudinal designs, and collecting information from patients at their illness onset, combined with multimodal MRI approaches, such as volumetric, DTI, fMRI, and MRS will help provide an understanding of the timing and course of brain changes in OCD, and provide greater insight into the mechanisms involved in various stages of OCD, including the long-term effects of medication.Supplementary material


The recent increase in the use of targeted molecular therapy including anti-angiogenetic agents in cancer treatment necessitate the use of robust tools to assess and guide treatment. Angiogenesis, the formation of new disorganized blood vessels, is used by tumor cells to grow and spread using different mechanisms that could be targeted by anti-angiogenetic agents. In this review, we discuss the biological principles of tumor angiogenesis and the imaging modalities that could provide information beyond gross tumor size and morphology to capture the efficacy of anti-angiogenetic therapeutic response.There have been rapid advancements in cancer treatment in recent years, including targeted molecular therapy and the emergence of anti-angiogenic agents, which necessitate the need to quickly and accurately assess treatment response. The ideal tool is robust and non-invasive so that the treatment can be rapidly adjusted or discontinued based on efficacy. Since targeted therapies primarily affect tumor angiogenesis, morphological assessment based on tumor size alone may be insufficient, and other imaging modalities and features may be more helpful in assessing response. This review aims to discuss the biological principles of tumor angiogenesis and the multi-modality imaging evaluation of anti-angiogenic therapeutic responses. In the early 1970s, researchers first proposed that tumor growth is limited by an adequate blood supply. This concept propagated investigative research to explore various angiogenic factors, including the discovery of the human vascular endothelial growth factor (VEGF) in the 1980s. Eventually, researchers translated their growing knowledge about angiogenesis to clinical medicine by introducing angiogenesis inhibitors as a therapy for angiogenic diseases . Due to The non-cytotoxic nature of anti-angiogenic drugs provides a therapeutic advantage over many traditional chemotherapy agents, which generally target disruption of cellular mitosis in both healthy and cancerous cells. However, a relative disadvantage of anti-angiogenic drug use is difficulty in determining the most effective dose and treatment duration since optimal regimens are difficult to establish . UnfortuSince 1980, imaging has played an important role in the evaluation of tumor angiogenesis in preclinical models . HoweverCurrent research is focused on determining imaging regimens for follow-up of anti-angiogenic therapy. Efforts are also dedicated to improving both imaging diagnostic accuracy and anti-angiogenic interventional therapies . SpecifiUnder normal physiological conditions, angiogenic factors are strictly controlled by cellular machinery . HoweverTissue hypoxia stimulates the production of VEGF and fibroblast growth factor (FGF). In response, endothelial cells produce proteases that dissolve cellular basement membranes. This process propagates the formation of vascular buds, which eventually evolve into functional new blood vessels. Subsequent recruitment of accessory cells produce the necessary supporting extracellular matrix for newly formed vessels ,13.Although de novo formation of blood vessels, known as neovascularization, commonly occurs in malignancies, additional angiogenic pathways can also result in new vessel formation . These aAngiogenesis is governed by multiple angiogenic factors that may represent targets for novel molecular-based therapies. Some of these angiogenic elements include hypoxia-induced factors, growth factors, VEGF, platelet-derived growth factors, FGF2, matrix metalloproteinases, alpha v beta 3 integrin, and E-selectin . Among tMany architectural and functional differences exist between vessels formed under physiologic and neoplastic conditions. For example, tumoral angiogenesis results in the rapid overproduction of angiogenic factors, which makes newly formed blood vessels often disorganized and highly permeable to macromolecules ,18,19. VKnowledge of the key differences between normal and abnormal vessels assists clinicians with the diagnosis, treatment, and prognosis of various neoplastic processes. The various structural and functional differences between physiologic and tumoral vessels are appreciated with various molecular and non-molecular imaging methods, including cross-sectional imaging and digital subtraction angiography (DSA) .There are two main methods for imaging angiogenesis in tumoral tissue, including structural and functional assessments. Structural or anatomic evaluations involve molecular and non-molecular imaging, which include CT, MRI, contrast-enhanced ultrasound (CEUS), whole body optic imaging (after tumor labeling with green fluorescence), and positron emission tomography (PET) scan . The molThe second method for evaluating angiogenesis focuses on cellular function and involves various perfusion parameters. These factors include blood flow, mean transient time, regional blood volume and assessment of vascular permeability. Vascular permeability is assessed with dynamic contrast-enhanced MRI or CT . This paNon-invasive imaging methods primarily involve MRI and CT. MRI has various types of contrast agents that can be used. Low-molecular-weight MRI contrast agents, such as the now discontinued gadofosveset trisodium, extravasates at sites of angiogenesis, but also can leak from normal blood vessels. The most commercially used MRI contrast agents are macromolecular, such as Gd-DTPA, and are retained within normal vasculature but leak from vessels formed through a pathologic angiogenesis process, typical of tumors . UltrasmHowever, CT imaging with iodinated contrast is utilized as well, but it introduces ionizing radiation. Ultimately, the decision of CT or MRI is usually made on a case-by-case basis and is often dependent on the region being imaged and institution preference.Catheter angiography/DSA is a more invasive method where contrast is injected directly in the artery proximal to the tumor. This can be achieved using iodinated contrast agents or carbon dioxide (CO2). CO2 has an advantage as it is not nephrotoxic and does not have the risk of allergic reactions like iodinated contrasts . RegardlEven more invasive imaging methods quantify tumor microvascular density (MVD), which is an angiogenesis biomarker calculated from pathologic specimens, usually obtained after biopsy. Theoretically, acquisition of multiple biopsies throughout the course of the disease would provide information regarding response to anti-angiogenic therapy. However, MVD values are not entirely reliable because most tumors are not homogenously vascular . For exaWhen comparing techniques, imaging biomarkers may be advantageous over molecular biomarkers as the former is less invasive and has the added benefit of providing global information about tumor heterogeneity ,32. Althtrans) values, which combine both blood flow and vascular permeability surface area product, were comparable between these two modalities [In various clinical trials evaluating angiogenesis, the imaging modality of choice (MRI versus CT) is based on the availability . The voldalities . Howeverdalities . Additiodalities . Howeverdalities . Perfusidalities . Perfusidalities . With thdalities . Howeverdalities .By comparing image intensity before and after the administration of contrast, blood volume at the region of interest can be calculated, which can be used for follow-up of drug therapy. However, a disadvantage of contrast-enhanced MRI and CT is that contrast may leak through the endothelial barrier in disorganized tumoral neo-vasculature, thus yielding inaccurate measures of blood volume. In comparison, microbubble contrast used in CEUS remains within the intravascular compartment and does not leak through vessel walls like traditional contrast agents, and thus is more accurate for measuring blood volume .A large amount of useful information can be derived from dynamic studies, such as blood volume and blood flow, which may be particularly useful in assessing treatment response and tumor vascular dynamics. Provided that contrast remains within the intravascular compartment during ultrafast imaging techniques, the amount of contrast in each voxel of the image can be tracked and plotted against time such that the area under the curve will correspond to the blood volume . This teFlow can be calculated in similar ways, and although there are several methods, it generally is a measure of how fast the tracer (contrast) diffuses through a tissue . It alsoTwo non-contrast MRI techniques utilized in the evaluation of angiogenesis include arterial spine labeling (ASL) and blood oxygenation level-dependent (BOLD) imaging. ASL is a non-invasive, non-contrast method in which blood is magnetically labeled as it passes towards the organ of interest using RF pulse. The labelled protons interact with the tissue of interest, generating information use to indirectly calculate blood hemodynamics . Using tThe applicability of ASL to extracranial tumors is limited due to transient time artifact, such as those produced by patient movement or respiratory motion. Nevertheless, this technique can be extrapolated to assess vascular changes in various extracranial tumors, like renal and ovarian cancers ,41. ThisThe BOLD imaging technique is based on the fact that oxy and de-oxy hemoglobin exhibit different magnetic characteristics. This technique was initially developed for functional brain imaging, yet due to arteriovenous differences in blood oxygenation, indirect calculation of blood flow can be measured. However, it should be noted that this technique is limited by poor spatial resolution and susceptibility artifacts .Leakage of injected contrast from tumor angiogenic vasculature provides the basis for contrast-enhanced MRI studies. Furthermore, the high spatial resolution of contrast-enhanced MR imaging detects vessels as small as 30–100 micrometers . DCE MRIDCE MRI is performed with a T1-weighted technique to determine perfusion parameters and contrast material leakage. On the other hand, a T2* imaging technique is utilized to calculate cerebral blood flow and is optimized for the assessment of malignancy . In a clThe parameters derived from DCE MRI include the volume of extravascular extracellular space per unit volume, volume of blood plasma per unit volume of tissue, blood vessel permeability, vascular surface area, and interstitial pressure ,49. ThesDynamic susceptibility contrast (DSC) perfusion MRI, also termed bolus-tracking MRI, depends on the first pass metabolism of the contrast agent in the tissue. This technique involves injecting gadolinium chelate into the vascular compartment and then rapidly acquiring images through the region of interest. As contrast passes through the tissue of interest, the perivascular magnetic field is distorted which leads to T2 de-phasing and loss of signal intensity. When the measured signal intensity is plotted against time, hemodynamic parameters like blood volume and flow are calculated .trans and Vp can be calculated from dynamic contrast-enhanced T1 MRI images  such as matrix metalloproteinase. However, these soluble proteins have to be present in sufficient concentration at the vascular compartment in order to bind to protein specific contrast agents .A similar sonographic technique for evaluating angiogenesis is molecular ultrasound imaging, which uses antibodies bound to the microbubble contrast rather than just altering the lipid shells. These antibodies bind vascular surface receptors, which are over-expressed by many tumors, including vascular endothelial growth factor receptor (VEGFR). When combined with subtracted images, regional areas of target overexpression are revealed. In one study, this method showed a significant reduction of contrast binding after antiangiogenic therapy. The suggestion of this finding is a resultant decreased level of VEGFR expression after therapy and, thus, decreased angiogenic activity in the tumor tissue .PET imaging is advantageous for patients as the tracers are generally less toxic compared to MRI and CT contrast agents . Additio18F-FDG-based PET is the most frequently used radiotracer in the clinical setting. Its use in evaluating malignant lesions is dependent on the increased glucose metabolism in malignant tumor cells. However, its role in predicting tumor response to anti-angiogenic therapy is promising but not yet fully validated. Previously, a study revealed a strong association between tumor angiogenesis-related gene expression and 18F-FDG uptake . There aThe targeting of VEGF or its tyrosine kinase receptor (VEGFR) via radiopharmaceuticals in PET or single-photon emission computerized tomography (SPECT) imaging is an exciting prospect. This technique may play an important role in the evaluation of anti-angiogenic treatment response since VEGFR-2 is upregulated in many neoplasms . PreclinFibronectin is an extracellular matrix protein that is expressed in normal tissues. Through alternative mRNA splicing, it can exist as an isoform that expresses an extra domain-B (ED-B). This isoform is found predominantly within fetal and neoplastic adult tissues and is often absent from healthy adult tissues. Malignant cells grow over a background of extracellular matrix proteins that expresses ED-B fibronectin. Thus, this isoform may be a potential target for molecular imaging evaluation of anti-angiogenic treatment response .Other targets for radionuclide-based imaging include the integrins, predominantly alpha v beta 3, which are adhesive glycoproteins for extracellular matrix molecules. Both the alpha and beta subunits play an important role during angiogenesis, as they are upregulated in tumor metastasis to activate matrix metalloproteinase (MMP) that breaks down extracellular matrix . Alpha vMatrix metalloproteinases (MMP) are a group of zinc-dependent proteolytic enzymes with both pro- and anti-angiogenic activities. They are released during angiogenesis to degrade cellular basement membranes and permit branching of newly formed blood vessels. Previously, MMP-2 has been successfully targeted and imaged in mouse models by attaching an MMP-2 inhibitor to Galium-68, a radioisotope currently used in clinical practice . These pAminopeptidase N (APN) is a membrane-bound enzyme that is responsible for amino acid cleavage and has previously been shown to be involved in tumor angiogenesis . ImportaAdvances in ultrasound technology have introduced ultrafast techniques that deliver very high resolution and sensitivity. This allows for detailed characterization of the microvascular environment as well as quantification of blood flow without contrast . AdditioUltrasound with contrast pulse sequences is a real-time perfusion imaging technique with a high specificity and spatial resolution. Multi-pulse contrast imaging suppresses linear back scattering from tissue (noise) while detecting a non-linear signal from the contrast agent (microbubbles). This allows for better assessment of small microvasculature where there is sluggish blood flow and a small concentration of microbubbles. In experimental studies, this technique produces results that are comparable to dynamic contrast-enhanced MRI in terms of perfusion parameters. In addition, contrast-enhanced ultrasound provides real-time imaging and detailed perfusion information during a single cardiac cycle .Photoacoustic imaging is a relatively new advancement demonstrating promising results in clinical imaging. This technique utilizes optical waves (light) to vibrate a tissue, which then emits sonographic waves. A detector can then pick up the emitted wave and convert it to a usable image. There are several inherent advantages to this modality, including high-spatial and -temporal resolution and its ability to use endogenous substances as a contrast . This inThis is a new method which has not yet made its way into clinical imaging but has had several successes in the research and pre-clinical settings. Much like conventional radiography, this method uses ionizing X-rays, but instead of only measuring the degree of beam attenuation, it also accounts for beam distortion and scatter caused by the imaged structure. This results in an image with much improved resolution. An early study performed using both a phantom and a rat kidney found that CO2 angiography resulted in a vessel resolution of around 60 µm with simulations predicting a resolution of around 20 µm in living tissue at reasonable radiation doses . A folloBy using anti-angiogenic drugs, it is postulated that tumor vascular density will decrease more significantly than tumor cells density . If the Assessment of microvascular density in tissue biopsies is a straightforward, yet invasive, evaluation of therapy response . HoweverIn summary, this review highlights various established and experimental methods for evaluating tumor response with anti-angiogenic therapy. While conventional imaging like CT, MRI, and US provide excellent anatomic characterization of tumor tissue, limited microvascular data are available. The addition of contrast agents to these conventional imaging modalities permits quantification of various perfusion parameters and provides insight on vascular function. Finally, molecular imaging utilizes a variety of targets to analyze anti-angiogenic therapy response on a microvascular level and provides a promising avenue for future advances. Regardless of the preferred imaging method, it is imperative to follow up anti-angiogenic treatment response. As these drugs pose many side effects to patients, it is essential to confirm a positive or negative therapeutic response to prevent the unnecessary continuation of these medications . The use